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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of the implication of the DBIRD complex (DBC1 and ZNF326/ZIRD) in gene expression and alternative splicing

ABSTRACT: Alternative mRNA splicing is the main reason vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription and the rate of transcript elongation has a profound effect on splicing. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle that represents its functional form, and the composition of which determines the fate of the mature transcript4. However, factors that connect the transcribing polymerase with the mRNP particle and help integrate transcript elongation with mRNA splicing remain obscure. Here, we characterized the interactome of chromatin-associated mRNP particles and thereby identified Deleted in Breast Cancer 1 (DBC1) and a protein we named ZIRD. These proteins are subunits of a novel protein complex, named DBIRD, which binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in A/T-rich DNA, and is present at the affected exons. RNAi-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. These data indicate that DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with regulation of alternative splicing. HEK 293 cells were transfected with CTR-, DBC1- and ZIRD (ZNF326)- specific stealth siRNAs. Total RNAs from three independent experiments were extracted after 48 hours. All conditions were analyzed in triplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: pierre close

PROVIDER: E-GEOD-35480 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Analysis of the implication of the DBIRD complex (DBC1 and ZNF326/ZIRD) in gene expression and alternative splicing

Project description:Alternative mRNA splicing is the main reason vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription and the rate of transcript elongation has a profound effect on splicing. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle that represents its functional form, and the composition of which determines the fate of the mature transcript4. However, factors that connect the transcribing polymerase with the mRNP particle and help integrate transcript elongation with mRNA splicing remain obscure. Here, we characterized the interactome of chromatin-associated mRNP particles and thereby identified Deleted in Breast Cancer 1 (DBC1) and a protein we named ZIRD. These proteins are subunits of a novel protein complex, named DBIRD, which binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in A/T-rich DNA, and is present at the affected exons. RNAi-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. These data indicate that DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with regulation of alternative splicing.

2012-03-20 | GSE35480 | GEO

H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells [RNA-seq]

Project description:Epigenetic regulation of chromatin plays a critical role in controlling embryonic stem (ES) cell self-renewal and pluripotency. However, the roles of histone demethylases and activating histone modifications such as trimethylated histone 3 lysine 4 (H3K4me3) in transcriptional events such as RNA polymerase II (RNAPII) elongation and alternative splicing are largely unknown. In this study, we show that KDM5B, which demethylates H3K4me3, plays an integral role in regulating RNAPII occupancy, transcriptional initiation and elongation, and alternative splicing events in ES cells. Depletion of KDM5B leads to altered RNAPII promoter occupancy, and decreased RNAPII initiation and elongation rates at active genes and at genes marked with broad H3K4me3 domains. Moreover, our results demonstrate that spreading of H3K4me3 from promoter to gene body regions, which is mediated by depletion of KDM5B, modulates RNAPII elongation rates and RNA splicing in ES cells. We further show that KDM5B is enriched nearby alternatively spliced exons, and depletion of KDM5B leads to altered levels of H3K4 methylation in alternatively spliced exon regions, which is accompanied by differential expression of these ASEs. Altogether, our data indicate an epigenetic role for KDM5B in regulating RNAPII elongation and alternative splicing, which may support the diverse mRNA repertoire in ES cells.

2017-04-13 | GSE95074 | GEO

H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells [ChIP-seq]

2017-04-13 | GSE94739 | GEO

Transcriptomic Analysis of PNN- and ESRP1-regulation in Human Corneal Epithelial Cells

Project description:Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In human corneal epithelial cells (HCET), ESRP1 and PNN displayed close localization in and around nuclear speckles and their physical association in protein complexes was identified. In this study, gene expression profiling was performed to identify PNN- and ESRP1-regulated alternative pre-mRNA splicing in human corneal epithelial cells. Immortalized human corneal epithelial cells harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript level and splicing pattern of specific subsets of genes with significant overlaps in their candidate targets. Our data suggest that ESRP1 and PNN modulate alternative splicing of a specific subset of exons, but not general splicing events. ESRP1 and PNN may together participate in the regulation of epithelial-specific splicing program in a genome-wide fashion. Parental HCET, shRNA-PNN HCET, and shRNA-ESRP1 HCET cells were cultured for 3 days with/without doxycycline. Total RNA was isolated from four biological replicates of each sample group and then subjected to hGlue3_0 transcriptome array analysis.

2013-01-24 | E-GEOD-41996 | biostudies-arrayexpress

Splicing Factor RBM22 Broadly Affects RNA Polymerase II 5' Pausing, Elongation Rate and Termination and Prevents Genome Instability [C3'-seq]

Project description:Splicing factors are vital for the regulation of RNA splicing, but the underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood. Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity and provokes transcriptional readthrough genome-wide, coupled with downstream-of-gene (DoG) transcript production and genome instability. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level. Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII, and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.

2024-03-10 | GSE220317 | GEO

Splicing Factor RBM22 Broadly Affects RNA Polymerase II 5' Pausing, Elongation Rate and Termination and Prevents Genome Instability [ChIP-seq]

2024-03-10 | GSE220316 | GEO

Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential.

Project description:Organismal growth and development rely on RNA Polymerase II (RNAPII) synthesizing the appropriate repertoire of messenger RNAs (mRNAs) from protein-coding genes. Productive elongation of full-length transcripts is essential for mRNA function, however what determines whether an engaged RNAPII molecule will terminate prematurely or transcribe processively remains poorly understood. Notably, despite a common process for transcription initiation across RNAPII-synthesized RNAs1, RNAPII is highly susceptible to termination when transcribing non-coding RNAs such as upstream antisense RNAs (uaRNAs) and enhancers RNAs (eRNAs)2, suggesting that differences arise during RNAPII elongation. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in uaRNAs and eRNAs, rather than specific sequence motifs, underlies the propensity for RNAPII termination on these transcripts. Further, we demonstrate that 5’ splice sites exert both splicing-dependent and autonomous, splicing-independent stimulation of processive transcription, even in the absence of polyadenylation signals. Together, our results reveal a potent role for transcribed sequence in dictating gene output at mRNA and non-coding RNA loci, and demonstrate the power of INSERT-seq towards illuminating these contributions.

2022-04-06 | GSE178230 | GEO

Expression profile of ypr045cM-bM-^HM-^F mutant

Project description:We report a new protein complex with a role in transcription elongation that is formed by Ypr045c (Thp3) and the Csn12 component of the COP9-signalosome. Thp3-Csn12 is recruited to transcribed genes. Their mutations suppress the gene expression defects of mutants of the THO complex involved in mRNP biogenesis and export and show defects in mRNA accumulation. In vivo transcription elongation impairment of thp3M-bM-^HM-^F mutants is shown by reduction of RNAPII recruitment throughout an active gene and in transcript run on analysis performed in G-less systems. This new complex establishes a novel link between transcription and mRNA processing. Tthree repeats of the wild type control and three of the ypr045cM-bM-^HM-^F mutant (BY4741 background).

2011-02-23 | E-GEOD-18994 | biostudies-arrayexpress

Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation (expression)

Project description:The rate of RNA polymerase II (pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing is not well understood. We performed experiments to perturb pol II elongation and then globally compared alternative splicing patterns with genome-wide pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with pol II elongation inhibition-dependent changes in alternative splicing. Under conditions that interfere with pol II elongation, including cell stress, increased pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, alternative splicing and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels. In order to identify alternative splicing events influenced by changes in pol II elongation, we performed quantitative alternative splicing microarray profiling (Pan et al., 2004 (PMID 15610736); Shai et al., 2006 (PMID 16403798)) of RNA isolated from stimulated Jurkat T lymphoma cells, cultured separately in the presence or absence of two different drugs that can inhibit pol II elongation: 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) and camptothecin.

2010-12-09 | E-GEOD-25287 | biostudies-arrayexpress

Expression profile of ypr045c∆ mutant

Project description:We report a new protein complex with a role in transcription elongation that is formed by Ypr045c (Thp3) and the Csn12 component of the COP9-signalosome. Thp3-Csn12 is recruited to transcribed genes. Their mutations suppress the gene expression defects of mutants of the THO complex involved in mRNP biogenesis and export and show defects in mRNA accumulation. In vivo transcription elongation impairment of thp3∆ mutants is shown by reduction of RNAPII recruitment throughout an active gene and in transcript run on analysis performed in G-less systems. This new complex establishes a novel link between transcription and mRNA processing.

2011-02-23 | GSE18994 | GEO

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