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Background The concentrations of distinct types of RNA in cells result from a dynamic equilibrium between RNA synthesis and decay. Despite the critical importance of RNA decay rates, current approaches for measuring them are generally labor-intensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here, we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On sequencing (PRO-seq) and RNA sequencing (RNA-seq). Results Our method treats PRO-seq as a measure of transcription rate and RNA-seq as a measure of RNA concentration, and estimates the rate of RNA decay required for a steady-state equilibrium. We show that this approach can be used to assay relative RNA half-lives genome-wide, with good accuracy and sensitivity for both coding and noncoding transcription units. Using a structural equation model (SEM), we test several features of transcription units, nearby DNA sequences, and nearby epigenomic marks for associations with RNA stability after controlling for their effects on transcription. We find that RNA splicing-related features are positively correlated with RNA stability, whereas features related to miRNA binding and DNA methylation are negatively correlated with RNA stability. Furthermore, we find that a measure based on U1 binding and polyadenylation sites distinguishes between unstable noncoding and stable coding transcripts but is not predictive of relative stability within the mRNA or lincRNA classes. We also identify several histone modifications that are associated with RNA stability. Conclusion We introduce an approach for estimating the relative half-lives of individual RNAs. Together, our estimation method and systematic analysis shed light on the pervasive impacts of RNA stability on cellular RNA concentrations.

Background Efforts to characterize regulatory elements in plant genomes traditionally rely on evolutionary conservation and chromatin accessibility. Recently, intergenic bi-directional nascent transcript has emerged as a putative hallmark of active enhancers. Here, we integrate these approaches to better define the cis -regulatory landscape of the rice genome. Results In juvenile leaf tissues of the Azucena rice variety, we analyze conserved noncoding sequences, intergenic bi-directional transcripts, and regions of open chromatin. These three features highlight distinct classes of regulatory targets, each exhibiting complexity and regulatory roles. Conserved noncoding sequences are associated with more complex regulatory interactions, while regions marked by chromatin accessibility or bi-directional nascent transcription tend to promote more stable regulatory activity. Some transcribed regulatory sites harbor elements linked to transposable element silencing, whereas others correlate with increased expression of nearby genes, pointing to candidate transcribed regulatory elements. We further identified molecular interactions between genic regions and intergenic transcribed regulatory elements using 3-dimensional chromatin contact data, we identify physical interactions between transcribed intergenic regions and genic regions. These interactions often co-localize with expression quantitative trait loci and coincide with increased transcription, further supporting a regulatory role. Conclusions Our integrative analysis reveals multiple distinct classes of regulatory elements in the rice genome, with overlapping but non-identical targets and signatures. Many candidate elements share features consistent with transcriptional enhancement, though the specific criteria for defining active enhancers in plants require further characterization. These findings underscore the importance of using complementary genomic signals to discover and characterize functionally diverse regulatory elements in plant genomes.

Leishmania spp. are pathogens of humans and animals and cause one of the most important neglected tropical diseases. Regulation of gene expression in Leishmania but also in the related Trypanosoma is radically different from all eukaryotic model organisms, dispensing with regulated, gene-specific transcription, and relying instead on highly regulated translation. Our work sheds light on the initiation, elongation, and termination of transcription, maps unidirectional, polycistronic transcription units, provides evidence for transcriptional pausing at or near starting points of RNA synthesis, and quantifies the varying transcription rates of the polycistronic transcription units. Our results will further the understanding of these important pathogens and should provide a valuable resource for researchers in the field of eukaryotic microbiology.

Background A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the signal within the resulting libraries. Results Using run-on transcription datasets generated from the same biological system, we show that a variety of GRO- and PRO-seq preparation methods leave identifiable signatures within each library. Specifically we show that the library preparation method results in differences in quality control metrics, as well as differences in the signal distribution at the 5 ′ end of transcribed regions. These shifts lead to disparities in eRNA identification, but do not impact analyses aimed at inferring the key regulators involved in changes to transcription. Conclusions Run-on sequencing protocol variations result in technical signatures that can be used to identify both the enrichment and library preparation method of a particular data set. These technical signatures are batch effects that limit detailed comparisons of pausing ratios and eRNAs identified across protocols. However, these batch effects have only limited impact on our ability to infer which regulators underlie the observed transcriptional changes.

Background Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive. Results Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells. Conclusions Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

Human cytomegalovirus infects more than half of humans, persists silently in virtually all tissues, and produces life-threatening disease in immunocompromised individuals. HCMV is also the most common infectious cause of birth defects and the leading nongenetic cause of sensorineural hearing loss in the United States. Because there is no vaccine and current drugs have problems with potency, toxicity, and antiviral drug resistance, alternative treatment strategies that target different points of viral control are needed. Our current study contributes to this goal by applying newly developed methods to examine transcription of the HCMV and host genomes at nucleotide resolution in an attempt to find targetable differences between the two. After a thorough analysis of productive elongation and of core promoter element usage, we found that some mechanisms of regulating transcription are shared between the host and HCMV but that others are distinctly different. This suggests that HCMV transcription may be a legitimate target for future antiviral therapies and this might translate to other herpesviruses. The large genome of human cytomegalovirus (HCMV) is transcribed by RNA polymerase II (Pol II). However, it is not known how closely this betaherpesvirus follows host transcriptional paradigms. We applied PRO-Seq and PRO-Cap methods to profile and quantify transcription initiation and productive elongation across the host and virus genomes in late infection. A major similarity between host transcription and viral transcription is that treatment of cells with the P-TEFb inhibitor flavopiridol preempts virtually all productive elongation, which otherwise covers most of the HCMV genome. The deep, nucleotide resolution identification of transcription start sites (TSSs) enabled an extensive analysis of core promoter elements. An important difference between host and viral transcription is that initiation is much more pervasive on the HCMV genome. The sequence preferences in the initiator region around the TSS and the utilization of upstream T/A-rich elements are different. Upstream TATA positions the TSS and boosts initiation in both the host and the virus, but upstream TATT has a significant stimulatory impact only on the viral template. The major immediate early (MIE) promoter remained active during late infection and was accompanied by transcription of both strands of the MIE enhancer from promoters within the enhancer. Surprisingly, we found that the long noncoding RNA4.9 is intimately associated with the viral origin of replication (oriLyt) and was transcribed to a higher level than any other viral or host promoter. Finally, our results significantly contribute to the idea that late in infection, transcription takes place on viral genomes that are not highly chromatinized. IMPORTANCE Human cytomegalovirus infects more than half of humans, persists silently in virtually all tissues, and produces life-threatening disease in immunocompromised individuals. HCMV is also the most common infectious cause of birth defects and the leading nongenetic cause of sensorineural hearing loss in the United States. Because there is no vaccine and current drugs have problems with potency, toxicity, and antiviral drug resistance, alternative treatment strategies that target different points of viral control are needed. Our current study contributes to this goal by applying newly developed methods to examine transcription of the HCMV and host genomes at nucleotide resolution in an attempt to find targetable differences between the two. After a thorough analysis of productive elongation and of core promoter element usage, we found that some mechanisms of regulating transcription are shared between the host and HCMV but that others are distinctly different. This suggests that HCMV transcription may be a legitimate target for future antiviral therapies and this might translate to other herpesviruses.

Control of gene expression is fundamental at every level of cell function. Promoter-proximal pausing and divergent transcription at promoters and enhancers, which are prominent features in animals, have only been studied in a handful of research experiments in plants. PRO-Seq analysis in cassava (Manihot esculenta) identified peaks of transcriptionally engaged RNA polymerase at both the 5′ and 3′ end of genes, consistent with paused or slowly moving Polymerase. In addition, we identified divergent transcription at intergenic sites. A full genome search for bi-directional transcription using an algorithm for enhancer detection developed in mammals (dREG) identified many intergenic regulatory element (IRE) candidates. These sites showed distinct patterns of methylation and nucleotide conservation based on genomic evolutionary rate profiling (GERP). SNPs within these IRE candidates explained significantly more variation in fitness and root composition than SNPs in chromosomal segments randomly ascertained from the same intergenic distribution, strongly suggesting a functional importance of these sites. Maize GRO-Seq data showed RNA polymerase occupancy at IREs consistent with patterns in cassava. Furthermore, these IREs in maize significantly overlapped with sites previously identified on the basis of open chromatin, histone marks, and methylation, and were enriched for reported eQTL. Our results suggest that bidirectional transcription can identify intergenic genomic regions in plants that play an important role in transcription regulation and whose identification has the potential to aid crop improvement.

Objectives The interferon-triggered innate immune response has been observed to be under strong diversifying selection to counteract the many pathogens hosts have to defend against. In particular, rewiring of gene transcription regulation allows organisms to rapidly acquire new phenotypes by removing and adding genes into the innate immune gene network. Dissecting the molecular processes by which this rewiring takes place, either by changing the DNA regulatory elements or by changing the activity of the regulators across species, is key to better understand this evolutionary process. Data description To better comprehend the evolutionary dynamics that have occurred in the initial transcriptional response to interferon in primates, we present Precision Run-On (PRO-seq) datasets made after 1 h of interferon-α2 stimulation on human and rhesus macaque lymphoblastoid cell lines. Further, we tested the difference between using either species’ cognate interferon versus using the other orthologous interferon to account for any potential impacts in the interaction of the orthologous interferons with their cellular membrane receptors. This data provides insights into the regulatory mechanisms that drive species-specific responses to environmental perturbations, such as the one driven by the interactions of pathogens and their hosts.

Heat shock response (HSR) maintains and restores protein homeostasis when cells are exposed to proteotoxic heat stress. Heat shock (HS) triggers a rapid and robust change in genome-wide transcription, protein synthesis, and chaperone activity; and therefore, the HSR has been widely used as a model system in these studies. The conventional method of performing instantaneous HS in the laboratory uses heated fresh media to induce HSR when added to cells. However, addition of fresh media to cells may evoke additional cellular responses and signaling pathways. Here, we compared the change in global transcription profile when HS is performed with either heated fresh media or heated conditioned media. We found that the use of heated fresh media induces transcription of hundreds of genes that HS alone does not induce, and masks or partially masks HS-mediated downregulation of thousands of genes. The fresh-media-dependent upregulated genes encode ribosomal subunit proteins involved in translation and RNA processing factors. More importantly, fresh media also induce transcription of several heat shock protein genes (Hsps) in a heat shock factor 1 (HSF1)-independent manner. Thus, we conclude that a conventional method of HS with heated fresh media causes changes in transcription regulation that confound the actual change caused solely by elevated temperature of cells.