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Genome-wide gene expression analysis in Thoc5- and CFIm68-depleted HeLa cells

ABSTRACT: We found that CFIm68, a mRNA cleavage and polyadenylation factor implicated for alternative polyadenylation site choice, was co-purified with Thoc5, a component of human THO/TREX. Microarray analysis using human HeLa cells reveals knockdown of Thoc5 affects the expression of a subset of non-heat shock genes. Notably, depletion of Thoc5 attenuated the expression of the mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68. Two independent experiments using different siRNAs against Thoc5 (siThoc5-1and siThoc5-2) and CFIm68 (siCFIm68-1and siCFIm68-2) were performed. In both experiments, siDsRed was used as negative control.

ORGANISM(S): Homo sapiens

SUBMITTER: Daisuke Okuzaki

PROVIDER: E-GEOD-42327 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Human TREX component Thoc5 affects alternative polyadenylation site choice by recruiting mammalian cleavage factor I.

Katahira Jun J Okuzaki Daisuke D Inoue Hitomi H Yoneda Yoshihiro Y Maehara Kazumitsu K Ohkawa Yasuyuki Y

Nucleic acids research 20130517 14

The transcription-export complex (TREX) couples mRNA transcription, processing and nuclear export. We found that CFIm68, a large subunit of a heterotetrameric protein complex mammalian cleavage factor I (CFIm), which is implicated in alternative polyadenylation site choice, co-purified with Thoc5, a component of human TREX. Immunoprecipitation using antibodies against different components of TREX indicated that most likely both complexes interact via an interaction between Thoc5 and CFIm68. Micr ...[more]

PMID: 23685434

Similar Datasets

Genome-wide gene expression analysis in Thoc5- and CFIm68-depleted HeLa cells

Project description:We found that CFIm68, a mRNA cleavage and polyadenylation factor implicated for alternative polyadenylation site choice, was co-purified with Thoc5, a component of human THO/TREX. Microarray analysis using human HeLa cells reveals knockdown of Thoc5 affects the expression of a subset of non-heat shock genes. Notably, depletion of Thoc5 attenuated the expression of the mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68.

2013-05-21 | GSE42327 | GEO

A member of transcription/export complex (TREX), THOC5 controls elongation rate by recruiting CDK12.

Project description:THOC5, a member of the THO complex that is a subcomplex of Transcription/export complex (TREX) 1, is essential for 3´processing of slow kinetics IEGs, and for export of only a subset of genes. Applying nanopore mRNA-sequence technology, we show here that upon depletion of THOC5, 50% of mRNAs showed alteration of 3´end cleavage sites. Moreover, polymerase-II (Pol II)-CHIP-sequencing data reveals that upon depletion of THOC5 Pol II density was increased at gene body and 3´UTR. THOC5 is recruited near to Pol II high density sites except promotor regions, suggesting that THOC5 is involved in transcription elongation. Indeed, 385 THOC5 independent genes in fast Pol II cells became THOC5 dependent in slow Pol II cells. Chromatin associated THOC5 in slow Pol II cells interacts with CDK12, RNA helicases DDX5 and DDX, and THOC6, but not with other members of THO complex. Notably, THOC5 did not form this complex in fast poly II cells. CDK12 was recruited to R-loop (DNA:RNA hybrid) in THOC5/THOC6 dependent manner. Here, THOC6, but not THOC5 directly interacts with R-loop then, recruited DDX5 resolves R-loop and then CDK12 enhances further transcription elongation. These data show for the first time the novel function of THOC5/THOC6 complex during transcription elongation.

2022-12-31 | GSE166115 | GEO

The THO complex regulates pluripotency gene mRNA export to control embryonic stem cell self-renewal and somatic cell reprogramming (microarray)

Project description:Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, post-transcriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5, and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, as its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of post-transcriptional regulation in stem cell fate specification. mouse J1 cells were transfected with non-targeting (NT), Thoc2, and Thoc5 siRNAs. Total RNA was isolated 96 hours after transfection.

2013-11-30 | E-GEOD-51496 | biostudies-arrayexpress

THOC5 controls 3M-BM-4end-processing of immediate early genes via interaction with CPSF100

Project description:Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. THOC5, a member of the TREX (transcription/export) complex plays a role in expression of only a subset of constitutively active genes. In this report, we show that THOC5 plays a role in the 3M-BM-4end-processing of more than 90% of IEGs induced by serum stimulation, however THOC5 depletion does not influence the expression of the most rapidly induced IEGs, such as Fos or Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts were not released from chromatin in THOC5 depleted cells. Another group of genes, such as Myc, and Smad7 transcripts, were released with shortening of 3M-BM-4UTR by alternative cleavage, and were spliced but were exported to a lesser extent in THOC5 depleted cells. Furthermore, upon stimulation with serum THOC5 forms a complex with polyadenylation specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3M-BM-4UTR of THOC5 target genes, but not for THOC5 independent genes. These data suggest the presence of a novel mechanism for the control of immediate early genes response via 3M-BM-4end-processing. In 2 dual-color microarray hybridizations, mRNA expression changes after 1h of FCS treatment were examined. MEF(Rosa26ERT2 Cre: THOC5 (flox/flox)) cells in which a THOC5 knockdown is inducible upon Tamoxifen treatment were analyzed. M4040 represents cells without Taxoxifen pre-treatment (wildtype reference) whereas M4041 represents the same celltype pre-treated for 2 days with Tamoxifen to induce a THOC5 knockdown.

2014-11-03 | E-GEOD-59561 | biostudies-arrayexpress

THOC5 controls 3´end-processing of immediate early genes via interaction with CPSF100

Project description:Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. THOC5, a member of the TREX (transcription/export) complex plays a role in expression of only a subset of constitutively active genes. In this report, we show that THOC5 plays a role in the 3´end-processing of more than 90% of IEGs induced by serum stimulation, however THOC5 depletion does not influence the expression of the most rapidly induced IEGs, such as Fos or Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts were not released from chromatin in THOC5 depleted cells. Another group of genes, such as Myc, and Smad7 transcripts, were released with shortening of 3´UTR by alternative cleavage, and were spliced but were exported to a lesser extent in THOC5 depleted cells. Furthermore, upon stimulation with serum THOC5 forms a complex with polyadenylation specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3´UTR of THOC5 target genes, but not for THOC5 independent genes. These data suggest the presence of a novel mechanism for the control of immediate early genes response via 3´end-processing.

2014-11-03 | GSE59561 | GEO

The THO complex regulates pluripotency gene mRNA export to control embryonic stem cell self-renewal and somatic cell reprogramming (RNA-IP)

Project description:Embryonic stem cell (ESC) self-renewal and differentiation is governed by a comprehensive regulatory network. Although the transcriptional regulation has been extensively investigated, post-transcriptional mechanisms controlling the ESC state are poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5, and is required for self-renewal by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, which leads to decreased expression of pluripotency proteins and facilitates differentiation. Finally, THO is also important for the establishment of pluripotency, as its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicates that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and uncovers a novel mechanism of post-transcriptional regulation in stem cell fate specification. RNA IP was conducted by use of antibody against Thoc2, the precipitated RNA was used to generate library using illumina Kit, and subsequently sequenced by miSeq

2013-11-30 | E-GEOD-48436 | biostudies-arrayexpress

Splicing-independent loading of TREX on nascent RNA is required for efficient transcription of dual-strand piRNA clusters in Drosophila

Project description:The conserved THO/TREX complex is critical for pre-mRNA processing and mRNA nuclear export. In Metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. Here we show that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small non-coding RNAs that control expression of transposable elements (TE) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and co-localize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depends on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, we show that TREX is required for accumulation of nascent piRNA precursors. Our study reveals a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis.

2016-03-18 | GSE79325 | GEO

The THO complex regulates pluripotency gene mRNA export to control embryonic stem cell self-renewal and somatic cell reprogramming (RNA-IP)

2013-11-30 | GSE48436 | GEO

The THO complex regulates pluripotency gene mRNA export to control embryonic stem cell self-renewal and somatic cell reprogramming (microarray)

2013-11-30 | GSE51496 | GEO

Identification and functional analysis of SHISA2 overexpressed in high-grade prostate cancer cells.

Project description:We identified a novel molecular target and diagnostic biomarker, SHISA2, as an overexpressed gene in high-grade prostate cancer (PC) cells. To understand the association of SHISA2 overexpression with the aggressiveness of high-grade PC, we performed gene expression analysis using a cDNA microarray. Gene expression patterns of PC-3 cells transfected with the two shRNA expression vectors (siSHISA2 and siCONTROL) were compared.

2013-09-13 | E-GEOD-48833 | biostudies-arrayexpress

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