Jie Wu

The lncRNA Malat1 Is Dispensable for Mouse Development but Its Transcription Plays a cis-Regulatory Role in the Adult

Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs) lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.

SpliceTrap: a method to quantify alternative splicing under single cellular conditions

MOTIVATIONnAlternative splicing (AS) is a pre-mRNA maturation process leading to the expression of multiple mRNA variants from the same primary transcript. More than 90% of human genes are expressed via AS. Therefore, quantifying the inclusion level of every exon is crucial for generating accurate transcriptomic maps and studying the regulation of AS.nnnRESULTSnHere we introduce SpliceTrap, a method to quantify exon inclusion levels using paired-end RNA-seq data. Unlike other tools, which focus on full-length transcript isoforms, SpliceTrap approaches the expression-level estimation of each exon as an independent Bayesian inference problem. In addition, SpliceTrap can identify major classes of alternative splicing events under a single cellular condition, without requiring a background set of reads to estimate relative splicing changes. We tested SpliceTrap both by simulation and real data analysis, and compared it to state-of-the-art tools for transcript quantification. SpliceTrap demonstrated improved accuracy, robustness and reliability in quantifying exon-inclusion ratios.nnnCONCLUSIONSnSpliceTrap is a useful tool to study alternative splicing regulation, especially for accurate quantification of local exon-inclusion ratios from RNA-seq data.nnnAVAILABILITY AND IMPLEMENTATIONnSpliceTrap can be implemented online through the CSH Galaxy server http://cancan.cshl.edu/splicetrap and is also available for download and installation at http://rulai.cshl.edu/splicetrap/.nnnCONTACTnmichael.zhang@utdallas.edu.nnnSUPPLEMENTARY INFORMATIONnSupplementary data are available at Bioinformatics online.

SRSF1-Regulated Alternative Splicing in Breast Cancer.

Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5 splice site generally promoted exon inclusion, whereas binding near the 3 splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1s oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development.

OLego: fast and sensitive mapping of spliced mRNA-Seq reads using small seeds

A crucial step in analyzing mRNA-Seq data is to accurately and efficiently map hundreds of millions of reads to the reference genome and exon junctions. Here we present OLego, an algorithm specifically designed for de novo mapping of spliced mRNA-Seq reads. OLego adopts a multiple-seed-and-extend scheme, and does not rely on a separate external aligner. It achieves high sensitivity of junction detection by strategic searches with small seeds (∼14 nt for mammalian genomes). To improve accuracy and resolve ambiguous mapping at junctions, OLego uses a built-in statistical model to score exon junctions by splice-site strength and intron size. Burrows–Wheeler transform is used in multiple steps of the algorithm to efficiently map seeds, locate junctions and identify small exons. OLego is implemented in C++ with fully multithreaded execution, and allows fast processing of large-scale data. We systematically evaluated the performance of OLego in comparison with published tools using both simulated and real data. OLego demonstrated better sensitivity, higher or comparable accuracy and substantially improved speed. OLego also identified hundreds of novel micro-exons (<30 nt) in the mouse transcriptome, many of which are phylogenetically conserved and can be validated experimentally in vivo. OLego is freely available at http://zhanglab.c2b2.columbia.edu/index.php/OLego.

Chd5 orchestrates chromatin remodelling during sperm development

One of the most remarkable chromatin remodelling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodelling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying this process of extensive chromatin remodelling.

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.

Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression

We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern for some C/D box snoRNAs and abundant expression of longer, non-coding RNAs in the cell that can potentially form dsRNAs.

DAZL Limits Pluripotency, Differentiation, and Apoptosis in Developing Primordial Germ Cells

Summary The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.

Distinct and Predictive Histone Lysine Acetylation Patterns at Promoters, Enhancers, and Gene Bodies

In eukaryotic cells, histone lysines are frequently acetylated. However, unlike modifications such as methylations, histone acetylation modifications are often considered redundant. As such, the functional roles of distinct histone acetylations are largely unexplored. We previously developed an algorithm RFECS to discover the most informative modifications associated with the classification or prediction of mammalian enhancers. Here, we used this tool to identify the modifications most predictive of promoters, enhancers, and gene bodies. Unexpectedly, we found that histone acetylation alone performs well in distinguishing these unique genomic regions. Further, we found the association of characteristic acetylation patterns with genic regions and association of chromatin state with splicing. Taken together, our work underscores the diverse functional roles of histone acetylation in gene regulation and provides several testable hypotheses to dissect these roles.

Abstract A50: Nonredundant functions of splicing factors in breast-cancer initiation and metastasis

Alternative splicing is a key control point in gene expression, whose misregulation contributes to cancer malignancy. Although certain splicing factors (SFs) and their targets are altered in human tumors, the functional significance of these alterations remains unclear. We previously demonstrated that the splicing factor SRSF1 is upregulated in human breast tumors and promotes transformation in vivo and in vitro. SRSF1 is a prototypical member of the SR protein family, composed of 12 structurally related proteins. However, little is known about differences and redundancies in their splicing targets and biological functions. Here, we investigated whether additional SFs also promoted breast cancer, using transformation models that mimic the relevant biological context. In parallel, we used RNA sequencing (RNA-seq) to systematically identify their oncogenic splicing targets. By mining a large collection of human tumors from the TCGA project, we defined the molecular portraits of SFs alterations in breast tumors. We identified five SFs amplified and/or overexpressed in at least 10% of breast tumors. We then used SF-overexpressing human mammary epithelial MCF-10A cells grown in organotypic 3-D culture; these cells form polarized growth-arrested acinar structures, similar to the terminal units of mammary ducts. Various breast-cancer oncogenes are known to disrupt acinar growth and/or architecture. Interestingly, only certain SFs were oncogenic in this context, differentially affecting cell proliferation, apoptosis, or acinar organization, suggesting non-redundant functions. We then characterized the splicing targets relevant for SF-mediated transformation. We developed a bioinformatics pipeline to identify and quantify splicing variation in RNA-seq data. We defined the global repertoire of SF-regulated splicing events in 3-D culture and compared the target specificities of various SR proteins. In addition, we identified splicing targets regulated both in 3-D culture as well as in human breast tumors. Strikingly, SFs that promoted similar phenotypic changes shared a significant number of splicing targets, suggesting that they regulate common genes to promote tumor initiation. Furthermore, specific SFs affected targets previously associated with epithelial to mesenchymal transition, and increased cell migration or invasion. Finally, we uncovered that the splicing regulator TRA2β is required for the maintenance of metastatic properties of human breast-cancer cells in 3-D culture and in mouse orthotopic models. Furthermore, TRA2β levels correlate with increased metastatic incidence in breast cancer patients. Thus TRA2β represent a potential target for therapeutics development. In summary, we gained new insights into the biological functions of SR proteins and identified novel oncogenic SF-regulated splicing events involved in tumor initiation and metastasis. Citation Format: Olga Anczukow, Shipra Das, Kuan-Ting Lin, Jie Wu, Martin Akerman, Senthil K. Muthuswamy, Adrian R. Krainer. Nonredundant functions of splicing factors in breast-cancer initiation and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A50.