Exon-Mediated Activation of Transcription Starts.

The processing of RNA transcripts from mammalian genes occurs in proximity to their transcription. Here, we describe a phenomenon affecting thousands of genes that we call exon-mediated activation of transcription starts (EMATS), in which the splicing of internal exons impacts promoter choice and the expression level of the gene. We observed that evolutionary gain of internal exons is associated with gain of new transcription start sites (TSSs) nearby and increased gene expression. Inhibiting exon splicing reduced transcription from nearby promoters, and creation of new spliced exons activated transcription from cryptic promoters. The strongest effects occurred for weak promoters located proximal and upstream of efficiently spliced exons. Together, our findings support a model in which splicing recruits transcription machinery locally to influence TSS choice and identify exon gain, loss, and regulatory change as major contributors to the evolution of alternative promoters and gene expression in mammals.

Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription.

Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.

Loss of LUC7L2 and U1 snRNP subunits shifts energy metabolism from glycolysis to OXPHOS.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.

SR proteins collaborate with 7SK and promoter-associated nascent RNA to release paused polymerase.

RNAP II is frequently paused near gene promoters in mammals, and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR-splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes.

Braveheart, a long noncoding RNA required for cardiovascular lineage commitment.

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.

Regulatory features aid interpretation of 3'UTR variants.

Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3' UTR variants from several datasets. Variants in putative regulatory elements, including RNA-binding protein motifs, eCLIP peaks, and microRNA sites, are up to 16 times more likely than variants not in these elements to have gene expression and phenotype associations. Variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3' UTR variants in ClinVar are up to 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3' UTR variant and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in individuals.

Revisiting global gene expression analysis.

Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease, and for many other purposes. Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data. We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms.

Transcriptional amplification in tumor cells with elevated c-Myc.

Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood but is widely thought to involve newly activated or repressed "Myc target genes." We report here that in tumor cells expressing high levels of c-Myc the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation.

Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins.

The muscleblind-like (Mbnl) family of RNA-binding proteins plays important roles in muscle and eye development and in myotonic dystrophy (DM), in which expanded CUG or CCUG repeats functionally deplete Mbnl proteins. We identified transcriptome-wide functional and biophysical targets of Mbnl proteins in brain, heart, muscle, and myoblasts by using RNA-seq and CLIP-seq approaches. This analysis identified several hundred splicing events whose regulation depended on Mbnl function in a pattern indicating functional interchangeability between Mbnl1 and Mbnl2. A nucleotide resolution RNA map associated repression or activation of exon splicing with Mbnl binding near either 3' splice site or near the downstream 5' splice site, respectively. Transcriptomic analysis of subcellular compartments uncovered a global role for Mbnls in regulating localization of mRNAs in both mouse and Drosophila cells, and Mbnl-dependent translation and protein secretion were observed for a subset of mRNAs with Mbnl-dependent localization. These findings hold several new implications for DM pathogenesis.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

Sequence, Structure, and Context Preferences of Human RNA Binding Proteins.

RNA binding proteins (RBPs) orchestrate the production, processing, and function of mRNAs. Here, we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of these proteins in vitro by deep sequencing of bound RNAs. These data enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays. We found an unexpectedly low diversity of RNA motifs, implying frequent convergence of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, however, we observed extensive preferences for contextual features distinct from short linear RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide composition, and bias away from or toward RNA structure. Our results emphasize the importance of contextual features in RNA recognition, which likely enable targeting of distinct subsets of transcripts by different RBPs that recognize the same linear motif.

c-Myc regulates transcriptional pause release.

Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.

RBP Image Database: A resource for the systematic characterization of the subcellular distribution properties of human RNA binding proteins.

RNA binding proteins (RBPs) are central regulators of gene expression implicated in all facets of RNA metabolism. As such, they play key roles in cellular physiology and disease etiology. Since different steps of post-transcriptional gene expression tend to occur in specific regions of the cell, including nuclear or cytoplasmic locations, defining the subcellular distribution properties of RBPs is an important step in assessing their potential functions. Here, we present the RBP Image Database, a resource that details the subcellular localization features of 301 RBPs in the human HepG2 and HeLa cell lines, based on the results of systematic immuno-fluorescence studies conducted using a highly validated collection of RBP antibodies and a panel of 12 markers for specific organelles and subcellular structures. The unique features of the RBP Image Database include: (i) hosting of comprehensive representative images for each RBP-marker pair, with ∼250,000 microscopy images; (ii) a manually curated controlled vocabulary of annotation terms detailing the localization features of each factor; and (iii) a user-friendly interface allowing the rapid querying of the data by target or annotation. The RBP Image Database is freely available at https://rnabiology.ircm.qc.ca/RBPImage/.

RNA Sequence Context Effects Measured In Vitro Predict In Vivo Protein Binding and Regulation.

Many RNA binding proteins (RBPs) bind specific RNA sequence motifs, but only a small fraction (∼15%-40%) of RBP motif occurrences are occupied in vivo. To determine which contextual features discriminate between bound and unbound motifs, we performed an in vitro binding assay using 12,000 mouse RNA sequences with the RBPs MBNL1 and RBFOX2. Surprisingly, the strength of binding to motif occurrences in vitro was significantly correlated with in vivo binding, developmental regulation, and evolutionary age of alternative splicing. Multiple lines of evidence indicate that the primary context effect that affects binding in vitro and in vivo is RNA secondary structure. Large-scale combinatorial mutagenesis of unfavorable sequence contexts revealed a consistent pattern whereby mutations that increased motif accessibility improved protein binding and regulatory activity. Our results indicate widespread inhibition of motif binding by local RNA secondary structure and suggest that mutations that alter sequence context commonly affect RBP binding and regulation.

Distal Alternative Last Exons Localize mRNAs to Neural Projections.

Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer.

Cotargeting among microRNAs in the brain.

MicroRNAs (miRNAs) play roles in diverse developmental and disease processes. Distinct miRNAs have hundreds to thousands of conserved mRNA binding sites but typically direct only modest repression via single sites. Cotargeting of individual mRNAs by different miRNAs could potentially achieve stronger and more complex patterns of repression. By comparing target sets of different miRNAs, we identified hundreds of pairs of miRNAs that share more mRNA targets than expected (often by twofold or more) relative to stringent controls. Genetic perturbations revealed a functional overlap in neuronal differentiation for the cotargeting pair miR-138/miR-137. Clustering of all cotargeting pairs revealed a group of nine predominantly brain-enriched miRNAs that share many targets. In reporter assays, subsets of these miRNAs together repressed gene expression by five- to 10-fold, often showing cooperative repression. Together, our results uncover an unexpected pattern in which combinations of miRNAs collaborate to robustly repress cotargets, and suggest important developmental roles for cotargeting.

RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins.

Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.