A catalog of stability-associated sequence elements in 3' UTRs of yeast mRNAs

BACKGROUND: In recent years, intensive computational efforts have been directed towards the discovery of promoter motifs that correlate with mRNA expression profiles. Nevertheless, it is still not always possible to predict steady-state mRNA expression levels based on promoter signals alone, suggesting that other factors may be involved. Other genic regions, in particular 3' UTRs, which are known to exert regulatory effects especially through controlling RNA stability and localization, were less comprehensively investigated, and deciphering regulatory motifs within them is thus crucial. RESULTS: By analyzing 3' UTR sequences and mRNA decay profiles of Saccharomyces cerevisiae genes, we derived a catalog of 53 sequence motifs that may be implicated in stabilization or destabilization of mRNAs. Some of the motifs correspond to known RNA-binding protein sites, and one of them may act in destabilization of ribosome biogenesis genes during stress response. In addition, we present for the first time a catalog of 23 motifs associated with subcellular localization. A significant proportion of the 3' UTR motifs is highly conserved in orthologous yeast genes, and some of the motifs are strikingly similar to recently published mammalian 3' UTR motifs. We classified all genes into those regulated only at transcription initiation level, only at degradation level, and those regulated by a combination of both. Interestingly, different biological functionalities and expression patterns correspond to such classification. CONCLUSION: The present motif catalogs are a first step towards the understanding of the regulation of mRNA degradation and subcellular localization, two important processes which - together with transcription regulation - determine the cell transcriptome

Widespread Inhibition of Posttranscriptional Splicing Shapes the Cellular Transcriptome following Heat Shock

During heat shock and other proteotoxic stresses, cells regulate multiple steps in gene expression in order to globally repress protein synthesis and selectively upregulate stress response proteins. Splicing of several mRNAs is known to be inhibited during heat stress, often meditated by SRp38, but the extent and specificity of this effect have remained unclear. Here, we examined splicing regulation genome-wide during heat shock in mouse fibroblasts. We observed widespread retention of introns in transcripts from ~1,700 genes, which were enriched for tRNA synthetase, nuclear pore, and spliceosome functions. Transcripts with retained introns were largely nuclear and untranslated. However, a group of 580+ genes biased for oxidation reduction and protein folding functions continued to be efficiently spliced. Interestingly, these unaffected transcripts are mostly cotranscriptionally spliced under both normal and stress conditions, whereas splicing-inhibited transcripts are mostly spliced posttranscriptionally. Altogether, our data demonstrate widespread repression of splicing in the mammalian heat stress response, disproportionately affecting posttranscriptionally spliced genes.Weizmann Institute of Science (Postdoctoral Award for Advancing Women in Science)European Molecular Biology Organization (Long-term Fellowship)Machiah FoundationNational Science Foundation (U.S.) (Grant 0821391)National Institutes of Health (U.S.

Global and Local Architecture of the Mammalian microRNA–Transcription Factor Regulatory Network

microRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. It is anticipated that, in combination with transcription factors (TFs), they span a regulatory network that controls thousands of mammalian genes. Here we set out to uncover local and global architectural features of the mammalian miR regulatory network. Using evolutionarily conserved potential binding sites of miRs in human targets, and conserved binding sites of TFs in promoters, we uncovered two regulation networks. The first depicts combinatorial interactions between pairs of miRs with many shared targets. The network reveals several levels of hierarchy, whereby a few miRs interact with many other lowly connected miR partners. We revealed hundreds of “target hubs” genes, each potentially subject to massive regulation by dozens of miRs. Interestingly, many of these target hub genes are transcription regulators and they are often related to various developmental processes. The second network consists of miR–TF pairs that coregulate large sets of common targets. We discovered that the network consists of several recurring motifs. Most notably, in a significant fraction of the miR–TF coregulators the TF appears to regulate the miR, or to be regulated by the miR, forming a diversity of feed-forward loops. Together these findings provide new insights on the architecture of the combined transcriptional–post transcriptional regulatory network

Widespread Regulation of Translation by Elongation Pausing in Heat Shock

Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation, although this mechanism may not always explain the full extent of repression. Here, using ribosome footprinting, we show that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells. The genome-wide nature of the phenomenon, its location, and features of protein N termini suggested the involvement of ribosome-associated chaperones. After severe heat shock, Hsp70’s interactions with the translational machinery were markedly altered and its association with ribosomes was reduced. Pretreatment with mild heat stress or overexpression of Hsp70 protected cells from heat shock-induced elongation pausing, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses.National Institute of General Medical Sciences (U.S.) (NIGMS fellowship number F32GM095060)Machiah FoundationEuropean Molecular Biology Organization (EMBO long-term fellowship)Weitzmann Institute of Science (National Postdoctoral Award Program for Advancing Women in Science

An IQSEC2 Mutation Associated With Intellectual Disability and Autism Results in Decreased Surface AMPA Receptors

We have recently described an A350V mutation in IQSEC2 associated with intellectual disability, autism and epilepsy. We sought to understand the molecular pathophysiology of this mutation with the goal of developing targets for drug intervention. We demonstrate here that the A350V mutation results in interference with the binding of apocalmodulin to the IQ domain of IQSEC2. We further demonstrate that this mutation results in constitutive activation of the guanine nucleotide exchange factor (GEF) activity of IQSEC2 resulting in increased production of the active form of Arf6. In a CRISPR generated mouse model of the A350V IQSEC2 mutation, we demonstrate that the surface expression of GluA2 AMPA receptors in mouse hippocampal tissue was significantly reduced in A350V IQSEC2 mutant mice compared to wild type IQSEC2 mice and that there is a significant reduction in basal synaptic transmission in the hippocampus of A350V IQSEC2 mice compared to wild type IQSEC2 mice. Finally, the A350V IQSEC2 mice demonstrated increased activity, abnormal social behavior and learning as compared to wild type IQSEC2 mice. These findings suggest a model of how the A350V mutation in IQSEC2 may mediate disease with implications for targets for drug therapy. These studies provide a paradigm for a personalized approach to precision therapy for a disease that heretofore has no therapy

Preferential regulation of miRNA targets by environmental chemicals in the human genome

<p>Abstract</p> <p>Background</p> <p>microRNAs (miRNAs) represent a class of small (typically 22 nucleotides in length) non-coding RNAs that can degrade their target mRNAs or block their translation. Recent disease research showed the exposure to some environmental chemicals (ECs) can regulate the expression patterns of miRNAs, which raises the intriguing question of how miRNAs and their targets cope with the exposure to ECs throughout the genome.</p> <p>Results</p> <p>In this study, we comprehensively analyzed the properties of genes regulated by ECs (EC-genes) and found miRNA targets were significantly enriched among the EC-genes. Compared with the non-miRNA-targets, miRNA targets were roughly twice as likely to be EC-genes. By investigating the collection methods and other properties of the EC-genes, we demonstrated that the enrichment of miRNA targets was not attributed to either the potential collection bias of EC-genes, the presence of paralogs, longer 3'UTRs or more conserved 3'UTRs. Finally, we identified 1,842 significant concurrent interactions between 407 miRNAs and 497 ECs. This association network of miRNAs-ECs was highly modular and could be separated into 14 interconnected modules. In each module, miRNAs and ECs were closely connected, providing a good method to design accurate miRNA markers for ECs in toxicology research.</p> <p>Conclusions</p> <p>Our analyses indicated that miRNAs and their targets played important roles in cellular responses to ECs. Association analyses of miRNAs and ECs will help to broaden the understanding of the pathogenesis of such chemical components.</p

DoGFinder: a software for the discovery and quantification of readthrough transcripts from RNA-seq

Abstract Background Recent studies have described a widespread induction of transcriptional readthrough as a consequence of various stress conditions in mammalian cells. This novel phenomenon, initially identified from analysis of RNA-seq data, suggests intriguing new levels of gene expression regulation. However, the mechanism underlying naturally occurring transcriptional readthrough, as well as its regulatory consequences, still remain elusive. Furthermore, the readthrough response to stress has thus far not been investigated outside of mammalian species, and the occurrence of readthrough in many physiological and disease conditions remains to be explored. Results To facilitate a wider investigation into transcriptional readthrough, we created the DoGFinder software package, for the streamlined identification and quantification of readthrough transcripts, also known as DoGs (Downstream of Gene-containing transcripts), from any RNA-seq dataset. Using DoGFinder, we explore the dependence of DoG discovery potential on RNA-seq library depth, and show that stress-induced readthrough induction discovery is robust to sequencing depth, and input parameter settings. We further demonstrate the use of the DoGFinder software package on a new publically available RNA-seq dataset, and discover DoG induction in human PME cells following hypoxia – a previously unknown readthrough inducing stress type. Conclusions DoGFinder will enable users to explore, in a few simple steps, the readthrough phenomenon in any condition and organism. DoGFinder is freely available at https://github.com/shalgilab/DoGFinder

Widespread PERK-dependent repression of ER targets in response to ER stress

Abstract The UPR (Unfolded Protein Response) is a well-orchestrated response to ER protein folding and processing overload, integrating both transcriptional and translational outputs. Its three arms in mammalian cells, the PERK translational response arm, together with the ATF6 and IRE1-XBP1-mediated transcriptional arms, have been thoroughly investigated. Using ribosome footprint profiling, we performed a deep characterization of gene expression programs involved in the early and late ER stress responses, within WT or PERK −/− Mouse Embryonic Fibroblasts (MEFs). We found that both repression and activation gene expression programs, affecting hundreds of genes, are significantly hampered in the absence of PERK. Specifically, PERK −/− cells do not show global translational inhibition, nor do they specifically activate early gene expression programs upon short exposure to ER stress. Furthermore, while PERK −/− cells do activate/repress late ER-stress response genes, the response is substantially weaker. Importantly, we highlight a widespread PERK-dependent repression program, consisting of ER targeted proteins, including transmembrane proteins, glycoproteins, and proteins with disulfide bonds. This phenomenon occurs in various different cell types, and has a major translational regulatory component. Moreover, we revealed a novel interplay between PERK and the XBP1-ATF6 arms of the UPR, whereby PERK attenuates the expression of a specific subset of XBP1-ATF6 targets, further illuminating the complexity of the integrated ER stress response