In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 ...insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.
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•We sequence resolve and annotate 99,604 common human structural variants•55% of VNTRs map to the end of chromosomes and correlate with double-strand breaks•Alternate alleles facilitate accurate genotyping with short reads and new associations•We patch the reference and add diversity needed for developing a pan human genome
Long-read sequencing allows generation of a large catalog of human structural variants and the development of an algorithm for genotyping SVs from short-read data, clarifying the spectrum and importance of structural variation in the human genome.
Alternative cleavage and polyadenylation (APA), an RNA processing event, occurs in over 70% of human protein-coding genes. APA results in mRNA transcripts with distinct 3' ends. Most APA occurs ...within 3' UTRs, which harbor regulatory elements that can impact mRNA stability, translation, and localization.
APA can be profiled using a number of established computational tools that infer polyadenylation sites from standard, short-read RNA-seq datasets. Here, we benchmarked a number of such tools-TAPAS, QAPA, DaPars2, GETUTR, and APATrap- against 3'-Seq, a specialized RNA-seq protocol that enriches for reads at the 3' ends of genes, and Iso-Seq, a Pacific Biosciences (PacBio) single-molecule full-length RNA-seq method in their ability to identify polyadenylation sites and quantify polyadenylation site usage. We demonstrate that 3'-Seq and Iso-Seq are able to identify and quantify the usage of polyadenylation sites more reliably than computational tools that take short-read RNA-seq as input. However, we find that running one such tool, QAPA, with a set of polyadenylation site annotations derived from small quantities of 3'-Seq or Iso-Seq can reliably quantify variation in APA across conditions, such asacross genotypes, as demonstrated by the successful mapping of alternative polyadenylation quantitative trait loci (apaQTL).
We envisage that our analyses will shed light on the advantages of studying APA with more specialized sequencing protocols, such as 3'-Seq or Iso-Seq, and the limitations of studying APA with short-read RNA-seq. We provide a computational pipeline to aid in the identification of polyadenylation sites and quantification of polyadenylation site usages using Iso-Seq data as input.
RNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence motifs in their target transcripts, but precisely defining their binding ...specificity remains challenging. Crosslinking and immunoprecipitation (CLIP) allows for mapping of the exact protein-RNA crosslink sites, which frequently reside at specific positions in RBP motifs at single-nucleotide resolution. 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 discovered motifs by genome-wide analysis of allelic binding sites. Our analyses revealed that the prototypical SR protein SRSF1 recognizes clusters of GGA half-sites in addition to its canonical GGAGGA motif. Therefore, SRSF1 regulates splicing of a much larger repertoire of transcripts than previously appreciated, including HNRNPD and HNRNPDL, which are involved in multivalent protein assemblies and phase separation.
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•mCross jointly models RBP binding motif and crosslink positions using CLIP data•mCross was applied to 112 RBPs with eCLIP data from ENCODE•SRSF1 predominantly recognizes clusters of GGA half-sites to regulate splicing•SRSF1 regulates HNRNPD and HNRNPDL splicing involved in phase separation
Feng et al. described an algorithm called mCross to accurately define RNA-binding protein specificity by precisely registering protein-RNA crosslink sites using CLIP data. This method was applied to >100 RBPs and identified a noncanoncial binding motif of SRSF1, which implicates the protein in modulating phase separation.
The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N6-methyladenosine (m6A), the most common internal RNA ...modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m6A modification on numerous transcripts. In vivo conditional inactivation of an essential m6A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts postmitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including those that encode the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination.
•Oligodendrocyte maturation is accompanied by modifications in m6A mRNA methylation•m6A mRNA methylation is required for oligodendrocyte maturation and CNS myelination•m6A mRNA methylation regulates the transcriptomes of oligodendrocyte lineage cells•Proper neurofascin mRNA splicing in oligodendrocytes requires m6A methylation
Xu et al. show that oligodendrocyte development is associated with dynamic changes in posttranscriptional mRNA methylation. Moreover, they demonstrate that the m6A epigenetic RNA mark has considerable impact on the myelinating cell’s transcriptome and is essential for normal CNS myelination.
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.
The software is implemented in Perl. The source code and detailed documentation are available at http://zhanglab.c2b2.columbia.edu/index.php/CTK .
cz2294@columbia.edu.
Genome-wide association studies (GWAS) have allowed us to successfully identify thousands of common genetic variants underlying a number of diseases, but it has been difficult to understand how ...mechanism of action because the vast majority of these loci are located in non-coding regions of the genome. Because it is estimated that only 25% of disease associated genetic variants contribute to disease by affecting steady-state gene expression levels, it will be important to establish a more comprehensive understanding of alternative mechanisms through which genetic variants act to contribute to disease, such as RNA processing and RNA modification. Motivated by this, this dissertation outlines the development of computational methods and assessment of existing tools to profiles various RNA processing and RNA modification events across individuals, cell types, and developmental stages, which can ultimately be applied to large disease-cohort datasets in future studies. In the first chapter, we provide primers on quantitative genetics and RNA processing and modifications to put this work in context. In the second chapter, we demonstrate that combining large quantities of RNA-seq data with small quantities of specialized data, including 3’-Seq and single-molecule real-time (SMRT) isoform sequencing (Iso-Seq), allows one to study alternative cleavage and polyadenylation, without compromising affordability or accuracy. We apply this approach to explore inter-individual variation in polyadenylation site choice. In the third chapter, we profile the role of the RNA modification N6-methyladenosine (m6A) in oligodendrocyte lineage progression and its potential impacts in human diseases, such as multiple sclerosis. Finally, in the fourth chapter, we develop a method to examine how genetic variants that increase risk of disease reduce the fidelity of RNA splicing.
Most complex traits, including diseases, have a large genetic component. Identifying the genetic variants and genes underlying phenotypic variation remains one of the most important objectives of ...current biomedical research. Unlike Mendelian or familial diseases, which are usually caused by mutations in the coding regions of individual genes, complex diseases are thought to result from the cumulative effects of a large number of variants, of which, the vast majority are noncoding. Therefore, to discern the genetic underpinnings of a complex trait, we must first understand the impact of noncoding variation, which presumably affects gene regulation. In this chapter, we outline the recent progress made and methods used to discover putative regulatory regions associated with complex traits. We will specifically focus on mapping splicing quantitative trait loci (sQTL) using Yoruba samples from GEUVADIS as a motivating example.
Abstract
Advanced-stage prostate cancer is characterized by osseous metastases whose establishment involves the dynamic interplay of factors and exchange of cellular contents by constituents of the ...tumor bony microenvironment. Among the factors that transport cellular cargoes and facilitate the transmission of signaling complexes for establishment of the bone metastatic lesions are large extracellular vesicles (LEVs), also known as large oncosomes. Information on the contents of LEVs and the mechanisms by which LEVs are formed and regulated is incomplete. In the course of studies aimed at elucidating the mechanisms of bony metastases using the LNCaP-C4-2B prostate cancer progression model, we show that the expression and cellular distribution of nicotinamide riboside: quinone oxidoreductase 2 (NQO2) and glutaminase (GLS, kidney type) both participate in the induction of membrane-localized oxidative stress and the formation of LEVs. The salient features of our findings are the following: The LNCaP model system exhibits gradually increasing oxidative stress levels as it progresses to the bone metastatic derivative C4-2B, while at the same time showing a progressive decrease in the expression level of NQO2. Reverse transcription PCR studies showed that NQO2 mRNA levels were unchanged between LNCaP and its metastatic derivatives C4, C4-2 and C4-2B. As a control, the expression level of the analogous enzyme NADP(H):quinone oxidoreductase 1 (NQO1) was also unchanged. These results suggested the possibility that the NQO2 enzyme in the bone metastatic C4-2B derivative could be localized elsewhere, possibly at an extracellular location. Further studies showed that a fraction of the NQO2 enzyme was associated with the detergent resistant membrane fraction of the C4-2B prostate cancer cells. Molecular modeling studies showed that the NQO2 enzyme had two caveolin-1 and NQO1 enzyme had one caveolin-1 binding sites. While the caveolin-1 binding site of NQO1 is buried inside the molecule and hence inaccessible to caveolin-1, one of the two binding sites available in NQO2 was external and accessible to caveolin-1. We have validated the specific interaction between NQO2 and caveolin-1 using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B cells showed increasing level of expression of the GLS enzyme, suggesting a correlation between increased oxidative stress, glutaminase activity, and metastatic potential. Moreover, we found that the metastatic C4-2B derivative extruded LEVs in which the enzyme NQO2 and caveolae specific caveolin-1 could be visualized as cargo using double immunofluorescence studies. The extrusion of these LEVs could be inhibited by the GLS specific inhibitor BP-TES. Characteristically, curcumin was also found to inhibit the extrusion of these LEVs. This naturally occurring plant-based compound was shown earlier to inhibit prostate cancer bone metastasis in vivo using the C4-2B model. These results indicate a role for the NQO2 enzyme at the membrane level, possibly increasing the oxidative stress in a localized manner by lipid peroxidation. These results also suggest that a glutamine/GLS-mediated metabolic reprogramming in the LNCaP model system as it progresses toward the bone metastatic C4-2B is an integral component of the force driving the metastatic process. Based on the results of our studies, a combinatorial strategy using antimetastatic therapies such as nitrogen-containing bisphosphonates (NBPs) and anti-GLS therapy or, alternatively, a naturally occurring plant compound-based anti-NQO2 and anti-GLS therapy, could be considered for treating advanced prostate cancer patients with bone metastatic complications.
Citation Format: Thambi Dorai, Ankeeta B. Shah, Faith Summers, Rajamma Mathew, Jing Huang, Tze-chen Hsieh, Joseph M. Wu. NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis abstract. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B003.
The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N
-methyladenosine (m
A), the most common internal RNA ...modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m
A modification on numerous transcripts. In vivo conditional inactivation of an essential m
A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts postmitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including those that encode the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination.
Abstract
Prostate cancer is characterized by an innate capacity to produce higher concentrations of reactive oxygen species (ROS). ROS is also a hallmark for highly aggressive disease. Our laboratory ...has focused on the molecular mechanisms of inhibition of prostate cancer bone metastasis by the naturally occurring plant based anti-oxidant compound curcumin, arising from the rhizomes of Curcuma Longa. These studies highlight the importance of studying the mechanisms of redox homeostasis in normal cells and how they are subverted in cancer cells. The LNCaP prostate cancer progression model system as exemplified by the development of C4, C4-2 and C4-2B ( bone metastatic) sublines derived from the parental LNCaP prostate cancer cells offer an unique avenue to study the role of redox systems in the progression to metastatic disease, culminating in the establishment of osseous metastases. The mechanisms of ROS production and destruction, the cellular machinery and the enzyme systems involved, the relationship of ROS to cellular oncogene activation and the antagonistic duality of oncogenes and tumor suppressor genes with respect to ROS homeostasis have been subjects of intense investigation. How the prostate cancer cells metabolize ROS to achieve their proliferative and metastatic potential is not completely understood. We chose to study one redox system orchestrated by the expression of NADPH:Quinone Oxidoreductase1 (NQO1) and the Nicotinamide Riboside (NRH): Quinone Oxidoreductase 2 (NQO2) in the LNCaP progression model system. Particularly, the enzyme NQO2 has been shown earlier to be the target of another polyphenolic chemopreventive antioxidant resveratrol. The role of NQO2 as a phase II detoxification enzyme acting on several xenobiotics is well characterized. However, the role of NQO2 in regulating ROS and hence cancer progression is only beginning to be understood. With this background, we investigated the potential role of NQO2/NQO1 expression in the transition from androgen dependence to androgen independence and to acquiring bone metastatic potential in C4-2B prostate cancer cells. Our studies indicate that 1) there is a down regulation of NQO2 as the androgen dependent LNCaP cells progress towards androgen independence in C4 and C4-2 cells. In C4-2B cells, there is a barely detectable level of NQO2. In contrast, there is very little change in the level of expression of NQO1; 2) Characteristically, the intracellular ROS production progressively increases from LNCaP to C4-2B; 3) In parallel, there is an up regulation of caveolin-1 as the LNCaP cells progress to bone metastatic C4-2B and 4) a small fraction of NQO2 could be detected in the detergent resistant membrane (DRM) fraction consisting of lipid rafts and caveolae. Our studies suggest that caveolin-1 may play a role in the membrane sequestration of the largely cytoplasmic NQO2 in LNCaP cells and that this interaction could be lost in bone metastatic C4-2B cells. The significance of these findings in relation to the metastatic potential of C4-2B cells will be discussed.
Citation Format: Ankeeta Shah, Tze-Chen Hsieh, Rajamma Mathew, Joseph M. Wu, Thambi Dorai. Downregulation of NRH:quinone oxidoreductase 2(NQO2) in the LNCaP prostate cancer progression model system: Implications to metastasis. abstract. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A11.
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