Abstract The intricate landscape of tRNA modification presents
persistent analytical challenges, which have impeded efforts
to simultaneously resolve sequence, modification, and
aminoacylation state at the level of individual tRNAs. To
address these challenges, we introduce “aa-tRNA-seq”, an
integrated method that uses chemical ligation to sandwich the
amino acid of a charged tRNA in between the body of the tRNA
and an adaptor oligonucleotide, followed by high throughput
nanopore sequencing. Our approach reveals the identity of the
amino acids attached to all tRNAs in a cellular sample, at the
single molecule level. We describe machine learning models
that enable the accurate identification of amino acid
identities based on the unique signal distortions generated by
the interactions between the amino acid in the RNA backbone
and the nanopore motor protein and reader head. We apply
aa-tRNA-seq to characterize the impact of the loss of specific
tRNA modification enzymes, confirming the
hypomodification-associated instability of specific tRNAs, and
identifying additional candidate targets of modification. Our
studies lay the groundwork for understanding the efficiency
and fidelity of tRNA aminoacylation as a function of tRNA
sequence, modification, and environmental conditions.
Big1 is a cell cycle regulator linking cell size to basal body
number
Alexander J
Stemm-Wolf, Adam W J
Soh, Lisa E
Mitchell, and
9 more authors
Cell size control in dividing cells coordinates cell growth with
cell division. In the ciliated protozoan, Tetrahymena, there is a
tight link between cell size and the cytoskeletal assemblies at
the cell cortex organized around basal bodies (BBs). BBs dictate
the distribution of ciliary units governing cell motility and are
organized into 18-22 ciliary rows. The number of BBs per cell
remains remarkably consistent even when the number and lengths of
ciliary rows vary. big1-1 mutant cells are large and have elevated
numbers of BBs, providing a system to investigate links between BB
number and cell size control. We discovered BIG1 encodes a protein
with an RRM3 RNA-binding domain similar to the fission yeast
meiotic entry gene, mei2 . The big1-1 mutation is a predicted null
allele. By extending the duration of specific cell cycle stages
conducive to new BB assembly, big1-1 promotes cell size increases
through BB amplification. In contrast, excess Big1 protein
localizes to BBs and drives cells into premature cell division,
resulting in small cells with fewer BBs. Thus, Tetrahymena Big1
localizes to BBs and controls cell cycle progression, indicating
BBs and Big1 link cell growth to the cell division cycle.
Chikungunya virus persists in joint associated macrophages and
promotes chronic disease
Thomas
Morrison, Kristen
Zarrella, Ryan
Sheridan, and
10 more authors
Abstract Arthritogenic alphaviruses including chikungunya, Mayaro,
and Ross River viruses cause long-lasting musculoskeletal pain and
inflammation. However, mechanisms driving chronic disease remain
poorly understood. Here, we investigated joint-associated tissues
in alphavirus-infected mice at a late stage of infection.
Utilizing scRNA-seq, spatial transcriptomics, and flow cytometry
we identified an accumulation of inflammatory macrophages in
joint-associated tissues with elevated Tnf, Nlrp3, Il1b, and H2-Aa
expression, and these cells harbored CHIKV RNA. Moreover, we
identified an accumulation of CD4+ T cells in joint-associated
tissues, which express Ifng. Depletion of CD4+ T cells diminished
MHC-II expression on joint macrophages, highlighting their
potential role in inflammation. In addition, treatment with a
small molecule inhibitor of CHIKV replication during chronic
disease reduced viral RNA and joint inflammation, suggesting that
viral RNA replication promotes chronic joint disease. Our data
suggest that macrophages harbor replicating viral RNA and
contribute to the sustained joint inflammation associated with
chronic alphavirus disease.
Systematic identification and characterization of eukaryotic
and viral 2A peptide-bond-skipping sequences
Deviyani M
Rao, Emma R
Horton, Chloe L
Barrington, and
10 more authors
2A peptides are 18- to 22-amino-acid sequences that cause an
unusual co-translational peptide-bond-skipping event.
Initially discovered in viruses, they allow multiple proteins
to be produced from a single open reading frame. Despite their
utility, their evolutionary prevalence and sequence diversit …
Systematic analysis of nonsense variants uncovers peptide
release rate as a novel modifier of nonsense-mediated mRNA
decay
Divya
Kolakada, Rui
Fu, Nikita
Biziaev, and
10 more authors
The phenotypic impact of nonsense variants is determined by
nonsense-mediated mRNA decay (NMD), which degrades transcripts
with premature termination codons (PTCs). Despite the clinical
importance of nonsense variants, transcript-specific and
context-dependent variations in NMD activity remain poorly
understood. Here, we show that the amino acid preceding the
PTC strongly influences NMD activity. Glycine codons promote
robust NMD efficiency and show striking enrichment before PTCs
but are depleted before normal termination codons. Glycine-PTC
enrichment is particularly pronounced in genes tolerant to
loss-of-function variants, suggesting efficient elimination of
truncated proteins from nonessential genes. We further
demonstrate that the peptide release rate during translation
termination is an important determinant of NMD activity. We
propose a "window of opportunity" model where translation
termination kinetics modulate NMD activity. By revealing how
sequence context shapes NMD activity through translation
termination dynamics, our findings provide a mechanistic
framework for improved clinical interpretation of nonsense
variants.
Activated polyreactive B cells are clonally expanded in
autoantibody positive and patients with recent-onset type 1
diabetes
Catherine A
Nicholas, Fatima A
Tensun, Spencer A
Evans, and
7 more authors
Autoreactive B cells play an important but ill-defined role in
autoimmune type 1 diabetes (T1D). We isolated pancreatic islet
antigen-reactive B cells from the peripheral blood of
non-diabetic autoantibody-negative first-degree relatives,
autoantibody-positive, and recent-onset T1D donors.
Single-cell RNA sequencing analysis revealed that islet
antigen-reactive B cells from autoantibody-positive and T1D
donors had altered gene expression in pathways associated with
B cell signaling and inflammation. Additionally, BCR
sequencing uncovered a similar shift in islet antigen-reactive
B cell repertoires among autoantibody-positive and T1D donors
where greater clonal expansion was also observed. Notably, a
substantial fraction of islet antigen-reactive B cells in
autoantibody-positive and T1D donors appeared to be
polyreactive, which was corroborated by analysis of
recombinant monoclonal antibodies. These results expand our
understanding of autoreactive B cell phenotypes during T1D and
identify unique BCR repertoire changes that may serve as
biomarkers for increased disease risk.
B cells shape naïve CD8 T cell programming
Cameron
Manes, Miguel
Guerrero Moreno, Jennifer
Cimons, and
14 more authors
The presence of B cells is essential for the formation of CD8
T cell memory after infection and vaccination. In this study,
we investigated whether B cells influence the programming of
naïve CD8 T cells prior to their involvement in an immune
response. RNA sequencing indicated that B cells are necessary
for sustaining the FOXO1-controlled transcriptional program,
which is critical for their homeostasis. Without an
appropriate B cell repertoire, mouse naïve CD8 T cells exhibit
a terminal, effector-skewed phenotype, which significantly
impacts their response to vaccination. A similar
effector-skewed phenotype with reduced FOXO1 expression was
observed in naïve CD8 T cells from human patients undergoing B
cell-depleting therapies. Furthermore, we show that patients
without B cells have a defect in generating long-lived CD8 T
cell memory following COVID vaccination. In summary, we
demonstrate that B cells promote the quiescence of naïve CD8 T
cells, poising them to become memory cells upon vaccination.
GPR182 is a lipoprotein receptor for dietary fat absorption
Zhiwei
Sun, Robert J
Torphy, Emily N
Miller, and
16 more authors
The lymphatic system plays a central role in lipid absorption,
which transports chylomicrons from the small intestine to the
circulation. However, the molecular mechanism by which
chylomicrons get into the intestinal lymphatics is unknown. Here
we demonstrated that GPR182, a receptor in lymphatic endothelial
cells (LECs), mediates dietary fat absorption. GPR182 knockout
mice are resistant to dietary-induced obesity. GPR182 ablation in
mice leads to poor lipid absorption and thereby a delay in growth
during development. GPR182 binds and endocytoses lipoproteins
broadly. Mechanistically, loss of GPR182 prevents chylomicrons
from entering the lacteal lumen of the small intestine. GPR182
blockage with a monoclonal antibody (mAb) protects mice from
dietary induced obesity. Together, our study identifies GPR182 as
a lipoprotein receptor that mediates dietary fat absorption.
2024
2024
Co-regulator activity of Mediator of DNA Damage Checkpoint 1
(MDC1) is associated with DNA repair dysfunction and PARP
inhibitor sensitivity in lobular carcinoma of the breast
Joseph L
Sottnik, Madeleine T
Shackleford, Camryn S
Nesiba, and
10 more authors
Invasive lobular carcinoma of the breast (ILC) are typically
estrogen receptor α (ER)-positive and present with biomarkers of
anti-estrogen sensitive disease, yet patients with ILC face
uniquely poor long-term outcomes with increased recurrence risk,
suggesting endocrine response and ER function are unique in ILC.
We previously found specifically in ILC cells that ER is
co-regulated by the DNA repair protein Mediator of DNA Damage
Checkpoint 1 (MDC1). This novel MDC1 activity, however, was
associated with dysfunction in the canonical DNA repair activity
of MDC1, but absent typical features of DNA repair deficiency. To
understand reciprocal activities of MDC1, we profiled the MDC1
interactome and found MDC1-associated proteins in ILC cells mirror
a "BRCA-like" state lacking key homologous recombination (HR)
proteins, consistent with HR dysfunction but distinct from classic
"BRCAness". HR dysfunction in ILC cells was mirrored in
single-cell transcriptome and DNA repair activity analyses, along
with DNA repair signaling and functional data, showing
dysfunctional HR induction and resolution. In parallel, ILC tumor
data are consistent with a distinct form of HR dysfunction via
impaired HR resolution, lacking BRCA-like genomic scarring but
with elevated signatures of PARP inhibitor sensitivity. We tested
whether this HR dysfunction could indeed be exploited using PARP
inhibition and found that talazoparib treatment produced a durable
growth suppression in vitro and in multiple ILC xenografts in
vivo. ILC-specific ER:MDC1 activity creates a new context for ER
and MDC1 function in ILC, at the cost of a DNA repair dysfunction
that is therapeutically targetable.
Nanopore sequencing of intact aminoacylated tRNAs
Laura K
White, Aleksandar
Radakovic, Marcin P
Sajek, and
5 more authors
Transfer RNAs (tRNA) are decorated during biogenesis with a
variety of modifications that modulate their stability,
aminoacylation, and decoding potential during translation. The
complex landscape of tRNA modification presents significant
analysis challenges and to date no single approach enables the
simultaneous measurement of important but disparate chemical
properties of individual, mature tRNA molecules. We developed a
new, integrated approach to analyze the sequence, modification,
and aminoacylation state of tRNA molecules in a high throughput
nanopore sequencing experiment, leveraging a chemical ligation
that embeds the charged amino acid in an adapted tRNA molecule.
During nanopore sequencing, the embedded amino acid generates
unique distortions in ionic current and translocation speed,
enabling application of machine learning approaches to classify
charging status and amino acid identity. Specific applications of
the method indicate it will be broadly useful for examining
relationships and dependencies between tRNA sequence,
modification, and aminoacylation.
Comparative analysis of 43 distinct RNA modifications by nanopore tRNA sequencing
Laura K
White, Kezia
Dobson, Samantha
Pozo, and
10 more authors
Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.Competing Interest StatementL.K.W. has received travel and accommodation expenses from Oxford Nanopore Technologies to present at scientific meetings, and has been a participant in ONT Early Access programs for SQK-RNA004. The remaining authors declare no conflicts of interest.
A specific and portable gene expression program underlies antigen
archiving by lymphatic endothelial cells
Ryan M
Sheridan, Thu A
Doan, Cormac
Lucas, and
5 more authors
Antigens from protein subunit vaccination traffic from the tissue
to the draining lymph node, either passively via the lymph or
carried by dendritic cells at the local injection site. Lymph
node (LN) lymphatic endothelial cells (LEC) actively acquire and
archive foreign antigens, and archived antigen can be released
during subsequent inflammatory stimulus to improve immune
responses. Here, we answer questions about how LECs achieve
durable antigen archiving and whether there are transcriptional
signatures associated with LECs containing high levels of
antigen. We used single cell sequencing in dissociated LN tissue
to quantify antigen levels in LEC and dendritic cell populations
at multiple timepoints after immunization, and used machine
learning to define a unique transcriptional program within
archiving LECs that can predict LEC archiving capacity in
independent data sets. Finally, we validated this modeling,
showing we could predict antigen archiving from a transcriptional
dataset of CHIKV infected mice and demonstrated in vivo the
accuracy of our prediction. Collectively, our findings establish
a unique transcriptional program in LECs that promotes antigen
archiving that can be translated to other systems. ###
Competing Interest Statement The authors have declared no
competing interest.
Immunization-induced antigen archiving enhances local memory
CD8+ T cell responses following an unrelated viral infection
Thu A
Doan, Tadg S
Forward, Johnathon B
Schafer, and
8 more authors
Antigens from viruses or immunizations can persist or are
archived in lymph node stromal cells such as lymphatic
endothelial cells (LEC) and fibroblastic reticular cells (FRC).
Here, we find that, during the time frame of antigen archiving,
LEC apoptosis caused by a second, but unrelated, innate immune
stimulus such as vaccina viral infection or CpG DNA
administration resulted in cross-presentation of archived
antigens and boosted memory CD8 + T cells specific to the
archived antigen. In contrast to “bystander” activation
associated with unrelated infections, the memory CD8 + T cells
specific to the archived antigen from the immunization were
significantly higher than memory CD8 + T cells of a different
antigen specificity. Finally, the boosted memory CD8 + T cells
resulted in increased protection against Listeria monocytogenes
expressing the antigen from the immunization, but only for the
duration that the antigen was archived. These findings outline an
important mechanism by which lymph node stromal cell archived
antigens, in addition to bystander activation, can augment memory
CD8 + T cell responses during repeated inflammatory insults.
Peptidyl-tRNA hydrolysis rate influences the efficiency of
nonsense-mediated mRNA decay
Divya
Kolakada, Rui
Fu, Nikita
Biziaev, and
9 more authors
Nonsense-mediated mRNA decay (NMD) is a quality control mechanism
that prevents the accumulation of harmful truncated proteins by
degrading transcripts with premature termination codons (PTCs).
NMD efficiency varies across many contexts, but the factors that
influence this variability remain poorly understood. Here, we
find an enrichment of glycine (Gly) codons preceding a PTC in
common nonsense variants in contrast with a depletion of Gly
codons preceding a normal termination codon (NTC). Gly-PTC
contexts have higher NMD activity compared to an alanine-PTC
context, and this effect is stronger on NMD substrates with long
3’UTRs. We used a massively parallel reporter assay to test all
possible combinations of -2 and -1 codons, the PTC, and the +4
nucleotide to assess comprehensively how PTC sequence context
affects NMD efficiency. A random forest classifier revealed that
peptidyl-tRNA hydrolysis rate during translation termination was
the most important feature in discriminating high and low NMD
activity. We show with in vitro biochemical assays that Gly-TC
contexts have the slowest termination rate compared to other
codons. Furthermore, Gly-PTC enrichment is most pronounced in
genes that tolerate loss-of-function variants, suggesting that
enhanced NMD of Gly-PTC context has shaped the evolution of PTCs.
Based on these findings, we propose that NMD efficiency is
modulated by the “window of opportunity” offered by peptidyl
tRNA hydrolysis rate and thus, translation termination kinetics.
Chikungunya virus infection disrupts lymph node lymphatic
endothelial cell composition and function via MARCO
Cormac J
Lucas, Ryan M
Sheridan, Glennys V
Reynoso, and
7 more authors
Infection with chikungunya virus (CHIKV) causes disruption of
draining lymph node (dLN) organization, including paracortical
relocalization of B cells, loss of the B cell-T cell border, and
lymphocyte depletion that is associated with infiltration of the
LN with inflammatory myeloid cells. Here, we found that, during
the first 24 hours of infection, CHIKV RNA accumulated in
MARCO-expressing lymphatic endothelial cells (LECs) in both the
floor and medullary LN sinuses. The accumulation of viral RNA in
the LN was associated with a switch to an antiviral and
inflammatory gene expression program across LN stromal cells, and
this inflammatory response - including recruitment of myeloid
cells to the LN - was accelerated by CHIKV-MARCO interactions. As
CHIKV infection progressed, both floor and medullary LECs
diminished in number, suggesting further functional impairment of
the LN by infection. Consistent with this idea, antigen
acquisition by LECs, a key function of LN LECs during infection
and immunization, was reduced during pathogenic CHIKV infection.
2023
2023
Vaccine-induced antigen archiving enhances local memory CD8+
T cell responses following an unrelated viral infection
Beth
Tamburini, Thu
Doan, Tadg
Forward, and
5 more authors
Viral and vaccine antigens persist or are archived in lymph node
stromal cells (LNSC) such as lymphatic endothelial cells (LEC)
and fibroblastic reticular cells (FRC). Here, we find that,
during the time frame of antigen archiving, LEC apoptosis caused
by a second, but unrelated, innate immune stimulus such as
vaccina viral infection or CpG DNA administration boosted memory
CD8+ T cells specific to the archived antigen. In contrast to
“bystander” activation associated with unrelated infections,
the memory CD8+ T cells specific to the vaccine archived antigen
were significantly higher than memory CD8+ T cells of a different
antigen specificity. Finally, the boosted memory CD8+ T cells
resulted in increased protection against Listeria monocytogenes
expressing the vaccine antigen, but only for the duration that
the vaccine antigen was archived. These findings outline a novel
mechanism by which LNSC archived antigens, in addition to
bystander activation, can augment memory CD8+ T cell responses
during repeated inflammatory insults.
Vaccine adjuvant-elicited CD8+ T cell immunity is
co-dependent on T-bet and FOXO1
Daria L
Ivanova, Scott B
Thompson, Jared
Klarquist, and
6 more authors
T-bet and FOXO1 are transcription factors canonically associated
with effector and memory T cell fates, respectively. During an
infectious response, these factors direct the development of CD8+
T cell fates, where T-bet deficiency leads to ablation of only
short-lived effector cells, while FOXO1 deficiency results in
selective loss of memory. In contrast, following adjuvanted
subunit vaccination in mice, both effector- and memory-fated T
cells are compromised in the absence of either T-bet or FOXO1.
Thus, unlike responses to challenge with Listeria monocytogenes,
productive CD8+ T cell responses to adjuvanted vaccination
require coordinated regulation of FOXO1 and T-bet transcriptional
programs. Single-cell RNA sequencing analysis confirms
simultaneous T-bet, FOXO1, and TCF1 transcriptional activity in
vaccine-elicited, but not infection-elicited, T cells undergoing
clonal expansion. Collectively, our data show that subunit
vaccine adjuvants elicit T cell responses dependent on
transcription factors associated with effector and memory cell
fates.
Nanopore sequencing of internal 2’-PO4 modifications installed
by RNA repair
Laura K
White, Saylor M
Strugar, Andrea
MacFadden, and
1 more author
Ligation by plant and fungal RNA ligases yields an internal
2’-phosphate group on each RNA ligation product. In budding
yeast, this covalent mark occurs at the splice junction of two
targets of ligation: intron-containing tRNAs and the messenger
RNA HAC1 The repertoire of RNA molecules repaired by RNA ligation
has not been explored due to a lack of unbiased approaches for
identifying RNA ligation products. Here, we define several unique
signals produced by 2’-phosphorylated RNAs during nanopore
sequencing. A 2’-phosphate at the splice junction of HAC1 mRNA
inhibits 5’ →3’ degradation, enabling detection of
decay intermediates in yeast RNA repair mutants by nanopore
sequencing. During direct RNA sequencing, intact
2’-phosphorylated RNAs on HAC1 and tRNAs produce diagnostic
changes in nanopore current properties and base calling features,
including stalls produced as the modified RNA translocates
through the nanopore motor protein. These approaches enable
directed studies to identify novel RNA repair events in other
contexts.
Next generation sequencing (NGS) has provided biologists with an
unprecedented view into biological processes and their regulation
over the past 2 decades, fueling a wave of development of high
throughput methods based on short read DNA and RNA sequencing.
For nucleic acid modifications, NGS has been coupled with
immunoprecipitation, chemical treatment, enzymatic treatment,
and/or the use of reverse transcriptase enzymes with fortuitous
activities to enrich for and to identify covalent modifications
of RNA and DNA. However, the majority of nucleic acid
modifications lack commercial monoclonal antibodies, and mapping
techniques that rely on chemical or enzymatic treatments to
manipulate modification signatures add additional technical
complexities to library preparation. Moreover, such approaches
tend to be specific to a single class of RNA or DNA modification,
and generate only indirect readouts of modification status. Third
generation sequencing technologies such as the commercially
available “long read” platforms from Pacific Biosciences and
Oxford Nanopore Technologies are an attractive alternative for
high throughput detection of nucleic acid modifications. While
the former can indirectly sense modified nucleotides through
changes in the kinetics of reverse transcription reactions,
nanopore sequencing can in principle directly detect any nucleic
acid modification that produces a signal distortion as the
nucleic acid passes through a nanopore sensor embedded within a
charged membrane. To date, more than a dozen endogenous DNA and
RNA modifications have been interrogated by nanopore sequencing,
as well as a number of synthetic nucleic acid modifications used
in metabolic labeling, structure probing, and other emerging
applications. This review is intended to introduce the reader to
nanopore sequencing and key principles underlying its use in
direct detection of nucleic acid modifications in unamplified DNA
or RNA samples, and outline current approaches for detecting and
quantifying nucleic acid modifications by nanopore sequencing. As
this technology matures, we anticipate advances in both
sequencing chemistry and analysis methods will lead to rapid
improvements in the identification and quantification of these
epigenetic marks.
scraps: an end-to-end pipeline for measuring alternative
polyadenylation at high resolution using single-cell RNA-seq
Rui
Fu, Kent A
Riemondy, Ryan M
Sheridan, and
3 more authors
Alternative cleavage and polyadenylation (APA) contributes to the
diversity of mRNA 3’ ends, affecting post-transcriptional
regulation by including or excluding cis -regulatory elements in
mRNAs, altering their stability and translational efficiency.
While APA analysis has been applied broadly in mixed populations
of cells, the heterogeneity of APA among single cells has only
recently begun to be explored. We developed an approach we termed
scraps (Single Cell RNA PolyA Site Discovery), implemented as a
user-friendly, scalable, and reproducible end-to-end workflow, to
identify polyadenylation sites at near-nucleotide resolution in
single cells using 10X Genomics and other TVN-primed single-cell
RNA-seq (scRNA-seq) libraries. Our approach, which performs best
with long (>100bp) read 1 sequencing and paired alignment to the
genome, is both unbiased relative to existing methods that
utilize only read 2 and recovers more sites at higher resolution,
despite the reduction in read quality observed on most modern DNA
sequencers following homopolymer stretches. For libraries
sequenced without long read 1, we implement a fallback approach
using read 2-only alignments that performs similarly to our
optimal approach, but recovers far fewer polyadenylation sites
per experiment. scraps also enables assessment of internal
priming capture events, which we demonstrate occur commonly but
at higher frequency during apoptotic 3’ RNA decay. We also
provide an R package, scrapR, that integrates the results of the
scaps pipeline with the popular Seruat single-cell analysis
package. Refinement and expanded application of these approaches
will further clarify the role of APA in single cells, as well as
the effects of internal priming on expression measurements in
scRNA-seq libraries. ### Competing Interest Statement The
authors have declared no competing interest.
2021
2021
Haploinsufficiency, Dominant Negative, and Gain-of-Function
Mechanisms in Epilepsy: Matching Therapeutic Approach to the
Pathophysiology
Gemma L
Carvill, Tyler
Matheny, Jay
Hesselberth, and
1 more author
This review summarizes the pathogenic mechanisms that underpin
the monogenic epilepsies and discusses the potential of novel
precision therapeutics to treat these disorders. Pathogenic
mechanisms of epilepsy include recessive (null alleles),
haploinsufficiency, imprinting, gain-of-function, and dominant
negative effects. Understanding which pathogenic mechanism(s)
that underlie each genetic epilepsy is pivotal to design
precision therapies that are most likely to be beneficial for the
patient. Novel therapeutics discussed include gene therapy, gene
editing, antisense oligonucleotides, and protein replacement.
Discussions are illustrated and reinforced with examples from the
literature.
MARCO+ lymphatic endothelial cells sequester arthritogenic
alphaviruses to limit viremia and viral dissemination
Kathryn S
Carpentier, Ryan M
Sheridan, Cormac J
Lucas, and
10 more authors
Viremia in the vertebrate host is a major determinant of
arboviral reservoir competency, transmission efficiency, and
disease severity. However, immune mechanisms that control
arboviral viremia are poorly defined. Here, we identify critical
roles for the scavenger receptor MARCO in controlling viremia
during arthritogenic alphavirus infections in mice. Following
subcutaneous inoculation, arthritogenic alphavirus particles
drain via the lymph and are rapidly captured by MARCO+ lymphatic
endothelial cells (LECs) in the draining lymph node (dLN),
limiting viral spread to the bloodstream. Upon reaching the
bloodstream, alphavirus particles are cleared from the
circulation by MARCO-expressing Kupffer cells in the liver,
limiting viremia and further viral dissemination. MARCO-mediated
accumulation of alphavirus particles in the draining lymph node
and liver is an important host defense mechanism as viremia and
viral tissue burdens are elevated in MARCO-/- mice and disease is
more severe. In contrast to prior studies implicating a key role
for lymph node macrophages in limiting viral dissemination, these
findings exemplify a previously unrecognized arbovirus-scavenging
role for lymphatic endothelial cells and improve our mechanistic
understanding of viremia control during arthritogenic alphavirus
infection.
Cell-level metadata are indispensable for documenting single-cell
sequencing datasets
Sidhant
Puntambekar, Jay R
Hesselberth, Kent A
Riemondy, and
1 more author
Single-cell RNA sequencing (scRNA-seq) provides an unprecedented
view of cellular diversity of biological systems. However, across
the thousands of publications and datasets generated using this
technology, we estimate that only a minority (<25%) of studies
provide cell-level metadata information containing identified
cell types and related findings of the published dataset.
Metadata omission hinders reproduction, exploration, validation,
and knowledge transfer and is a common problem across journals,
data repositories, and publication dates. We encourage
investigators, reviewers, journals, and data repositories to
improve their standards and ensure proper documentation of these
valuable datasets.
Neoplastic and immune single cell transcriptomics define
subgroup-specific intra-tumoral heterogeneity of childhood
medulloblastoma
Kent A
Riemondy, Sujatha
Venkataraman, Nicholas
Willard, and
18 more authors
BACKGROUND: Medulloblastoma (MB) is a heterogeneous disease in
which neoplastic cells and associated immune cells contribute to
disease progression. We aimed to determine the influence of
neoplastic and immune cell diversity on MB biology in patient
samples and animal models. METHODS: To better characterize
cellular heterogeneity in MB we used single-cell RNA sequencing,
immunohistochemistry and deconvolution of transcriptomic data to
profile neoplastic and immune populations in patient samples and
animal models across childhood MB subgroups. RESULTS: Neoplastic
cells cluster primarily according to individual sample of origin
which is influenced by chromosomal copy number variance. Harmony
alignment reveals novel MB subgroup/subtype-associated
subpopulations that recapitulate neurodevelopmental processes,
including photoreceptor and glutamatergic neuron-like cells in
molecular subgroups GP3 and GP4, and a specific nodule-associated
neuronally-differentiated subpopulation in subgroup molecular
SHH. We definitively chart the spectrum of MB immune cell
infiltrates, which include subpopulations that recapitulate
developmentally-related neuron-pruning and antigen presenting
myeloid cells. MB cellular diversity matching human samples is
mirrored in subgroup-specific mouse models of MB. CONCLUSIONS:
These findings provide a clearer understanding of the diverse
neoplastic and immune cell subpopulations that constitute the MB
microenvironment.
Molecular tracking devices quantify antigen distribution and
archiving in the murine lymph node
Shannon M
Walsh, Ryan M
Sheridan, Erin D
Lucas, and
7 more authors
The detection of foreign antigens in vivo has relied on
fluorescent conjugation or indirect read-outs such as antigen
presentation. In our studies, we found that these widely used
techniques had several technical limitations that have precluded
a complete picture of antigen trafficking or retention across
lymph node cell types. To address these limitations, we
developed a ’molecular tracking device’ to follow the
distribution, acquisition, and retention of antigen in the lymph
node. Utilizing an antigen conjugated to a nuclease-resistant
DNA tag, acting as a combined antigen-adjuvant conjugate, and
single-cell mRNA sequencing, we quantified antigen abundance in
the lymph node. Variable antigen levels enabled the
identification of caveolar endocytosis as a mechanism of antigen
acquisition or retention in lymphatic endothelial cells. Thus,
these molecular tracking devices enable new approaches to study
dynamic tissue dissemination of antigen-adjuvant conjugates and
identify new mechanisms of antigen acquisition and retention at
cellular resolution in vivo.
2020
2020
Enhancing Hematopoiesis from Murine Embryonic Stem Cells through
MLL1-Induced Activation of a Rac/Rho/Integrin Signaling Axis
Weiwei
Yang, G Devon
Trahan, Elizabeth D
Howell, and
7 more authors
The Mixed Lineage Leukemia (MLL1, KMT2A) gene is critical for
development and maintenance of hematopoietic stem cells (HSCs),
however, whether this protein is limiting for HSC development is
unknown due to lack of physiologic model systems. Here, we
develop an MLL1-inducible embryonic stem cell (ESC) system and
show that induction of wild-type MLL1 during ESC differentiation
selectively increases hematopoietic potential from a transitional
c-Kit+/Cd41+ population in the embryoid body and also at sites of
hematopoiesis in embryos. Single-cell sequencing analysis
illustrates inherent heterogeneity of the c-Kit+/Cd41+ population
and demonstrates that MLL1 induction shifts its composition
toward multilineage hematopoietic identities. Surprisingly, this
does not occur through increasing Hox or other canonical MLL1
targets but through an enhanced Rac/Rho/integrin signaling state,
which increases responsiveness to Vla4 ligands and enhances
hematopoietic commitment. Together, our data implicate a
Rac/Rho/integrin signaling axis in the endothelial to
hematopoietic transition and demonstrate that MLL1 actives this
axis.
Dynamic RNA Regulation in the Brain Underlies Physiological
Plasticity in a Hibernating Mammal
Rui
Fu, Austin E
Gillen, Katharine R
Grabek, and
5 more authors
Hibernation is a physiological and behavioral phenotype that
minimizes energy expenditure. Hibernators cycle between profound
depression and rapid hyperactivation of multiple physiological
processes, challenging our concept of mammalian homeostasis. How
the hibernator orchestrates and survives these extremes while
maintaining cell to organismal viability is unknown. Here, we
enhance the genome integrity and annotation of a model
hibernator, the 13-lined ground squirrel. Our new assembly brings
this genome to near chromosome-level contiguity and adds
thousands of previously unannotated genes. These new genomic
resources were used to identify 6,505 hibernation-related,
differentially-expressed and processed transcripts using RNA-seq
data from three brain regions in animals whose physiological
status was precisely defined using body temperature telemetry. A
software tool, squirrelBox, was developed to foster further data
analyses and visualization. SquirrelBox includes a comprehensive
toolset for rapid visualization of gene level and cluster group
dynamics, sequence scanning of k-mer and domains, and interactive
exploration of gene lists. Using these new tools and data, we
deconvolute seasonal from temperature-dependent effects on the
brain transcriptome during hibernation for the first time,
highlighting the importance of carefully timed samples for
studies of differential gene expression in hibernation. The
identified genes include a regulatory network of RNA binding
proteins that are dynamic in hibernation along with the
composition of the RNA pool. In addition to passive effects of
temperature, we provide evidence for regulated transcription and
RNA turnover during hibernation. Significant alternative
splicing, largely temperature dependent, also occurs during
hibernation. These findings form a crucial first step and provide
a roadmap for future work toward defining novel mechanisms of
tissue protection and metabolic depression that may 1 day be
applied toward improving human health.
clustifyr: an R package for automated single-cell RNA
sequencing cluster classification
Rui
Fu, Austin E
Gillen, Ryan M
Sheridan, and
5 more authors
Assignment of cell types from single-cell RNA sequencing
(scRNA-seq) data remains a time-consuming and error-prone
process. Current packages for identity assignment use limited
types of reference data and often have rigid data structure
requirements. We developed the clustifyr R package to leverage
several external data types, including gene expression profiles
to assign likely cell types using data from scRNA-seq, bulk
RNA-seq, microarray expression data, or signature gene lists. We
benchmark various parameters of a correlation-based approach and
implement gene list enrichment methods. clustifyr is a
lightweight and effective cell-type assignment tool developed for
compatibility with various scRNA-seq analysis workflows.
clustifyr is publicly available at
https://github.com/rnabioco/clustifyr.
Simultaneous measurement of biochemical phenotypes and gene
expression in single cells
Amanda L
Richer, Kent A
Riemondy, Lakotah
Hardie, and
1 more author
Methods to measure heterogeneity among cells are rapidly
transforming our understanding of biology but are currently
limited to molecular abundance measurements. We developed an
approach to simultaneously measure biochemical activities and
mRNA abundance in single cells to understand the heterogeneity
of DNA repair activities across thousands of human lymphocytes,
identifying known and novel cell-type-specific DNA repair
phenotypes.
Physiologic RNA targets and refined sequence specificity of
coronavirus EndoU
Rachel
Ancar, Yize
Li, Eveline
Kindler, and
6 more authors
Coronavirus EndoU inhibits dsRNA-activated antiviral responses;
however, the physiologic RNA substrates of EndoU are unknown. In
this study, we used mouse hepatitis virus (MHV)-infected bone
marrow-derived macrophage (BMM) and cyclic phosphate cDNA
sequencing to identify the RNA targets of EndoU. EndoU targeted
viral RNA, cleaving the 3’ side of pyrimidines with a strong
preference for U A and C A sequences (endoY A).
EndoU-dependent cleavage was detected in every region of MHV RNA,
from the 5’ NTR to the 3’ NTR, including transcriptional
regulatory sequences (TRS). Cleavage at two CA dinucleotides
immediately adjacent to the MHV poly(A) tail suggests a mechanism
to suppress negative-strand RNA synthesis and the accumulation of
viral dsRNA. MHV with EndoU (EndoUmut) or 2’-5’ phosphodiesterase
(PDEmut) mutations provoked the activation of RNase L in BMM,
with corresponding cleavage of RNAs by RNase L. The physiologic
targets of EndoU are viral RNA templates required for
negative-strand RNA synthesis and dsRNA accumulation. Coronavirus
EndoU cleaves U A and C A sequences (endoY A) within viral
(+) strand RNA to evade dsRNA-activated host responses.
Single-Cell RNA Sequencing of Childhood Ependymoma Reveals
Neoplastic Cell Subpopulations That Impact Molecular
Classification and Etiology
Austin E
Gillen, Kent A
Riemondy, Vladimir
Amani, and
14 more authors
Ependymoma (EPN) is a brain tumor commonly presenting in
childhood that remains fatal in most children. Intra-tumoral
cellular heterogeneity in bulk-tumor samples significantly
confounds our understanding of EPN biology, impeding development
of effective therapy. We, therefore, use single-cell RNA
sequencing, histology, and deconvolution to catalog cellular
heterogeneity of the major childhood EPN subgroups. Analysis of
PFA subgroup EPN reveals evidence of an undifferentiated
progenitor subpopulation that either differentiates into
subpopulations with ependymal cell characteristics or transitions
into a mesenchymal subpopulation. Histological analysis reveals
that progenitor and mesenchymal subpopulations co-localize in
peri-necrotic zones. In conflict with current classification
paradigms, relative PFA subpopulation proportions are shown to
determine bulk-tumor-assigned subgroups. We provide an
interactive online resource that facilitates exploration of the
EPN single-cell dataset. This atlas of EPN cellular heterogeneity
increases understanding of EPN biology.
Custom-designed, degradation-resistant messenger RNAs in yeast
Ana L
Franklin, Andrea
Macfadden, Jeffrey S
Kieft, and
2 more authors
Structured RNA elements that protect RNA transcripts from
5’\rightarrow3’ degradation are becoming useful research tools.
Here we show that exonuclease-resistant RNA structures (xrRNAs)
from Flaviviruses can be used to protect heterologous messenger
RNAs (mRNAs) from 5’\rightarrow3’ degradation in budding yeast.
Installation of xrRNAs ahead of a downstream internal ribosome
entry site (IRES) leads to the accumulation of partially-degraded
mRNAs that are substrates for cap-independent translation of a
LacZ reporter, yielding a 30-fold increase in measured
β-galactosidase activity. Additionally, by monitoring the
translation of dual-luciferase reporters we show that xrRNA
sequences do not interfere with the progression of an elongating
ribosome. Combined these data indicate that xrRNA elements can be
used in creative ways to stabilize RNAs with potentially useful
applications. ### Competing Interest Statement AM, JSK, JRH
and EGC are listed as inventors of PCT/US2016/066723 that
describes Protecting RNAs From Degradation Using Engineered Viral
RNAs. This patent is owned by the Reagents of the University of
Colorado.
Single cell profiling identifies novel B cell populations that
drive chronic rejection after lung transplantation
N
Smirnova, K
Riemondy, S
Collins, and
2 more authors
Methods of measuring multiple enzyme activities in parallel in a
sequencing-based assay to characterize enzyme activities in
individual mammalian cells. In preferred implementations, the
methods involve forming microfluidic droplets containing
oligonucleotide functionalized microbeads and single mammalian
cells, lysing the cells, and allowing enzyme activity on enzyme
substrates present in the oligonucleotides, isolating the
individual microbeads, and determining the enzymatic activity to
quantitate and evaluate the enzymatic activity or capacity of the
cells.
Single-cell RNAseq of childhood ependymoma reveals distinct
neoplastic cell subpopulations that impact etiology, molecular
classification and outcome
Austin
Gillen, Kent
Riemondy, Vladimir
Amani, and
14 more authors
Liver lymphatic vessels support liver function by draining
interstitial fluid, cholesterol, fat, and immune cells for
surveillance in the liver draining lymph node. Chronic liver
disease is associated with increased inflammation and immune
cell infiltrate. However, it is currently unknown if or how
lymphatic vessels respond to increased inflammation and immune
cell infiltrate in the liver during chronic disease. Here we
demonstrate that lymphatic vessel abundance increases in
patients with chronic liver disease and is associated with areas
of fibrosis and immune cell infiltration. Using single-cell mRNA
sequencing and multi-spectral immunofluorescence analysis we
identified liver lymphatic endothelial cells and found that
chronic liver disease results in lymphatic endothelial cells
(LECs) that are in active cell cycle with increased expression
of CCL21. Additionally, we found that LECs from patients with
NASH adopt a transcriptional program associated with increased
IL13 signaling. Moreover, we found that oxidized low density
lipoprotein, associated with NASH pathogenesis, induced the
transcription and protein production of IL13 in LECs both in
vitro and in a mouse model. Finally, we show that oxidized low
density lipoprotein reduced the transcription of PROX1 and
decreased lymphatic stability. Together these data indicate that
LECs are active participants in the liver, expanding in an
attempt to maintain tissue homeostasis. However, when
inflammatory signals, such as oxidized low density lipoprotein
are increased, as in NASH, lymphatic function declines and liver
homeostasis is impeded.
Single cell RNA sequencing identifies TGFβ as a key
regenerative cue following LPS-induced lung injury
Kent A
Riemondy, Nicole L
Jansing, Peng
Jiang, and
8 more authors
Many lung diseases result from a failure of efficient
regeneration of damaged alveolar epithelial cells (AECs) after
lung injury. During regeneration, AEC2s proliferate to replace
lost cells, after which proliferation halts and some AEC2s
transdifferentiate into AEC1s to restore normal alveolar
structure and function. Although the mechanisms underlying AEC2
proliferation have been studied, the mechanisms responsible for
halting proliferation and inducing transdifferentiation are
poorly understood. To identify candidate signaling pathways
responsible for halting proliferation and inducing
transdifferentiation, we performed single cell RNA sequencing on
AEC2s during regeneration in a murine model of lung injury
induced by intratracheal LPS. Unsupervised clustering revealed
distinct subpopulations of regenerating AEC2s: proliferating,
cell cycle arrest, and transdifferentiating. Gene expression
analysis of these transitional subpopulations revealed that
TGFβsignaling was highly upregulated in the cell cycle
arrest subpopulation and relatively downregulated in
transdifferentiating cells. In cultured AEC2s, TGFβwas
necessary for cell cycle arrest but impeded
transdifferentiation. We conclude that during regeneration after
LPS-induced lung injury, TGFβis a critical signal halting
AEC2 proliferation but must be inactivated to allow
transdifferentiation. This study provides insight into the
molecular mechanisms regulating alveolar regeneration and the
pathogenesis of diseases resulting from a failure of
regeneration.
Recovery and analysis of transcriptome subsets from pooled
single-cell RNA-seq libraries
Kent A
Riemondy, Monica
Ransom, Christopher
Alderman, and
8 more authors
Single-cell RNA sequencing (scRNA-seq) methods generate sparse
gene expression profiles for thousands of single cells in a
single experiment. The information in these profiles is
sufficient to classify cell types by distinct expression
patterns but the high complexity of scRNA-seq libraries often
prevents full characterization of transcriptomes from individual
cells. To extract more focused gene expression information from
scRNA-seq libraries, we developed a strategy to physically
recover the DNA molecules comprising transcriptome subsets,
enabling deeper interrogation of the isolated molecules by
another round of DNA sequencing. We applied the method in
cell-centric and gene-centric modes to isolate cDNA fragments
from scRNA-seq libraries. First, we resampled the transcriptomes
of rare, single megakaryocytes from a complex mixture of
lymphocytes and analyzed them in a second round of DNA
sequencing, yielding up to 20-fold greater sequencing depth per
cell and increasing the number of genes detected per cell from a
median of 1313 to 2002. We similarly isolated mRNAs from
targeted T cells to improve the reconstruction of their
VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D
mRNA fragments expressed across cells in a scRNA-seq library
prepared from a clonal T cell line, increasing the number of
cells with detected CD3D expression from 59.7% to 100%.
Transcriptome resampling is a general approach to recover
targeted gene expression information from single-cell RNA
sequencing libraries that enhances the utility of these costly
experiments, and may be applicable to the targeted recovery of
molecules from other single-cell assays.
Multiple decay events target HAC1 mRNA during splicing to
regulate the unfolded protein response
Patrick D
Cherry, Sally E
Peach, and Jay R
Hesselberth
In the unfolded protein response (UPR), stress in the
endoplasmic reticulum (ER) activates a large transcriptional
program to increase ER folding capacity. During the budding
yeast UPR, Ire1 excises an intron from the HAC1 mRNA and the
exon products of cleavage are ligated, and the translated
protein induces hundreds of stress-response genes. Using cells
with mutations in RNA repair and decay enzymes, we show that
phosphorylation of two different HAC1 splicing intermediates is
required for their degradation by the 5’\rightarrow3’
exonuclease Xrn1 to enact opposing effects on the UPR. We also
found that ligated but 2’-phosphorylated HAC1 mRNA is cleaved,
yielding a decay intermediate with both 5’- and 2’-phosphates at
its 5’-end that inhibit 5’\rightarrow3’ decay and suggesting
that Ire1 degrades incompletely processed HAC1. These decay
events expand the scope of RNA-based regulation in the budding
yeast UPR and have implications for the control of the metazoan
UPR.
Although Cdc7 protein kinase is important for regulating DNA
replication in all eukaryotes and is a target for cancer therapy,
it has never been localized in cells. Recently, a novel molecular
genomic method used by our laboratory to localize Cdc7 to regions
of chromosomes. Originally, mutations in the CDC7 gene were found
in the classic cdc mutant collection of Hartwell et al. (Genetics
74:267-286, 1973). The CDC7 gene was found to encode a protein
kinase called DDK that has been studied for many years,
establishing its precise role in the initiation of DNA
replication at origins. Recently, clinical studies are underway
with DDK inhibitors against DDK in cancer patients. However, the
conundrum is that Cdc7 has never been detected at origins of
replication even though many studies have suggested it should be
there. We used “Calling Card” system in which DNA binding
proteins are localized to the genome via retrotransposon
insertion and deep-sequencing methods. We have shown that Cdc7
localizes at many regions of the genome and was enriched at
functional origins of replication. These results are consistent
with DDK’s role in many additional genomic processes including
mutagenesis, chromatid cohesion, and meiotic recombination. Thus,
the main conclusion from our studies is that Cdc7 kinase is found
at many locations in the genome, but is enriched at functional
origins of replication. Furthermore, we propose that application
of the Calling Card system to other eukaryotes should be useful
in identification of functional origins in other eukaryotic
cells.
High-Resolution Mapping of Modified DNA Nucleobases Using
Excision Repair Enzymes
Monica
Ransom, D Suzi
Bryan, and Jay R
Hesselberth
Modification of DNA nucleobases has a profound effect on genome
function. We developed a method that maps the positions of the
modified DNA nucleobases throughout genomic DNA. This method
couples in vitro nucleobase excision with massively parallel DNA
sequencing to determine the location of modified DNA nucleobases
with single base precision. This protocol was used to map uracil
incorporation and UV photodimers in DNA, and a modification of
the protocol has been used to map sparse modification events in
cells. The Excision-seq protocol is broadly applicable to a
variety of base modifications for which an excision enzyme is
available.
TCR signal strength controls thymic differentiation of iNKT
cell subsets
Kathryn D
Tuttle, S Harsha
Krovi, Jingjing
Zhang, and
13 more authors
During development in the thymus, invariant natural killer T
(iNKT) cells commit to one of three major functionally different
subsets, iNKT1, iNKT2, and iNKT17. Here, we show that T cell
antigen receptor (TCR) signal strength governs the development of
iNKT cell subsets, with strong signaling promoting iNKT2 and
iNKT17 development. Altering TCR diversity or signaling
diminishes iNKT2 and iNKT17 cell subset development in a
cell-intrinsic manner. Decreased TCR signaling affects the
persistence of Egr2 expression and the upregulation of PLZF. By
genome-wide comparison of chromatin accessibility, we identify a
subset of iNKT2-specific regulatory elements containing NFAT and
Egr binding motifs that is less accessible in iNKT2 cells that
develop from reduced TCR signaling. These data suggest that
variable TCR signaling modulates regulatory element activity at
NFAT and Egr binding sites exerting a determinative influence on
the dynamics of gene enhancer accessibility and the developmental
fate of iNKT cells.
Venetoclax with azacitidine disrupts energy metabolism and
targets leukemia stem cells in patients with acute myeloid
leukemia
Daniel A
Pollyea, Brett M
Stevens, Courtney L
Jones, and
14 more authors
Acute myeloid leukemia (AML) is the most common acute leukemia in
adults. Leukemia stem cells (LSCs) drive the initiation and
perpetuation of AML, are quantifiably associated with worse
clinical outcomes, and often persist after conventional
chemotherapy resulting in relapse1-5. In this report, we show
that treatment of older patients with AML with the B cell
lymphoma 2 (BCL-2) inhibitor venetoclax in combination with
azacitidine results in deep and durable remissions and is
superior to conventional treatments. We hypothesized that these
promising clinical results were due to targeting LSCs. Analysis
of LSCs from patients undergoing treatment with venetoclax +
azacitidine showed disruption of the tricarboxylic acid (TCA)
cycle manifested by decreased α-ketoglutarate and
increased succinate levels, suggesting inhibition of electron
transport chain complex II. In vitro modeling confirmed
inhibition of complex II via reduced glutathionylation of
succinate dehydrogenase. These metabolic perturbations suppress
oxidative phosphorylation (OXPHOS), which efficiently and
selectively targets LSCs. Our findings show for the first time
that a therapeutic intervention can eradicate LSCs in patients
with AML by disrupting the metabolic machinery driving energy
metabolism, resulting in promising clinical activity in a patient
population with historically poor outcomes.
Dynamic temperature-sensitive A-to-I RNA editing in the
brain of a heterothermic mammal during hibernation
Kent A
Riemondy, Austin E
Gillen, Emily A
White, and
3 more authors
RNA editing diversifies genomically encoded information to
expand the complexity of the transcriptome. In ectothermic
organisms, including Drosophila and Cephalopoda, where body
temperature mirrors ambient temperature, decreases in
environmental temperature lead to increases in A-to-I RNA
editing and cause amino acid recoding events that are thought to
be adaptive responses to temperature fluctuations. In contrast,
endothermic mammals, including humans and mice, typically
maintain a constant body temperature despite environmental
changes. Here, A-to-I editing primarily targets repeat elements,
rarely results in the recoding of amino acids, and plays a
critical role in innate immune tolerance. Hibernating ground
squirrels provide a unique opportunity to examine RNA editing in
a heterothermic mammal whose body temperature varies over 30C
and can be maintained at 5C for many days during torpor. We
profiled the transcriptome in three brain regions at six
physiological states to quantify RNA editing and determine
whether cold-induced RNA editing modifies the transcriptome as a
potential mechanism for neuroprotection at low temperature
during hibernation. We identified 5165 A-to-I editing sites in
1205 genes with dynamically increased editing after prolonged
cold exposure. The majority (99.6%) of the cold-increased
editing sites are outside of previously annotated coding
regions, 82.7% lie in SINE-derived repeats, and 12 sites are
predicted to recode amino acids. Additionally, A-to-I editing
frequencies increase with increasing cold-exposure,
demonstrating that ADAR remains active during torpor. Our
findings suggest that dynamic A-to-I editing at low body
temperature may provide a neuroprotective mechanism to limit
aberrant dsRNA accumulation during torpor in the mammalian
hibernator.
TCR signal strength controls thymic differentiation of iNKT
cell subsets
Kathryn D
Tuttle, S Harsha
Krovi, Jingjing
Zhang, and
13 more authors
During development in the thymus, invariant natural killer T
(iNKT) cells commit to one of three major functionally different
subsets, iNKT1, iNKT2, and iNKT17. Here, we show that T cell
antigen receptor (TCR) signal strength governs the development of
iNKT cell subsets, with strong signaling promoting iNKT2 and
iNKT17 development. Altering TCR diversity or signaling
diminishes iNKT2 and iNKT17 cell subset development in a
cell-intrinsic manner. Decreased TCR signaling affects the
persistence of Egr2 expression and the upregulation of PLZF. By
genome-wide comparison of chromatin accessibility, we identify a
subset of iNKT2-specific regulatory elements containing NFAT and
Egr binding motifs that is less accessible in iNKT2 cells that
develop from reduced TCR signaling. These data suggest that
variable TCR signaling modulates regulatory element activity at
NFAT and Egr binding sites exerting a determinative influence on
the dynamics of gene enhancer accessibility and the developmental
fate of iNKT cells.
Genetic bypass of essential RNA repair enzymes in budding yeast
Patrick D
Cherry, Laura K
White, Kerri
York, and
1 more author
RNA repair enzymes catalyze rejoining of an RNA molecule after
cleavage of phosphodiester linkages. RNA repair in budding yeast
is catalyzed by two separate enzymes that process tRNA exons
during their splicing andHAC1mRNA exons during activation of the
unfolded protein response (UPR). The RNA ligase Trl1 joins
2’,3’-cyclic phosphate and 5’-hydroxyl RNA fragments, creating a
phosphodiester linkage with a 2’-phosphate at the junction. The
2’-phosphate is removed by the 2’-phosphotransferase Tpt1. We
bypassed the essential functions ofTRL1andTPT1in budding yeast by
expressing “prespliced,” intronless versions of the 10 normally
intron-containing tRNAs, indicating this repair pathway does not
have additional essential functions. Consistent with previous
studies, expression of intronless tRNAs failed to rescue the
growth of cells with deletions in components of the SEN complex,
implying an additional essential role for the splicing
endonuclease. Thetrl1∆andtpt1∆mutants accumulate
tRNA andHAC1splicing intermediates indicative of RNA repair
defects and are hypersensitive to drugs that inhibit translation.
Failure to induce the unfolded protein response intrl1∆cells grown with tunicamycin is lethal owing to their inability
to ligateHAC1after its cleavage by Ire1. In contrast,tpt1∆mutants grow in the presence of tunicamycin despite reduced
accumulation of splicedHAC1mRNA. We optimized a PCR-based method
to detect RNA 2’-phosphate modifications and show they are
present on ligatedHAC1mRNA. These RNA repair mutants enable new
studies of the role of RNA repair in cellular physiology.
2017
2017
Localization of Cdc7 Protein Kinase During DNA Replication in
Saccharomyces cerevisiae
Daniel
Rossbach, D Suzi
Bryan, Jay R
Hesselberth, and
1 more author
DDK, a conserved serine-threonine protein kinase composed of a
regulatory subunit, Dbf4, and a catalytic subunit, Cdc7, is
essential for DNA replication initiation during S phase of the
cell cycle through MCM2-7 helicase phosphorylation. The
biological significance of DDK is well characterized, but the
full mechanism of how DDK associates with substrates remains
unclear. Cdc7 is bound to chromatin in the Saccharomyces
cerevisiae genome throughout the cell cycle, but there is little
empirical evidence as to specific Cdc7 binding locations. Using
biochemical and genetic techniques, this study investigated the
specific localization of Cdc7 on chromatin. The Calling Cards
method, using Ty5 retrotransposons as a marker for DNA-protein
binding, suggests Cdc7 kinase is preferentially bound to genomic
DNA known to replicate early in S phase, including centromeres
and origins of replication. We also discovered Cdc7 binding
throughout the genome, which may be necessary to initiate other
cellular processes, including meiotic recombination and
translesion synthesis. A kinase dead Cdc7 point mutation
increases the Ty5 retrotransposon integration efficiency and a
55-amino acid C-terminal truncation of Cdc7, unable to bind Dbf4,
reduces Cdc7 binding suggesting a requirement for Dbf4 to
stabilize Cdc7 on chromatin during S phase. Chromatin
immunoprecipitation demonstrates that Cdc7 binding near specific
origins changes during S phase. Our results suggest a model where
Cdc7 is loosely bound to chromatin during G1 At the G1/S
transition, Cdc7 binding to chromatin is increased and
stabilized, preferentially at sites that may become origins, in
order to carry out a variety of cellular processes.
Alternative Polyadenylation of PRELID1 Regulates Mitochondrial
ROS Signaling and Cancer Outcomes
Austin E
Gillen, Heather M
Brechbuhl, Tomomi M
Yamamoto, and
4 more authors
Disruption of posttranscriptional gene regulation is a critical
step in oncogenesis that can be difficult to observe using
traditional molecular techniques. To overcome this limitation, a
modified polyadenylation site sequencing (PAS-seq) protocol was
used to generate a genome-wide map of alternative polyadenylation
(APA) events in human primary breast tumor specimens and matched
normal tissue. This approach identified an APA event in the
PRELID1 mRNA that enhances its steady-state level and
translational efficiency, and is a strong breast cancer
subtype-dependent predictor of patient clinical outcomes.
Furthermore, it has been demonstrated that PRELID1 regulates
stress response and mitochondrial reactive oxygen species (ROS)
production in a cell type-specific manner. Modulation of PRELID1
expression, including its posttranscriptional control, appears to
be a common stress response across different cancer types. These
data reveal that PRELID1 mRNA processing is an important
regulator of cell type-specific responses to stress used by
multiple cancers and is associated with patient
outcomes.Implications: This study suggests that the regulation of
PRELID1 expression, by APA and other mechanisms, plays a role in
mitochondrial ROS signaling and represents a novel prognostic
factor and therapeutic target in cancer. Mol Cancer Res; 15(12);
1741-51. \copyright2017 AACR.
valr: Reproducible genome interval analysis in R
Kent A
Riemondy, Ryan M
Sheridan, Austin
Gillen, and
3 more authors
New tools for reproducible exploratory data analysis of large
datasets are important to address the rising size and complexity
of genomic data. We developed the valr R package to enable
flexible and efficient genomic interval analysis. valr leverages
new tools available in the “tidyverse”, including dplyr.
Benchmarks of valr show it performs similar to BEDtools and can
be used for interactive analyses and incorporated into existing
analysis pipelines.
2016
2016
Improvements to the HITS-CLIP protocol eliminate widespread
mispriming artifacts
Austin E
Gillen, Tomomi M
Yamamoto, Enos
Kline, and
2 more authors
BACKGROUND: High-throughput sequencing of RNA isolated by
crosslinking immunoprecipitation (HITS-CLIP) allows for high
resolution, genome-wide mapping of RNA-binding proteins. This
methodology is frequently used to validate predicted targets of
microRNA binding, as well as direct targets of other RNA-binding
proteins. Hence, the accuracy and sensitivity of binding site
identification is critical. RESULTS: We found that substantial
mispriming during reverse transcription results in the
overrepresentation of sequences complementary to the primer used
for reverse transcription. Up to 45 % of peaks in publicly
available HITS-CLIP libraries are attributable to this mispriming
artifact, and the majority of libraries have detectable levels of
mispriming. We also found that standard techniques for validating
microRNA-target interactions fail to differentiate between
artifactual peaks and physiologically relevant peaks.
CONCLUSIONS: Here, we present a modification to the HITS-CLIP
protocol that effectively eliminates this artifact and improves
the sensitivity and complexity of resulting libraries.
Diverse fates of uracilated HIV-1 DNA during infection of
myeloid lineage cells
Erik C
Hansen, Monica
Ransom, Jay R
Hesselberth, and
9 more authors
We report that a major subpopulation of monocyte-derived
macrophages (MDMs) contains high levels of dUTP, which is
incorporated into HIV-1 DNA during reverse transcription (U/A
pairs), resulting in pre-integration restriction and
post-integration mutagenesis. After entering the nucleus,
uracilated viral DNA products are degraded by the uracil base
excision repair (UBER) machinery with less than 1% of the
uracilated DNA successfully integrating. Although uracilated
proviral DNA showed few mutations, the viral genomic RNA was
highly mutated, suggesting that errors occur during
transcription. Viral DNA isolated from blood monocytes and
alveolar macrophages (but not T cells) of drug-suppressed
HIV-infected individuals also contained abundant uracils. The
presence of viral uracils in short-lived monocytes suggests their
recent infection through contact with virus producing cells in a
tissue reservoir. These findings reveal new elements of a viral
defense mechanism involving host UBER that may be relevant to the
establishment and persistence of HIV-1 infection.
2015
2015
Coordination between Drosophila Arc1 and a specific population of
brain neurons regulates organismal fat
Jeremy
Mosher, Wei
Zhang, Rachel Z
Blumhagen, and
5 more authors
The brain plays a critical yet incompletely understood role in
regulating organismal fat. We performed a neuronal silencing
screen in Drosophila larvae to identify brain regions required to
maintain proper levels of organismal fat. When used to modulate
synaptic activity in specific brain regions, the enhancer-trap
driver line E347 elevated fat upon neuronal silencing, and
decreased fat upon neuronal activation. Unbiased sequencing
revealed that Arc1 mRNA levels increase upon E347 activation. We
had previously identified Arc1 mutations in a high-fat screen.
Here we reveal metabolic changes in Arc1 mutants consistent with
a high-fat phenotype and an overall shift toward energy storage.
We find that Arc1-expressing cells neighbor E347 neurons, and
manipulating E347 synaptic activity alters Arc1 expression
patterns. Elevating Arc1 expression in these cells decreased fat,
a phenocopy of E347 activation. Finally, loss of Arc1 prevented
the lean phenotype caused by E347 activation, suggesting that
Arc1 activity is required for E347 control of body fat.
Importantly, neither E347 nor Arc1 manipulation altered
energy-related behaviors. Our results support a model wherein
E347 neurons induce Arc1 in specific neighboring cells to prevent
excess fat accumulation.
Enhanced stability and polyadenylation of select mRNAs support
rapid thermogenesis in the brown fat of a hibernator
Katharine R
Grabek, Cecilia
Diniz Behn, Gregory S
Barsh, and
2 more authors
During hibernation, animals cycle between torpor and arousal.
These cycles involve dramatic but poorly understood mechanisms of
dynamic physiological regulation at the level of gene expression.
Each cycle, Brown Adipose Tissue (BAT) drives periodic arousal
from torpor by generating essential heat. We applied digital
transcriptome analysis to precisely timed samples to identify
molecular pathways that underlie the intense activity cycles of
hibernator BAT. A cohort of transcripts increased during torpor,
paradoxical because transcription effectively ceases at these low
temperatures. We show that this increase occurs not by elevated
transcription but rather by enhanced stabilization associated
with maintenance and/or extension of long poly(A) tails.
Mathematical modeling further supports a temperature-sensitive
mechanism to protect a subset of transcripts from ongoing bulk
degradation instead of increased transcription. This subset was
enriched in a C-rich motif and genes required for BAT activation,
suggesting a model and mechanism to prioritize translation of key
proteins for thermogenesis.
RNase L targets distinct sites in influenza A virus RNAs
Daphne A
Cooper, Shuvojit
Banerjee, Arindam
Chakrabarti, and
4 more authors
UNLABELLED: Influenza A virus (IAV) infections are influenced by
type 1 interferon-mediated antiviral defenses and by viral
countermeasures to these defenses. When IAV NS1 protein is
disabled, RNase L restricts virus replication; however, the RNAs
targeted for cleavage by RNase L under these conditions have not
been defined. In this study, we used deep-sequencing methods to
identify RNase L cleavage sites within host and viral RNAs from
IAV PR8\DeltaNS1-infected A549 cells. Short hairpin RNA
knockdown of RNase L allowed us to distinguish between RNase
L-dependent and RNase L-independent cleavage sites. RNase
L-dependent cleavage sites were evident at discrete locations in
IAV RNA segments (both positive and negative strands). Cleavage
in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are
susceptible to cleavage by RNase L. Prominent amounts of
cleavage mapped to specific regions within IAV RNAs, including
some areas of increased synonymous-site conservation. Among
cellular RNAs, RNase L-dependent cleavage was most frequent at
precise locations in rRNAs. Our data show that RNase L targets
specific sites in both host and viral RNAs to restrict influenza
virus replication when NS1 protein is disabled. IMPORTANCE:
RNase L is a critical component of interferon-regulated and
double-stranded-RNA-activated antiviral host responses. We
sought to determine how RNase L exerts its antiviral activity
during influenza virus infection. We enhanced the antiviral
activity of RNase L by disabling a viral protein, NS1, that
inhibits the activation of RNase L. Then, using deep-sequencing
methods, we identified the host and viral RNAs targeted by RNase
L. We found that RNase L cleaved viral RNAs and rRNAs at very
precise locations. The direct cleavage of IAV RNAs by RNase L
highlights an intimate battle between viral RNAs and an
antiviral endonuclease.
Global analysis of RNA cleavage by 5’-hydroxyl RNA sequencing
RNA cleavage by some endoribonucleases and self-cleaving
ribozymes produces RNA fragments with 5’-hydroxyl (5’-OH) and
2’,3’-cyclic phosphate termini. To identify 5’-OH RNA fragments
produced by these cleavage events, we exploited the unique
ligation mechanism of Escherichia coli RtcB RNA ligase to attach
an oligonucleotide linker to RNAs with 5’-OH termini, followed by
steps for library construction and analysis by massively parallel
DNA sequencing. We applied the method to RNA from budding yeast
and captured known 5’-OH fragments produced by tRNA Splicing
Endonuclease (SEN) during processing of intron-containing
pre-tRNAs and by Ire1 cleavage of HAC1 mRNA following induction
of the unfolded protein response (UPR). We identified numerous
novel 5’-OH fragments derived from mRNAs: some 5’-OH mRNA
fragments were derived from single, localized cleavages, while
others were likely produced by multiple, distributed cleavages.
Many 5’-OH fragments derived from mRNAs were produced upstream of
codons for highly electrostatic peptides, suggesting that the
fragments may be generated by co-translational mRNA decay.
Several 5’-OH RNA fragments accumulated during the induction of
the UPR, some of which share a common sequence motif that may
direct cleavage of these mRNAs. This method enables specific
capture of 5’-OH termini and complements existing methods for
identifying RNAs with 2’,3’-cyclic phosphate termini.
Ribose-seq: global mapping of ribonucleotides embedded in genomic
DNA
Kyung Duk
Koh, Sathya
Balachander, Jay R
Hesselberth, and
1 more author
Abundant ribonucleotide incorporation in DNA during replication
and repair has profound consequences for genome stability, but
the global distribution of ribonucleotide incorporation is
unknown. We developed ribose-seq, a method for capturing unique
products generated by alkaline cleavage of DNA at embedded
ribonucleotides. High-throughput sequencing of these fragments in
DNA from the yeast Saccharomyces cerevisiae revealed widespread
ribonucleotide distribution, with a strong preference for
cytidine and guanosine, and identified hotspots of ribonucleotide
incorporation in nuclear and mitochondrial DNA. Ribonucleotides
were primarily incorporated on the newly synthesized leading
strand of nuclear DNA and were present upstream of (G+C)-rich
tracts in the mitochondrial genome. Ribose-seq is a powerful tool
for the systematic profiling of ribonucleotide incorporation in
genomic DNA.
Global analysis of RNA cleavage by 5’-hydroxyl RNA sequencing
RNA cleavage by some endoribonucleases and self-cleaving
ribozymes produces RNA fragments with 5’-hydroxyl (5’-OH) and
2’,3’-cyclic phosphate termini. To identify 5’-OH RNA fragments
produced by these cleavage events, we exploited the unique
ligation mechanism of Escherichia coli RtcB RNA ligase to attach
an oligonucleotide linker to RNAs with 5’-OH termini, followed by
steps for library construction and analysis by massively parallel
DNA sequencing. We applied the method to RNA from budding yeast
and captured known 5’-OH fragments produced by tRNA Splicing
Endonuclease (SEN) during processing of intron-containing
pre-tRNAs and by Ire1 cleavage of HAC1 mRNA following induction
of the unfolded protein response (UPR). We identified numerous
novel 5’-OH fragments derived from mRNAs: some 5’-OH mRNA
fragments were derived from single, localized cleavages, while
others were likely produced by multiple, distributed cleavages.
Many 5’-OH fragments derived from mRNAs were produced upstream of
codons for highly electrostatic peptides, suggesting that the
fragments may be generated by co-translational mRNA decay.
Several 5’-OH RNA fragments accumulated during the induction of
the UPR, some of which share a common sequence motif that may
direct cleavage of these mRNAs. This method enables specific
capture of 5’-OH termini and complements existing methods for
identifying RNAs with 2’,3’-cyclic phosphate termini.
2014
2014
A homolog of lariat-debranching enzyme modulates turnover of
branched RNA
Stephen M
Garrey, Adam
Katolik, Mantas
Prekeris, and
7 more authors
Turnover of the branched RNA intermediates and products of
pre-mRNA splicing is mediated by the lariat-debranching enzyme
Dbr1. We characterized a homolog of Dbr1 from Saccharomyces
cerevisiae, Drn1/Ygr093w, that has a
pseudo-metallophosphodiesterase domain with primary sequence
homology to Dbr1 but lacks essential active site residues found
in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing
broadly to turnover, loss of Drn1 causes low levels of
lariat-intron accumulation. Conserved residues in the Drn1
C-terminal CwfJ domains, which are not present in Dbr1, are
required for efficient intron turnover. Drn1 interacts with Dbr1,
components of the Nineteen Complex, U2 snRNA, branched
intermediates, and products of splicing. Drn1 enhances
debranching catalyzed by Dbr1 in vitro, but does so without
significantly improving the affinity of Dbr1 for branched RNA.
Splicing carried out in in vitro extracts in the absence of Drn1
results in an accumulation of branched splicing intermediates and
products released from the spliceosome, likely due to less active
debranching, as well as the promiscuous release of cleaved
5’-exon. Drn1 enhances Dbr1-mediated turnover of
lariat-intermediates and lariat-intron products, indicating that
branched RNA turnover is regulated at multiple steps during
splicing.
The RtcB RNA ligase is an essential component of the metazoan
unfolded protein response
Sara Guckian
Kosmaczewski, Tyson James
Edwards, Sung Min
Han, and
6 more authors
EMBO Rep., Aug 2014
High resolution mapping of modified DNA nucleobases using
excision repair enzymes
D Suzi
Bryan, Monica
Ransom, Biniam
Adane, and
2 more authors
The incorporation and creation of modified nucleobases in DNA
have profound effects on genome function. We describe methods for
mapping positions and local content of modified DNA nucleobases
in genomic DNA. We combined in vitro nucleobase excision with
massively parallel DNA sequencing (Excision-seq) to determine the
locations of modified nucleobases in genomic DNA. We applied the
Excision-seq method to map uracil in E. coli and budding yeast
and discovered significant variation in uracil content, wherein
uracil is excluded from the earliest and latest replicating
regions of the genome, possibly driven by changes in nucleotide
pool composition. We also used Excision-seq to identify sites of
pyrimidine dimer formation induced by UV light exposure, where
the method could distinguish between sites of cyclobutane and 6-4
photoproduct formation. These UV mapping data enabled analysis of
local sequence bias around pyrimidine dimers and suggested a
preference for an adenosine downstream from 6-4 photoproducts.
The Excision-seq method is broadly applicable for high precision,
genome-wide mapping of modified nucleobases with cognate repair
enzymes.
Ribonuclease L and metal-ion-independent endoribonuclease
cleavage sites in host and viral RNAs
Daphne A
Cooper, Babal K
Jha, Robert H
Silverman, and
2 more authors
Ribonuclease L (RNase L) is a metal-ion-independent
endoribonuclease associated with antiviral and antibacterial
defense, cancer and lifespan. Despite the biological significance
of RNase L, the RNAs cleaved by this enzyme are poorly defined.
In this study, we used deep sequencing methods to reveal the
frequency and location of RNase L cleavage sites within host and
viral RNAs. To make cDNA libraries, we exploited the 2’,
3’-cyclic phosphate at the end of RNA fragments produced by RNase
L and other metal-ion-independent endoribonucleases. We optimized
and validated 2’, 3’-cyclic phosphate cDNA synthesis and Illumina
sequencing methods using viral RNAs cleaved with purified RNase
L, viral RNAs cleaved with purified RNase A and RNA from
uninfected and poliovirus-infected HeLa cells. Using these
methods, we identified (i) discrete regions of hepatitis C virus
and poliovirus RNA genomes that were profoundly susceptible to
RNase L and other single-strand specific endoribonucleases, (ii)
RNase L-dependent and RNase L-independent cleavage sites within
ribosomal RNAs (rRNAs) and (iii) 2’, 3’-cyclic phosphates at the
ends of 5S rRNA and U6 snRNA. Monitoring the frequency and
location of metal-ion-independent endoribonuclease cleavage sites
within host and viral RNAs reveals, in part, how these enzymes
contribute to health and disease.
The RtcB RNA ligase is an essential component of the metazoan
unfolded protein response
Sara Guckian
Kosmaczewski, Tyson James
Edwards, Sung Min
Han, and
6 more authors
RNA ligation can regulate RNA function by altering RNA sequence,
structure and coding potential. For example, the function of XBP1
in mediating the unfolded protein response requires RNA ligation,
as does the maturation of some tRNAs. Here, we describe a novel
in vivo model in Caenorhabditis elegans for the conserved RNA
ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the
IRE-1 branch of the unfolded protein response. Without RtcB,
protein stress results in the accumulation of unligated xbp-1
mRNA fragments, defects in the unfolded protein response, and
decreased lifespan. RtcB also ligates endogenous pre-tRNA halves,
and RtcB mutants have defects in growth and lifespan that can be
bypassed by expression of pre-spliced tRNAs. In addition, animals
that lack RtcB have defects that are independent of tRNA
maturation and the unfolded protein response. Thus, RNA ligation
by RtcB is required for the function of multiple endogenous
target RNAs including both xbp-1 and tRNAs. RtcB is uniquely
capable of performing these ligation functions, and RNA ligation
by RtcB mediates multiple essential processes in vivo.
After transcription of a eukaryotic pre-mRNA, its introns are
removed by the spliceosome, joining exons for translation. The
intron products of splicing have long been considered ’junk’ and
destined only for destruction. But because they are large in size
and under weak selection constraints, many introns have been
evolutionarily repurposed to serve roles after splicing. Some
spliced introns are precursors for further processing of other
encoded RNAs such as small nucleolar RNAs, microRNAs, and long
noncoding RNAs. Other intron products have long half-lives and
can be exported to the cytoplasm, suggesting that they have roles
in translation. Some viruses encode introns that accumulate after
splicing and play important but mysterious roles in viral
latency. Turnover of most lariat-introns is initiated by cleavage
of their internal 2’-5’ phosphodiester bonds by a unique
debranching endonuclease, and the linear products are further
degraded by exoribonucleases. However, several introns appear to
evade this turnover pathway and the determinants of their
stability are largely unknown. Whereas many stable intron
products were discovered serendipitously, new experimental and
computational tools will enable their direct identification and
study. Finally, the origins and mechanisms of mobility of
eukaryotic introns are mysterious, and mechanistic studies of the
intron life cycle may yield new insights into how they arose and
became widespread.
2010
2010
Capture and sequence analysis of RNAs with terminal
2’,3’-cyclic phosphates
Kevin
Schutz, Jay R
Hesselberth, and Stanley
Fields
The combination of ligation-based RNA capture methods and
high-throughput sequencing has facilitated the characterization
of transcriptomes and the identification of novel noncoding RNAs.
However, current ligation-based RNA capture methods require RNA
substrates with terminal 3’-hydroxyl groups, limiting their
utility for identifying RNAs with modified termini like
2’,3’-cyclic phosphates. Cyclic phosphate-terminated RNAs are
generated by endonucleolytic cleavages and self-cleaving
ribozymes and are found as stable modifications on cellular RNAs
such as the U6 spliceosomal RNA. We developed a method that uses
the Arabidopsis thaliana tRNA ligase to add an adaptor
oligonucleotide to RNAs that terminate in 2’,3’-cyclic
phosphates. The adaptor allows specific priming by reverse
transcriptase, which is followed by additional steps for PCR
amplification and high-throughput DNA sequencing. Applying the
method to total human RNA, we found 2836 sequencing reads
corresponding to the 3’ terminus of U6 snRNA, validating the
method. In addition to a large background of reads that map
throughout abundantly transcribed RNAs, we also found 42,324
reads of specific fragments from several tRNA isoacceptor
families, suggesting that this method may identify processing
events previously undetected by other RNA cloning techniques.
High-throughput profiling of amino acids in strains of the
Saccharomyces cerevisiae deletion collection
S J
Cooper, G L
Finney, S L
Brown, and
4 more authors
The combination of ligation-based RNA capture methods and
high-throughput sequencing has facilitated the characterization
of transcriptomes and the identification of novel noncoding RNAs.
However, current ligation-based RNA capture methods require RNA
substrates with terminal 3’-hydroxyl groups, limiting their
utility for identifying RNAs with modified termini like
2’,3’-cyclic phosphates. Cyclic phosphate-terminated RNAs are
generated by endonucleolytic cleavages and self-cleaving
ribozymes and are found as stable modifications on cellular RNAs
such as the U6 spliceosomal RNA. We developed a method that uses
the Arabidopsis thaliana tRNA ligase to add an adaptor
oligonucleotide to RNAs that terminate in 2’,3’-cyclic
phosphates. The adaptor allows specific priming by reverse
transcriptase, which is followed by additional steps for PCR
amplification and high-throughput DNA sequencing. Applying the
method to total human RNA, we found 2836 sequencing reads
corresponding to the 3’ terminus of U6 snRNA, validating the
method. In addition to a large background of reads that map
throughout abundantly transcribed RNAs, we also found 42,324
reads of specific fragments from several tRNA isoacceptor
families, suggesting that this method may identify processing
events previously undetected by other RNA cloning techniques.
2009
2009
Global mapping of protein-DNA interactions in vivo by digital
genomic footprinting
Jay R
Hesselberth, Xiaoyu
Chen, Zhihong
Zhang, and
9 more authors
The orchestrated binding of transcriptional activators and
repressors to specific DNA sequences in the context of chromatin
defines the regulatory program of eukaryotic genomes. We
developed a digital approach to assay regulatory protein
occupancy on genomic DNA in vivo by dense mapping of individual
DNase I cleavages from intact nuclei using massively parallel DNA
sequencing. Analysis of >23 million cleavages across the
Saccharomyces cerevisiae genome revealed thousands of protected
regulatory protein footprints, enabling de novo derivation of
factor binding motifs and the identification of hundreds of new
binding sites for major regulators. We observed striking
correspondence between single-nucleotide resolution DNase I
cleavage patterns and protein-DNA interactions determined by
crystallography. The data also yielded a detailed view of larger
chromatin features including positioned nucleosomes flanking
factor binding regions. Digital genomic footprinting should be a
powerful approach to delineate the cis-regulatory framework of
any organism with an available genome sequence.
2007
2007
Genome-wide identification of spliced introns using a tiling
microarray
Background The WW domain is found in a large number of
eukaryotic proteins implicated in a variety of cellular
processes. WW domains bind proline-rich protein and peptide
ligands, but the protein interaction partners of many WW
domain-containing proteins in
2005
2005
A protein interaction network of the malaria parasite Plasmodium
falciparum
Douglas J
LaCount, Marissa
Vignali, Rakesh
Chettier, and
9 more authors
Plasmodium falciparum causes the most severe form of malaria and
kills up to 2.7 million people annually. Despite the global
importance of P. falciparum, the vast majority of its proteins
have not been characterized experimentally. Here we identify P.
falciparum protein-protein interactions using a high-throughput
version of the yeast two-hybrid assay that circumvents the
difficulties in expressing P. falciparum proteins in
Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid
screens with P. falciparum protein fragments, we identified 2,846
unique interactions, most of which include at least one
previously uncharacterized protein. Informatic analyses of
network connectivity, coexpression of the genes encoding
interacting fragments, and enrichment of specific protein domains
or Gene Ontology annotations were used to identify groups of
interacting proteins, including one implicated in chromatin
modification, transcription, messenger RNA stability and
ubiquitination, and another implicated in the invasion of host
cells. These data constitute the first extensive description of
the protein interaction network for this important human
pathogen.
A protein interaction network of the malaria parasite Plasmodium
falciparum
Douglas J
LaCount, Marissa
Vignali, Rakesh
Chettier, and
9 more authors
Plasmodium falciparum causes the most severe form of malaria and
kills up to 2.7 million people annually. Despite the global
importance of P. falciparum, the vast majority of its proteins
have not been characterized experimentally. Here we identify P.
falciparum protein-protein interactions using a high-throughput
version of the yeast two-hybrid assay that circumvents the
difficulties in expressing P. falciparum proteins in
Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid
screens with P. falciparum protein fragments, we identified 2,846
unique interactions, most of which include at least one
previously uncharacterized protein. Informatic analyses of
network connectivity, coexpression of the genes encoding
interacting fragments, and enrichment of specific protein domains
or Gene Ontology annotations were used to identify groups of
interacting proteins, including one implicated in chromatin
modification, transcription, messenger RNA stability and
ubiquitination, and another implicated in the invasion of host
cells. These data constitute the first extensive description of
the protein interaction network for this important human
pathogen.
A protein interaction network of the malaria parasite Plasmodium
falciparum
Douglas J
LaCount, Marissa
Vignali, Rakesh
Chettier, and
9 more authors
Plasmodium falciparum causes the most severe form of malaria and
kills up to 2.7 million people annually. Despite the global
importance of P. falciparum, the vast majority of its proteins
have not been characterized experimentally. Here we identify P.
falciparum protein-protein interactions using a high-throughput
version of the yeast two-hybrid assay that circumvents the
difficulties in expressing P. falciparum proteins in
Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid
screens with P. falciparum protein fragments, we identified 2,846
unique interactions, most of which include at least one
previously uncharacterized protein. Informatic analyses of
network connectivity, coexpression of the genes encoding
interacting fragments, and enrichment of specific protein domains
or Gene Ontology annotations were used to identify groups of
interacting proteins, including one implicated in chromatin
modification, transcription, messenger RNA stability and
ubiquitination, and another implicated in the invasion of host
cells. These data constitute the first extensive description of
the protein interaction network for this important human
pathogen.
2004
2004
Aptamer database
Jennifer F
Lee, Jay R
Hesselberth, Lauren Ancel
Meyers, and
1 more author
The aptamer database is designed to contain comprehensive
sequence information on aptamers and unnatural ribozymes that
have been generated by in vitro selection methods. Such data are
not normally collected in ’natural’ sequence databases, such as
GenBank. Besides serving as a storehouse of sequences that may
have diagnostic or therapeutic utility, the database serves as a
valuable resource for theoretical biologists who describe and
explore fitness landscapes. The database is updated monthly and
is publicly available at http://aptamer. icmb.utexas.edu/.
2003
2003
Simultaneous detection of diverse analytes with an aptazyme
ligase array
Jay R
Hesselberth, Michael P
Robertson, Scott M
Knudsen, and
1 more author
Allosteric ribozymes (aptazymes) can transduce the noncovalent
recognition of analytes into the catalytic generation of readily
observable signals. Aptazymes are easily engineered, can detect
diverse classes of biologically relevant molecules, and have high
signal-to-noise ratios. These features make aptazymes useful
candidates for incorporation into biosensor arrays. Allosteric
ribozyme ligases that can recognize a variety of analytes ranging
from small organics to proteins have been generated. Upon
incorporation into an array format, multiple different aptazyme
ligases were able to simultaneously detect their cognate analytes
with high specificity. Analyte concentrations could be accurately
measured into the nanomolar range. The fact that analytes induced
the formation of new covalent bonds in aptazyme ligases (as
opposed to noncovalent bonds in antibodies) potentiated stringent
washing of the array, leading to improved signal-to-noise ratios
and limits of detection.
2002
2002
Automated selection of aptamers against protein targets
translated in vitro: from gene to aptamer
J Colin
Cox, Andrew
Hayhurst, Jay
Hesselberth, and
3 more authors
Reagents for proteome research must of necessity be generated by
high throughput methods. Aptamers are potentially useful as
reagents to identify and quantitate individual proteins, yet are
currently produced for the most part by manual selection
procedures. We have developed automated selection methods, but
must still individually purify protein targets. Therefore, we
have attempted to select aptamers against protein targets
generated by in vitro transcription and translation of individual
genes. In order to specifically immobilize the protein targets
for selection, they are also biotinylated in vitro. As a proof of
this method, we have selected aptamers against translated human
U1A, a component of the nuclear spliceosome. Selected sequences
demonstrated exquisite mimicry of natural binding sequences and
structures. These results not only reveal a potential path to the
high throughput generation of aptamers, but also yield insights
into the incredible specificity of the U1A protein for its
natural RNA ligands.
2001
2001
Optimization and optimality of a short ribozyme ligase that joins
non-Watson–Crick base pairings
Michael P
Robertson, Jay R
Hesselberth, and Andrew D
Ellington
A small ribozyme ligase (L1) selected from a random sequence
population appears to utilize non-Watson-Crick base pairs at its
ligation junction. Mutational and selection analyses confirmed
the presence of these base pairings. Randomization of the L1 core
and selection of active ligases yielded highly active variants
whose rates were on the order of 1 min(-1). Base-pairing
covariations confirmed the general secondary structure of the
ligase, and the most active ligases contained a novel pentuple
sequence covariation. The optimized L1 ligases may be optimal
within their sequence spaces, and minimal ligases that span less
than 60 nt in length have been engineered based on these results.
2000
2000
In vitro selection of nucleic acids for diagnostic applications
J
Hesselberth, M P
Robertson, S
Jhaveri, and
1 more author
In vitro selection methods have proven to be extraordinarily
adept at generating a wide variety of nucleic acid-binding
species (aptamers) and catalysts (ribozymes). To date, selected
nucleic acids have primarily been of academic interest. However,
just as antibodies have proven utility as ’universal receptors’
that can be crafted against a huge variety of ligands and can be
readily adapted to diagnostic assays, aptamers may yet find
application in assays. A new class of research reagents,
aptazymes, are not mere mimics of antibodies but in fact allow
the direct transduction of molecular recognition to catalysis.
Aptamers and aptazymes may prove to be uniquely useful for the
development of chip arrays for the detection and quantitation of
a wide range of molecules in organismal proteomes and
metabolomes.
In vitro selection of RNA molecules that inhibit the activity
of ricin A-chain
J R
Hesselberth, D
Miller, J
Robertus, and
1 more author
The cytotoxin ricin disables translation by depurinating a
conserved site in eukaryotic rRNA. In vitro selection has been
used to generate RNA ligands (aptamers) specific for the
catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no
resemblance to the normal RTA substrate, the sarcin-ricin loop
(SRL), and were not depurinated by RTA. An initial 80-nucleotide
RNA ligand was minimized to a 31-nucleotide aptamer that
contained all sequences and structures necessary for interacting
with RTA. This minimal RNA formed high affinity complexes with
RTA (K(d) = 7.3 nM) which could compete directly with the SRL for
binding to RTA. The aptamer inhibited RTA depurination of the SRL
and could partially protect translation from RTA inhibition. The
IC(50) of the aptamer for RTA in an in vitro translation assay is
100 nM, roughly 3 orders of magnitude lower than a small molecule
inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower
than the best known RNA inhibitor. The novel anti-RTA aptamers
may find application as diagnostic reagents for a potential
biological warfare agent and hold promise as scaffolds for the
development of strong ricin inhibitors.
