Processing of Endogenous Pre-mRNAs in Association with SC-35 Domains Is Gene Specific

Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript “trees” of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional “tracks” of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.

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Computational studies on RNA processing in higher eukaryotes

10.21248/gups.61384 ◽

2021 ◽

Author(s):

◽

Mirko Brüggemann

Keyword(s):

Beta Cell ◽

Rna Processing ◽

Binding Sites ◽

Pancreatic Beta Cells ◽

Splicing Factor ◽

Sequencing Data ◽

Diabetes Susceptibility ◽

Protein Coding ◽

Protein Coding Genes ◽

Phd Thesis

Most cellular processes are regulated by RNA-binding proteins (RBPs). These RBPs usually use defined binding sites to recognize and directly interact with their target RNA molecule. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments are an important tool to de- scribe such interactions in cell cultures in-vivo. This experimental protocol yields millions of individual sequencing reads from which the binding spec- trum of the RBP under study can be deduced. In this PhD thesis I studied how RNA processing is driven from RBP binding by analyzing iCLIP-derived sequencing datasets. First, I described a complete data analysis pipeline to detect RBP binding sites from iCLIP sequencing reads. This workflow covers all essential process- ing steps, from the first quality control to the final annotation of binding sites. I described the accurate integration of biological iCLIP replicates to boost the initial peak calling step while ensuring high specificity through replicate re- producibility analysis. Further I proposed a routine to level binding site width to streamline downstream analysis processes. This was exemplified in the re- analysis of the binding spectrum of the U2 small nuclear RNA auxiliary factor 2 (U2AF2, U2AF65). I recaptured the known dominance of U2AF65 to bind to intronic sequences of protein-coding genes, where it likely recognizes the polypyrimidine tract as part of the core spliceosome machinery. In the second part of my thesis, I analyzed the binding spectrum of the serine and arginine rich splicing factor 6 (SRSF6) in the context of diabetes. In pancreatic beta-cells, the expression of SRSF6 is regulated by the transcription factor GLIS3, which encodes for a diabetes susceptibility gene. It is known that SRSF6 promotes beta-cell death through the splicing dysregulation of genes essential to beta-cell function and survival. However, the exact mechanism of how these RNAs are targeted by SRSF6 remains poorly understood. Here, I applied the defined iCLIP processing pipeline to describe the binding landscape of the splicing factor SRSF6 in the human pancreatic beta-cell line EndoC-H1. The initial binding sites definition revealed a predominant binding to coding sequences (CDS) of protein-coding genes. This was followed up by extensive motif analysis which revealed a so far, in human, unknown purine-rich binding motif. SRSF6 seemed to specifically recognize repetitions of the triplet GAA. I also showed that the number of contiguous triplets correlated with increasing binding site strength. I further integrated RNA-sequencing data from the same cell type, with SRSF6 in KD and in basal conditions, to analyze SRSF6- related splicing changes. I showed that the exact positioning of SRSF6 on alternatively spliced exons regulates the produced transcript isoforms. This mechanism seemed to control exons in several known susceptibility genes for diabetes. In summary, in my PhD thesis, I presented a comprehensive workflow for the processing of iCLIP-derived sequencing data. I applied this pipeline on a dataset from pancreatic beta-cells to unveil the impact of SRSF6-mediated splicing changes. Thus, my analysis provides novel insights into the regulation of diabetes susceptibility genes.

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Integrative Analysis Reveals the Prognostic Value and Functions of Splicing Factors Implicated in Hepatocellular Carcinoma

10.21203/rs.3.rs-408292/v1 ◽

2021 ◽

Author(s):

Yue Wang ◽

Fan Yang ◽

Jiaqi Shang ◽

Haitao He ◽

Qing Yang

Keyword(s):

Hepatocellular Carcinoma ◽

Prognostic Model ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Splicing Factors ◽

Biological Functions ◽

Clinical Value ◽

Multiple Cancer ◽

Protein Coding ◽

Protein Coding Genes

Abstract Splicing factors (SFs) play critical roles in the pathogenesis of various cancers through regulating tumor-associated alternative splicing (AS) events. However, the clinical value and biological functions of SFs in hepatocellular carcinoma (HCC) remain obscure. In this study, we identified 40 dysregulated SFs in HCC and established a prognostic model composed of four SFs (DNAJC6, ZC3H13, IGF2BP3, DDX19B). The predictive efficiency and independence of the prognostic model were confirmed to be satisfactory. Gene Set Enrichment Analysis (GSEA) illustrated the risk score calculated by our prognostic model was significantly associated with multiple cancer-related pathways and metabolic processes. Furthermore, we constructed the SFs-AS events regulatory network and extracted 108 protein-coding genes from the network for following functional explorations. Protein-protein interaction (PPI) network delineated the potential interactions among these 108 protein-coding genes. GO and KEGG pathway analyses investigated ontology gene sets and canonical pathways enriched by these 108 protein-coding genes. Overlapping the results of GSEA and KEGG, seven pathways were identified to be potential pathways regulated by our prognostic model through triggering aberrant AS events in HCC. In conclusion, the present study established an effective prognostic model based on SFs for HCC patients. Functional explorations of SFs and SFs-associated AS events provided directions to explore biological functions and mechanisms of SFs in HCC tumorigenesis.

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Visualizing transcription in real-time

Open Life Sciences ◽

10.2478/s11535-008-0001-1 ◽

2008 ◽

Vol 3 (1) ◽

pp. 11-18 ◽

Cited By ~ 1

Author(s):

Yehuda Brody ◽

Yaron Shav-Tal

Keyword(s):

Rna Polymerase Ii ◽

Living Cells ◽

Messenger Rnas ◽

Protein Coding ◽

Upstream Gene ◽

Structural Modifications ◽

Protein Coding Genes ◽

Transcriptional Process ◽

Kinetics Of

AbstractThe transcriptional process is at the center of the gene expression pathway. In eukaryotes, the transcription of protein-coding genes into messenger RNAs is performed by RNA polymerase II. This enzyme is directed to bind at upstream gene sequences by the aid of transcription factors that assemble transcription-competent complexes. A series of biochemical and structural modifications render the polymerase transcriptionally active so that it can proceed from an initiation state into a functional elongating phase. Recent experimental efforts have attempted to visualize these processes as they take place on genes in living cells and to quantify the kinetics of in vivo transcription.

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Nuclear pre-mRNA Compartmentalization: Trafficking of Released Transcripts to Splicing Factor Reservoirs

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.497 ◽

2000 ◽

Vol 11 (2) ◽

pp. 497-510 ◽

Cited By ~ 64

Author(s):

Ivo Melčák ◽

Štěpánka Cermanová ◽

Kateřina Jirsová ◽

Karel Koberna ◽

Jan Malı́nský ◽

...

Keyword(s):

Spatial Organization ◽

Raji Cell ◽

Splicing Factor ◽

Chromosome 1 ◽

Viral Gene ◽

Splicing Factors ◽

Electron Microscopic ◽

Viral Transcript ◽

Nascent Transcript ◽

Nascent Transcripts

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein–Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.

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Reduced Mobility of the Alternate Splicing Factor (Asf) through the Nucleoplasm and Steady State Speckle Compartments

The Journal of Cell Biology ◽

10.1083/jcb.150.1.41 ◽

2000 ◽

Vol 150 (1) ◽

pp. 41-52 ◽

Cited By ~ 113

Author(s):

Michael J. Kruhlak ◽

Melody A. Lever ◽

Wolfgang Fischle ◽

Eric Verdin ◽

David P. Bazett-Jones ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Binding Sites ◽

Time Lapse ◽

Splicing Factor ◽

Nuclear Space ◽

Splicing Factors ◽

Transcription Inhibitor ◽

Spatial Differences ◽

Active Transcription ◽

Green Fluorescent

Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF–GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF–GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.

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Rbfox contributes to a conservative program of self-antigen splicing in thymic epithelial cells

10.1101/2020.03.06.980870 ◽

2020 ◽

Author(s):

Kathrin Jansen ◽

Noriko Shikama-Dorn ◽

Moustafa Attar ◽

Stefano Maio ◽

Maria Lopopolo ◽

...

Keyword(s):

Epithelial Cells ◽

Splicing Factor ◽

Thymic Epithelial Cells ◽

Splicing Factors ◽

Gene Repertoire ◽

Cell Repertoire ◽

Protein Coding ◽

Peripheral Tissues ◽

Splice Isoform ◽

Self Antigen

AbstractThymic epithelial cells (TEC) guide selection of a T-cell repertoire that is reactive to pathogens but tolerant to self. While this process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire by TEC, the extent to which these cells reproduce peripheral isoform structures is unknown. We performed a transcriptomic investigation of transcript structures and splicing factor expression in medullary TEC and 21 peripheral tissues. Our results indicate that TEC re-use a small number of peripheral splicing factors to recreate a limited subset of the peripheral splice isoform repertoire. We found, for example, that TEC lacked both neuronal micro-exons and the splicing factor, Srrm4, which promotes their inclusion. We demonstrate a functional role for the Rbfox splicing factors in promoting medullary TEC development and the alternative splicing of promiscuously expressed genes. Our findings have implications for understanding autoimmune diseases and the development of antigen-specific immunotherapies.

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Integrative analysis reveals the prognostic value and functions of splicing factors implicated in hepatocellular carcinoma

Scientific Reports ◽

10.1038/s41598-021-94701-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yue Wang ◽

Fan Yang ◽

Jiaqi Shang ◽

Haitao He ◽

Qing Yang

Keyword(s):

Hepatocellular Carcinoma ◽

Prognostic Model ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Splicing Factors ◽

Biological Functions ◽

Clinical Value ◽

Multiple Cancer ◽

Protein Coding ◽

Protein Coding Genes

AbstractSplicing factors (SFs) play critical roles in the pathogenesis of various cancers through regulating tumor-associated alternative splicing (AS) events. However, the clinical value and biological functions of SFs in hepatocellular carcinoma (HCC) remain obscure. In this study, we identified 40 dysregulated SFs in HCC and established a prognostic model composed of four SFs (DNAJC6, ZC3H13, IGF2BP3, DDX19B). The predictive efficiency and independence of the prognostic model were confirmed to be satisfactory. Gene Set Enrichment Analysis (GSEA) illustrated the risk score calculated by our prognostic model was significantly associated with multiple cancer-related pathways and metabolic processes. Furthermore, we constructed the SFs-AS events regulatory network and extracted 108 protein-coding genes from the network for following functional explorations. Protein–protein interaction (PPI) network delineated the potential interactions among these 108 protein-coding genes. GO and KEGG pathway analyses investigated ontology gene sets and canonical pathways enriched by these 108 protein-coding genes. Overlapping the results of GSEA and KEGG, seven pathways were identified to be potential pathways regulated by our prognostic model through triggering aberrant AS events in HCC. In conclusion, the present study established an effective prognostic model based on SFs for HCC patients. Functional explorations of SFs and SFs-associated AS events provided directions to explore biological functions and mechanisms of SFs in HCC tumorigenesis.

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