Sebastien M. Weyn-Vanhentenryck

MBNL Sequestration by Toxic RNAs and RNA Misprocessing in the Myotonic Dystrophy Brain

For some neurological disorders, disease is primarily RNA mediated due to expression of non-coding microsatellite expansion RNAs (RNA(exp)). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain, resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound-knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNA(exp) in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases.

Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators

Significance Alternative splicing (AS) plays an important role in the mammalian brain, but our atlas of AS events is incomplete. Here, we conducted comprehensive analysis of deep RNA-Seq data of mouse cortex to identify new AS events and evaluate their functionality. We expanded the number of annotated AS events more than 10-fold and demonstrated that, like many known events, thousands of newly discovered events are regulated, conserved, and likely functional. In particular, some can regulate gene expression levels through nonsense-mediated decay, a known mechanism for RNA binding protein autoregulation. Surprisingly, we discovered a number of chromatin regulators as novel targets of this mechanism, revealing a new regulatory link between epigenetics and AS that primarily emerged in the mammalian lineage. Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue- or cell type-specific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23–42% of all cassette exons under tissue- or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome.

CLIP Tool Kit (CTK): a flexible and robust pipeline to analyze CLIP sequencing data.

Summary: UV cross‐linking and immunoprecipitation (CLIP), followed by high‐throughput sequencing, is a powerful biochemical assay that maps in vivo protein‐RNA interactions on a genome‐wide scale. The CLIP Tool Kit (CTK) aims at providing a set of tools for flexible, streamlined and comprehensive CLIP data analysis. This software package extends the scope of our original CIMS package. Availability and Implementation: The software is implemented in Perl. The source code and detailed documentation are available at http://zhanglab.c2b2.columbia.edu/index.php/CTK. Contact: cz2294@columbia.edu

mCarts: Genome-Wide Prediction of Clustered Sequence Motifs as Binding Sites for RNA-Binding Proteins.

RNA-binding proteins (RBPs) are critical components of post-transcriptional gene expression regulation. However, their binding sites have until recently been difficult to determine due to the apparent low specificity of RBPs for their target transcripts and the lack of high-throughput assays for analyzing binding sites genome wide. Here we present a bioinformatics method for predicting RBP binding motif sites on a genome-wide scale that leverages motif conservation, RNA secondary structure, and the tendency of RBP binding sites to cluster together. A probabilistic model is learned from bona fide binding sites determined by CLIP and applied genome wide to generate high specificity binding site predictions.

Precise temporal regulation of alternative splicing during neural development

Alternative splicing (AS) is one crucial step of gene expression that must be tightly regulated during neurodevelopment. However, the precise timing of developmental splicing switches and the underlying regulatory mechanisms are poorly understood. Here we systematically analyze the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis reveals early-switch and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl, and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the “immature” splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.The precise timing of neurodevelopmental splicing switches and the underlying regulatory mechanisms remain poorly understood. This study identifies two major waves of developmental switches under the control of distinct combinations of RNA-binding proteins in central and peripheral nervous systems.

Microexons: discovery, regulation, and function

The importance of RNA splicing in numerous cellular processes is well established. However, an underappreciated aspect is the ability of the spliceosome to recognize a set of very small (3–30 nucleotide, 1–10 amino acid) exons named microexons. Despite their small size, microexons and their regulation through alternative splicing have now been shown to play critical roles in protein and system function. Here we review the discovery of microexons over time and the mechanisms by which their splicing is regulated, including recent progress made through deep RNA sequencing. We also discuss the functional role of microexons in biology and disease. WIREs RNA 2017, 8:e1418. doi: 10.1002/wrna.1418

Dazl regulates germ cell survival through a network of polyA proximal mRNA interactions

The RNA binding protein Dazl is essential for gametogenesis, but its direct in vivo functions, RNA targets, and the molecular basis for germ cell loss in DAZL KO mice are unknown. Here, we generated transcriptome-wide maps of Dazl-RNA interactions in adult and juvenile mouse testes. In parallel, we used transgenic mice and fluorescence activated cell sorting to isolate DAZL knockout germ cells and identify mRNAs sensitive to Dazl deletion. Integrative analyses reveal that Dazl functions as a master regulator of germ cell survival by post-transcriptionally enhancing a vast network of genes necessary for cell cycle regulation and spermatogenesis. Strikingly, Dazl displays a strong positional bias for binding near polyA tails and multimerizes on a subset of its targets. These results reveal a mechanism for Dazl recruitment to its RNA targets and delineate a Dazl-dependent mRNA regulatory program essential for postnatal mammalian germ cell survival.

Modeling RNA-binding protein specificity in vivo by precisely registering protein-RNA crosslink sites

RNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence elements in their target transcripts. Despite the expanding list of RBPs with in vivo binding sites mapped genomewide using crosslinking and immunoprecipitation (CLIP), defining precise RBP binding specificity remains challenging. We previously demonstrated that the exact protein-RNA crosslink sites can be mapped using CLIP data at single-nucleotide resolution and observed that crosslinking frequently occurs at specific positions in RBP motifs. Here we have developed a computational method, named mCross, to jointly model RBP binding specificity while precisely registering the crosslinking position in motif sites. We applied mCross to 112 RBPs using ENCODE eCLIP data and validated the reliability of the resulting motifs by genome-wide analysis of allelic binding sites also detected by CLIP. We found that the prototypical SR protein SRSF1 recognizes GGA clusters to regulate splicing in a much larger repertoire of transcripts than previously appreciated.