Publications by authors named "Christopher B Burge"

105 Publications

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

Mol Cell 2021 05 13;81(9):1905-1919.e12. Epub 2021 Apr 13.

Department of Molecular Biology and Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2021.02.033DOI Listing
May 2021

Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity.

Nat Struct Mol Biol 2020 10 17;27(10):901-912. Epub 2020 Aug 17.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.

The Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity and specificity, but just half of Rbfox binding sites contain a GCAUG motif in vivo. We incubated recombinant RBFOX2 with over 60,000 mouse and human transcriptomic sequences to reveal substantial binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX2 concentrations. We find that these 'secondary motifs' bind Rbfox robustly in cells and that several together can exert regulation comparable to GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.
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http://dx.doi.org/10.1038/s41594-020-0475-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554199PMC
October 2020

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

Nature 2020 07 29;583(7818):699-710. Epub 2020 Jul 29.

Department of Biological Science, Florida State University, Tallahassee, FL, USA.

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 ENCODE and Roadmap Epigenomics 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.
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http://dx.doi.org/10.1038/s41586-020-2493-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410828PMC
July 2020

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

Nature 2020 07 29;583(7818):711-719. Epub 2020 Jul 29.

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome. 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.
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http://dx.doi.org/10.1038/s41586-020-2077-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410833PMC
July 2020

Exon-Mediated Activation of Transcription Starts.

Cell 2019 12 28;179(7):1551-1565.e17. Epub 2019 Nov 28.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02138, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2019.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351029PMC
December 2019

Cotargeting among microRNAs in the brain.

Genome Res 2019 11 24;29(11):1791-1804. Epub 2019 Oct 24.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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.
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http://dx.doi.org/10.1101/gr.249201.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836737PMC
November 2019

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

Cell 2019 06;178(1):107-121.e18

Department of Cellular and Molecular Medicine, Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2019.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760001PMC
June 2019

Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells.

Proc Natl Acad Sci U S A 2019 04 25;116(15):7353-7362. Epub 2019 Mar 25.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142;

Carcinoma cells residing in an intermediate phenotypic state along the epithelial-mesenchymal (E-M) spectrum are associated with malignant phenotypes, such as invasiveness, tumor-initiating ability, and metastatic dissemination. Using the recently described CD104/CD44 antigen marker combination, we isolated highly tumorigenic breast cancer cells residing stably-both in vitro and in vivo-in an intermediate phenotypic state and coexpressing both epithelial (E) and mesenchymal (M) markers. We demonstrate that tumorigenicity depends on individual cells residing in this E/M hybrid state and cannot be phenocopied by mixing two cell populations that reside stably at the two ends of the spectrum, i.e., in the E and in the M state. Hence, residence in a specific intermediate state along the E-M spectrum rather than phenotypic plasticity appears critical to the expression of tumor-initiating capacity. Acquisition of this E/M hybrid state is facilitated by the differential expression of EMT-inducing transcription factors (EMT-TFs) and is accompanied by the expression of adult stem cell programs, notably, active canonical Wnt signaling. Furthermore, transition from the highly tumorigenic E/M state to a fully mesenchymal phenotype, achieved by constitutive ectopic expression of Zeb1, is sufficient to drive cells out of the E/M hybrid state into a highly mesenchymal state, which is accompanied by a substantial loss of tumorigenicity and a switch from canonical to noncanonical Wnt signaling. Identifying the gatekeepers of the various phenotypic states arrayed along the E-M spectrum is likely to prove useful in developing therapeutic approaches that operate by shifting cancer cells between distinct states along this spectrum.
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http://dx.doi.org/10.1073/pnas.1812876116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462070PMC
April 2019

Allele-specific binding of RNA-binding proteins reveals functional genetic variants in the RNA.

Nat Commun 2019 03 22;10(1):1338. Epub 2019 Mar 22.

Department of Integrative Biology and Physiology, UCLA, Los Angeles, CA, 90095, USA.

Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants (GVs) mediating post-transcriptional regulation. Recently, genome-wide detection of in vivo binding of RNA-binding proteins is greatly facilitated by the enhanced crosslinking and immunoprecipitation (eCLIP) method. We developed a new computational approach, called BEAPR, to identify allele-specific binding (ASB) events in eCLIP-Seq data. BEAPR takes into account crosslinking-induced sequence propensity and variations between replicated experiments. Using simulated and actual data, we show that BEAPR largely outperforms often-used count analysis methods. Importantly, BEAPR overcomes the inherent overdispersion problem of these methods. Complemented by experimental validations, we demonstrate that the application of BEAPR to ENCODE eCLIP-Seq data of 154 proteins helps to predict functional GVs that alter splicing or mRNA abundance. Moreover, many GVs with ASB patterns have known disease relevance. Overall, BEAPR is an effective method that helps to address the outstanding challenge of functional interpretation of GVs.
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http://dx.doi.org/10.1038/s41467-019-09292-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430814PMC
March 2019

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

Sci Rep 2019 03 13;9(1):4330. Epub 2019 Mar 13.

Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096, Israel.

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.
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http://dx.doi.org/10.1038/s41598-019-38705-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416471PMC
March 2019

Acidification of Tumor at Stromal Boundaries Drives Transcriptome Alterations Associated with Aggressive Phenotypes.

Cancer Res 2019 04 12;79(8):1952-1966. Epub 2019 Feb 12.

Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts.

Acidosis is a fundamental feature of the tumor microenvironment, which directly regulates tumor cell invasion by affecting immune cell function, clonal cell evolution, and drug resistance. Despite the important association of tumor microenvironment acidosis with tumor cell invasion, relatively little is known regarding which areas within a tumor are acidic and how acidosis influences gene expression to promote invasion. Here, we injected a labeled pH-responsive peptide to mark acidic regions within tumors. Surprisingly, acidic regions were not restricted to hypoxic areas and overlapped with highly proliferative, invasive regions at the tumor-stroma interface, which were marked by increased expression of matrix metalloproteinases and degradation of the basement membrane. RNA-seq analysis of cells exposed to low pH conditions revealed a general rewiring of the transcriptome that involved RNA splicing and enriched for targets of RNA binding proteins with specificity for AU-rich motifs. Alternative splicing of Mena and CD44, which play important isoform-specific roles in metastasis and drug resistance, respectively, was sensitive to histone acetylation status. Strikingly, this program of alternative splicing was reversed and through neutralization experiments that mitigated acidic conditions. These findings highlight a previously underappreciated role for localized acidification of tumor microenvironment in the expression of an alternative splicing-dependent tumor invasion program. SIGNIFICANCE: This study expands our understanding of acidosis within the tumor microenvironment and indicates that acidosis induces potentially therapeutically actionable changes to alternative splicing.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467770PMC
April 2019

Widespread Accumulation of Ribosome-Associated Isolated 3' UTRs in Neuronal Cell Populations of the Aging Brain.

Cell Rep 2018 11;25(9):2447-2456.e4

Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA. Electronic address:

Particular brain regions and cell populations exhibit increased susceptibility to aging-related stresses. Here, we describe the age-specific and brain-region-specific accumulation of ribosome-associated 3' UTR RNAs that lack the 5' UTR and open reading frame. Our study reveals that this phenomenon impacts hundreds of genes in aged D1 spiny projection neurons of the mouse striatum and also occurs in the aging human brain. Isolated 3' UTR accumulation is tightly correlated with mitochondrial gene expression and oxidative stress, with full-length mRNA expression that is reduced but not eliminated, and with production of short 3' UTR-encoded peptides. Depletion of the oxidation-sensitive Fe-S cluster ribosome recycling factor ABCE1 induces the accumulation of 3' UTRs, consistent with a model in which ribosome stalling and mRNA cleavage by No-Go decay yields isolated 3' UTR RNAs protected by ribosomes. Isolated 3' UTR accumulation is a hallmark of brain aging, likely reflecting regional differences in metabolism and oxidative stress.
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http://dx.doi.org/10.1016/j.celrep.2018.10.094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354779PMC
November 2018

Numerous recursive sites contribute to accuracy of splicing in long introns in flies.

PLoS Genet 2018 08 27;14(8):e1007588. Epub 2018 Aug 27.

Department of Biology, Massachusetts Institute of Technology, Cambridge, United States of America.

Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates that are inherently unstable, making it difficult to study. Here we developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. A subset of recursive sites were validated by RT-PCR and sequencing. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, we observe that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.
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http://dx.doi.org/10.1371/journal.pgen.1007588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110457PMC
August 2018

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

Mol Cell 2018 06 7;70(5):854-867.e9. Epub 2018 Jun 7.

Department of Biology, MIT, Cambridge, MA, USA; Department of Biological Engineering, MIT, Cambridge, MA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2018.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062212PMC
June 2018

Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow.

Elife 2018 01 2;7. Epub 2018 Jan 2.

Koch Institute for Integrative Cancer Research, MIT, Cambridge, United States.

Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of , the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation.
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http://dx.doi.org/10.7554/eLife.29494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771670PMC
January 2018

The kinetics of pre-mRNA splicing in the genome and the influence of gene architecture.

Elife 2017 12 27;6. Epub 2017 Dec 27.

Departments of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States.

Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning ('intron definition') or exon-spanning ('exon definition') pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in , using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60-70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. We observed unexpectedly low variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing.
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http://dx.doi.org/10.7554/eLife.32537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762160PMC
December 2017

A Requirement for Mena, an Actin Regulator, in Local mRNA Translation in Developing Neurons.

Neuron 2017 Aug 20;95(3):608-622.e5. Epub 2017 Jul 20.

The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

During neuronal development, local mRNA translation is required for axon guidance and synaptogenesis, and dysregulation of this process contributes to multiple neurodevelopmental and cognitive disorders. However, regulation of local protein synthesis in developing axons remains poorly understood. Here, we uncover a novel role for the actin-regulatory protein Mena in the formation of a ribonucleoprotein complex that involves the RNA-binding proteins HnrnpK and PCBP1 and regulates local translation of specific mRNAs in developing axons. We find that translation of dyrk1a, a Down syndrome- and autism spectrum disorders-related gene, is dependent on Mena, both in steady-state conditions and upon BDNF stimulation. We identify hundreds of additional mRNAs that associate with the Mena complex, suggesting that it plays broader role(s) in post-transcriptional gene regulation. Our work establishes a dual role for Mena in neurons, providing a potential link between regulation of actin dynamics and local translation.
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http://dx.doi.org/10.1016/j.neuron.2017.06.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5616167PMC
August 2017

Protein-RNA Networks Regulated by Normal and ALS-Associated Mutant HNRNPA2B1 in the Nervous System.

Neuron 2016 Nov 20;92(4):780-795. Epub 2016 Oct 20.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Stem Cell Program and Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Bioinformatics and Systems Biology, University of California, San Diego, La Jolla, CA 92093, USA; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore; Molecular Engineering Laboratory, A(∗)STAR, Singapore 138673, Singapore. Electronic address:

HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide crosslinking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within ∼2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2016.09.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123850PMC
November 2016

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

Mol Cell 2016 10 6;64(2):294-306. Epub 2016 Oct 6.

Departments of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.

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.
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http://dx.doi.org/10.1016/j.molcel.2016.08.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107313PMC
October 2016

Widespread Shortening of 3' Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection.

PLoS Genet 2016 Sep 30;12(9):e1006338. Epub 2016 Sep 30.

Sainte-Justine Hospital Research Centre, Montreal, QC, Canada.

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3' UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3' UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3' UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.
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http://dx.doi.org/10.1371/journal.pgen.1006338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045211PMC
September 2016

Identification of new branch points and unconventional introns in Saccharomyces cerevisiae.

RNA 2016 10 29;22(10):1522-34. Epub 2016 Jul 29.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5' splice site (5'SS), branch point (BP), and 3' splice site (3'SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BPs and 5'SSs of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We performed RNA-seq to confirm associated 3'SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3'SS choice, protein coding potential, or RNA stability, and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings show unanticipated complexity of splicing in yeast.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029451PMC
http://dx.doi.org/10.1261/rna.057216.116DOI Listing
October 2016

Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses.

Nat Commun 2016 07 5;7:12143. Epub 2016 Jul 5.

Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA.

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.
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http://dx.doi.org/10.1038/ncomms12143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935974PMC
July 2016

Enhanced CLIP Uncovers IMP Protein-RNA Targets in Human Pluripotent Stem Cells Important for Cell Adhesion and Survival.

Cell Rep 2016 Apr 7;15(3):666-679. Epub 2016 Apr 7.

Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92037, USA; Stem Cell Program and Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA 92037, USA; Molecular Engineering Laboratory, A(∗)STAR, Singapore 1190777, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 1190777, Singapore. Electronic address:

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP), we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels, IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and increase in cell death. For cell adhesion, we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally, we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles.
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http://dx.doi.org/10.1016/j.celrep.2016.03.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839292PMC
April 2016

Distal Alternative Last Exons Localize mRNAs to Neural Projections.

Mol Cell 2016 Mar 18;61(6):821-33. Epub 2016 Feb 18.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2016.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4798900PMC
March 2016

Meta-analysis of RNA-seq expression data across species, tissues and studies.

Genome Biol 2015 Dec 22;16:287. Epub 2015 Dec 22.

Department of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA.

Background: Differences in gene expression drive phenotypic differences between species, yet major organs and tissues generally have conserved gene expression programs. Several comparative transcriptomic studies have observed greater similarity in gene expression between homologous tissues from different vertebrate species than between diverse tissues of the same species. However, a recent study by Lin and colleagues reached the opposite conclusion. These studies differed in the species and tissues analyzed, and in technical details of library preparation, sequencing, read mapping, normalization, gene sets, and clustering methods.

Results: To better understand gene expression evolution we reanalyzed data from four studies, including that of Lin, encompassing 6-13 tissues each from 11 vertebrate species using standardized mapping, normalization, and clustering methods. An analysis of independent data showed that the set of tissues chosen by Lin et al. were more similar to each other than those analyzed by previous studies. Comparing expression in five common tissues from the four studies, we observed that samples clustered exclusively by tissue rather than by species or study, supporting conservation of organ physiology in mammals. Furthermore, inter-study distances between homologous tissues were generally less than intra-study distances among different tissues, enabling informative meta-analyses. Notably, when comparing expression divergence of tissues over time to expression variation across 51 human GTEx tissues, we could accurately predict the clustering of expression for arbitrary pairs of tissues and species.

Conclusions: These results provide a framework for the design of future evolutionary studies of gene expression and demonstrate the utility of comparing RNA-seq data across studies.
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http://dx.doi.org/10.1186/s13059-015-0853-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699362PMC
December 2015

Interactome analysis brings splicing into focus.

Genome Biol 2015 Jul 7;16:135. Epub 2015 Jul 7.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA.

The spliceosome is a huge molecular machine that assembles dynamically onto its pre-mRNA substrates. A new study based on interactome analysis provides clues about how splicing-regulatory proteins modulate assembly of the spliceosome to either activate or repress splicing.Please see related Research article: http://www.genomebiology.com/2015/16/1/119/abstract.
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http://dx.doi.org/10.1186/s13059-015-0707-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493805PMC
July 2015

RNA Bind-n-Seq: Measuring the Binding Affinity Landscape of RNA-Binding Proteins.

Methods Enzymol 2015 12;558:465-493. Epub 2015 May 12.

Department of Biology, MIT, Cambridge Massachusetts, USA; Program in Computational and Systems Biology, MIT, Cambridge, Massachusetts, USA. Electronic address:

RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function of these factors will require the identification of each RBP's distinct RNA binding specificity. RNA Bind-n-Seq (RBNS) is a high-throughput, cost-effective in vitro method capable of resolving sequence and secondary structure preferences of RBPs. Dissociation constants can also be inferred from RBNS data when provided with additional experimental information. Here, we describe the experimental procedures to perform RBNS and discuss important parameters of the method and ways that the experiment can be tailored to the specific RBP under study. Additionally, we present the conceptual framework and execution of the freely available RBNS computational pipeline and describe the outputs of the pipeline. Different approaches to quantify binding specificity, quality control metrics, and estimation of binding constants are also covered.
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http://dx.doi.org/10.1016/bs.mie.2015.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576890PMC
March 2016

Antagonistic regulation of mRNA expression and splicing by CELF and MBNL proteins.

Genome Res 2015 Jun 16;25(6):858-71. Epub 2015 Apr 16.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA;

RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their genome-wide functions, we analyzed the transcriptome dynamics following induction of CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3' UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3' UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3' UTR isoforms was altered following CELF1 induction, with 3' UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes.
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http://dx.doi.org/10.1101/gr.184390.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448682PMC
June 2015

Origins and impacts of new mammalian exons.

Cell Rep 2015 Mar 19;10(12):1992-2005. Epub 2015 Mar 19.

Departments of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Program in Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

Mammalian genes are composed of exons, but the evolutionary origins and functions of new internal exons are poorly understood. Here, we analyzed patterns of exon gain using deep cDNA sequencing data from five mammals and one bird, identifying thousands of species- and lineage-specific exons. Most new exons derived from unique rather than repetitive intronic sequence. Unlike exons conserved across mammals, species-specific internal exons were mostly located in 5' UTRs and alternatively spliced. They were associated with upstream intronic deletions, increased nucleosome occupancy, and RNA polymerase II pausing. Genes containing new internal exons had increased gene expression, but only in tissues in which the exon was included. Increased expression correlated with the level of exon inclusion, promoter proximity, and signatures of cotranscriptional splicing. Altogether, these findings suggest that increased splicing at the 5' ends of genes enhances expression and that changes in 5' end splicing alter gene expression between tissues and between species.
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http://dx.doi.org/10.1016/j.celrep.2015.02.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715653PMC
March 2015