Analyzing the role of transcription elongation factors Spt4-5 in splicing
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE1610 | GEO | 2005/11/01
SECONDARY ACCESSION(S): PRJNA90709
REPOSITORIES: GEO
Similar Datasets
Project description:A set of 22 expts. aimed at identifying splicing events dependent upon on the Spt4-5 transcription elongation factors in yeast. Four spt mutants and an mRNA capping mutant were analyzed four times each, including biological and technical (dye-swap) replicates. Two wt vs wt expts. were also performed. Keywords = transcription/spt5/spt4/splicing/DEDS
2010-06-09 | E-GEOD-1610 | biostudies-arrayexpress
Project description:Aim and Methods: To understand the precise role of Spt4 in budding yeast, various high throughput sequencing techniques were used. Nucleosome positions were studied using MNase-seq in WT and spt4∆ cells. The position of RNAPII was examined using NET-seq in spt4∆ cells or cells in which Spt4 has been anchored away to the cytoplasm (Spt4 AA). RNAPII was also mapped in cells in which Spt5 has been anchored away to the cytoplasm (Spt5 AA). Steady-state transcript levels were assessed using RNA-seq in samples coupled with NET-seq. The position of the Spt4 on RNAPII was mapped at single nucleotide resolution using TEF-seq. With the same technique, the position of Spt5 on RNAPII was investigated in wild type (WT) and spt4 knock-out (spt4∆) cells. The position and levels of the pre-initiation complex were assessed using ChIP-seq on Sua7(TFIIB) in WT and spt4∆ cells. Results: The interaction between the Spt4/5 complex and RNAPII periodically changes as RNAPII transitions through nucleosomes. The dynamic interaction of Spt5 with RNAPII is dependent on Spt4. In spt4∆ and Spt4 AA cells, RNAPII distribution is altered in a similar way compared to their respective controls. After transcribing into nucleosomes, RNAPII accumulates upstream of nucleosome dyads, especially around the +2 nucleosome. In Spt5 AA cells, the distribution and amount of RNAPII on genes are severely affected. Nucleosome positions are altered in spt4∆ cells. Although the position of the +1 nucleosome remains unchanged, nucleosome spacing from this point is increased, and the level of increase in nucleosome spacing correlates with the level of RNAPII accumulation. Conclusion: This work suggests that Spt4 regulates nucleosome positioning by a mechanism related to transcription and promotes RNAPII movement through nucleosome barriers, especially the barrier set by the +2 nucleosome.
2021-09-27 | GSE159291 | GEO
Project description:Class-switch recombination (CSR), induced by activation-induced cytidine deaminase, can be divided into two phases: DNA cleavage of the switch (S) regions, and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in CH12F3-2A cells, and Spt4 and Spt5, carry out independent functions in CSR. Depleting Spt5 reduced the H3K4me3 levels and DNA cleavage at the S? region, whereas Spt4 knockdown did not perturb the H3K4me3 status or S region cleavage. Thus, we conclude that Spt5 plays a role similar to the histone chaperone FACT by regulating histone modification and DNA cleavage in CSR. Assay systems using synthetic repair substrates revealed that both Spt4 and Spt5 are required for non-homologous end joining and homologous recombination. Taken together, Spt4 and Spt5 are involved in multiple CSR steps, and can function independently. Spt4 or Spt5 were knocked down in CH12F3-2A cells in the presence of CD40L, IL4, TGFb (CIT) stimulation. RNA from each samples were extracted and relative difference in transcript level were compared with control RNAi-treated, CIT-stimulated samples.
2013-10-22 | E-GEOD-33206 | biostudies-arrayexpress
Project description:Class-switch recombination (CSR), induced by activation-induced cytidine deaminase, can be divided into two phases: DNA cleavage of the switch (S) regions, and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in CH12F3-2A cells, and Spt4 and Spt5, carry out independent functions in CSR. Depleting Spt5 reduced the H3K4me3 levels and DNA cleavage at the Sα region, whereas Spt4 knockdown did not perturb the H3K4me3 status or S region cleavage. Thus, we conclude that Spt5 plays a role similar to the histone chaperone FACT by regulating histone modification and DNA cleavage in CSR. Assay systems using synthetic repair substrates revealed that both Spt4 and Spt5 are required for non-homologous end joining and homologous recombination. Taken together, Spt4 and Spt5 are involved in multiple CSR steps, and can function independently.
2013-10-22 | GSE33206 | GEO
Project description:The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline (micronucleus - MIC) and somatic nuclei (macronucleus - MAC), massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. As we were mostly interested in transcription of the germline genome in the MIC during meiosis, we decided to probe the composition of the Spt5-Spt4 complex characteristic to this nucleus. Experiments in which Spt4mB-3xFLAG was used as bait demonstrated its interaction with either Spt5m or Spt5v, which is consistent with the Spt4mB-GFP localization pattern. Interestingly, mass spectrometry revealed that Spt5m-3xFLAG (which is present only in the MIC at the analyzed stage) interacts either with the meiotic Spt4mB or with Spt4vA. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.
2022-04-27 | PXD027748 | Pride
Project description:The goal of this study was to determine the effect of Spt4 on transcription by Pol I in vivo. Therefore, Native Elongating Transcript Sequencing (NET-seq) was used to evaluate the occupancy of Pol I in wild-type and spt4△ strains. We determined that in spt4△ yeast, there was a processivity defect and Pol I accumulated at the 5' end of the 35S gene. Additionally, we observed that there was an enrichment for G-rich sequences just downstream of a high A content in the spt4△ strain, indicating that perhaps Spt4 plays a role in helping to propel Pol I through these regions of the rDNA. Altogether, this study suggests that Spt4 is an important transcription factor for Pol I by promoting polymerase processivity.
2021-02-18 | GSE166983 | GEO
Project description:We have done RNAseq analyses in yeast and in rat and mouse striatal neurons. The experiments in yeast examine globally the physiological effects of spt4 deletion on the transcriptome. The experiments in neurons examine the global effects of supt4 knockdown to a cellular level (i.e., 50% knockdown) that we have shown prevents toxicity of variant Htt protein by interfering with transcription of extended CAG repeats. The results of both sets of experiments indicate that absent spt4 function has limited effects on overall transcription. Moreover, the mammalian cell experiments indicate that supt4h knockdown to a cellular level that alters production of transcripts containing long CAG repeats has minimal effects on normal genes, supporting the notion that anti-supt4h measures may prove useful in combating trinucleotide repeat diseases. RNA-seq was done for two yeast WT strain W303 and two spt4 deletion strain spt4M-bM-^HM-^F as biological repeats, and rat ST14A cells and mouse HdhQ7/Q7 cells after transfection with NC control siRNA or Supt4h siRNA
2012-02-15 | E-GEOD-33497 | biostudies-arrayexpress
Project description:To study the relevance of the phosphorylation of Spt4 in residues T42 and S43, we assessed the genome-wide transcriptional response of the non-phosphorylatable Spt4-T42A/S43A mutant (Spt4-AA) upon osmotic stress. Results point out that the unphosphorylatable Spt4-T42A/S43A protein showed an impaired stress responsive gene expression indicating that these specific residues play a relevant role in osmotic stress responsive transcriptional regulation.
2017-11-20 | GSE98352 | GEO
Project description:Many proteins associate with elongating RNA polymerase II (RNAPII), although the functions of most of them are still not well understood. Spt5 is an essential transcription elongation factor that binds directly to RNA polymerase and is conserved in prokaryotes, archaea, and eukaryotes1. In eukaryotes, evidence suggests that Spt5 functions in transcription elongation by both RNA polymerases I and II, mRNA capping, mRNA splicing, and mRNA 3’ end formation2. However, the genome-wide requirement for Spt5 in transcription has not been extensively studied. To address this issue, we have comprehensively analyzed the consequences of Spt5 depletion on transcription by RNAPII in Schizosaccharomyces pombe using four genome-wide approaches: ChIP-seq, NET-seq, 4tU-seq, and RNA-seq. Our results demonstrate that, in the absence of Spt5, RNAPII accumulates at a high level over the first ~500 bp of transcription units, suggesting that Spt5 is globally required to elongate past a barrier to transcription. This possibility is strongly supported by results showing that Spt5 is required for a normal level of RNA synthesis. In addition to these effects on sense-strand transcription, Spt5 depletion results in widespread convergent antisense transcription that initiates at approximately the same position as the sense-strand barrier. While the role of these antisense transcripts is unknown, the two that were tested control the distribution of RNAPII. Our results also show that Spt5 represses divergent antisense transcription, explaining why it has not been previously observed in S. pombe. Taken together, our results reveal a global and critical role for Spt5 in multiple classes of transcription by RNAPII.
2017-03-30 | GSE85181 | GEO
Project description:Many proteins associate with elongating RNA polymerase II (RNAPII), although the functions of most of them are still not well understood. Spt5 is an essential transcription elongation factor that binds directly to RNA polymerase and is conserved in prokaryotes, archaea, and eukaryotes1. In eukaryotes, evidence suggests that Spt5 functions in transcription elongation by both RNA polymerases I and II, mRNA capping, mRNA splicing, and mRNA 3’ end formation2. However, the genome-wide requirement for Spt5 in transcription has not been extensively studied. To address this issue, we have comprehensively analyzed the consequences of Spt5 depletion on transcription by RNAPII in Schizosaccharomyces pombe using four genome-wide approaches: ChIP-seq, NET-seq, 4tU-seq, and RNA-seq. Our results demonstrate that, in the absence of Spt5, RNAPII accumulates at a high level over the first ~500 bp of transcription units, suggesting that Spt5 is globally required to elongate past a barrier to transcription. This possibility is strongly supported by results showing that Spt5 is required for a normal level of RNA synthesis. In addition to these effects on sense-strand transcription, Spt5 depletion results in widespread convergent antisense transcription that initiates at approximately the same position as the sense-strand barrier. While the role of these antisense transcripts is unknown, the two that were tested control the distribution of RNAPII. Our results also show that Spt5 represses divergent antisense transcription, explaining why it has not been previously observed in S. pombe. Taken together, our results reveal a global and critical role for Spt5 in multiple classes of transcription by RNAPII.
2017-03-30 | GSE85127 | GEO