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Linking Key Steps of MicroRNA Biogenesis by TREX-2 and the Nuclear Pore Complex in Arabidopsis [mRNA-Seq]

ABSTRACT: Unlike in metazoans, the stepwise biogenesis of microRNAs (miRNAs) occurs within the nucleus in plants. Whether or how the major steps in miRNA biogenesis are coordinated is largely unknown. Here, we show that the plant TREX-2 complex promotes multiple steps in miRNA biogenesis, including transcription, processing and nuclear export. Core subunits of TREX-2, THP1 and SAC3A, interact and co-localize with RNA Polymerase II to promote the transcription of MIR genes in the nucleoplasm. TREX-2 interacts with the microprocessor component SERRATE and promotes the formation of Dicing bodies in the nucleoplasm. THP1 also interacts and co-localizes with a nucleoporin protein NUP1 at the nuclear envelope. NUP1 and THP1 promote the nuclear export of miRNAs and AGO1. These results suggest that TREX-2 coordinates the transcription, processing and export steps in miRNA biogenesis to ensure efficient miRNA production.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE150472 | GEO | 2020/08/07

REPOSITORIES: GEO

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Linking Key Steps of MicroRNA Biogenesis by TREX-2 and the Nuclear Pore Complex in Arabidopsis [miRNA-Seq]

Project description:Unlike in metazoans, the stepwise biogenesis of microRNAs (miRNAs) occurs within the nucleus in plants. Whether or how the major steps in miRNA biogenesis are coordinated is largely unknown. Here, we show that the plant TREX-2 complex promotes multiple steps in miRNA biogenesis, including transcription, processing and nuclear export. Core subunits of TREX-2, THP1 and SAC3A, interact and co-localize with RNA Polymerase II to promote the transcription of MIR genes in the nucleoplasm. TREX-2 interacts with the microprocessor component SERRATE and promotes the formation of Dicing bodies in the nucleoplasm. THP1 also interacts and co-localizes with a nucleoporin protein NUP1 at the nuclear envelope. NUP1 and THP1 promote the nuclear export of miRNAs and AGO1. These results suggest that TREX-2 coordinates the transcription, processing and export steps in miRNA biogenesis to ensure efficient miRNA production.

2020-08-07 | GSE150471 | GEO

The nuclear pore-associated TREX-2 complex employs the Mediator Med31 submodule as a docking site to regulate gene expression

Project description:Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with its Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing at the Mediator level. In sum, we provide insight into how NPC-associated adaptor complexes can access the core transcription machinery. RNAseq was performed from WT, sac3∆, cdk8∆ and Sac3 R288D mutant cells. For each strain triplicates were analyzed. WT strain was sac3∆ transformed with pRS315 SAC3 WT

2015-08-27 | E-GEOD-67431 | biostudies-arrayexpress

The nuclear pore-associated TREX-2 complex employs the Mediator Med31 submodule as a docking site to regulate gene expression

2015-08-27 | GSE67431 | GEO

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

TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome

Project description:THOC6 is the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants do not alter the expression of TREX dimer component proteins in human cells, but reduced binding affinity to ALYREF implicates impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for regulation of key signaling pathways in human corticogenesis that dictate the transition from proliferative to neurogenic divisions, developmental biology implicated in TIDS neuropathology.

2023-10-17 | GSE245121 | GEO

Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis

Project description:Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells while we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3â² end, yielding miRNAs from the 5â² strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research. Two independent sequencing experiments (set 1 and set 2, respectively) were performed using 9 samples.

2016-03-17 | E-GEOD-77989 | biostudies-arrayexpress

Global RNA-binding profile of the mRNA export adaptor ALYREF

Project description:The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. As expected, iCLIP reads are mainly mapped to exons of mRNAs. Globally, ALYREF binding shows two apparent enrichments on the mRNA, one is near the 5’ end and the other is very close to the 3’ end. In addition, numerous middle exons harbor ALYREF binding sites. CBP80 and PABPN1 mainly affect ALYREF binding at the 5’ and the 3’ region, respectively. Interestingly, we found that the 3’ processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. Sequence analysis led to the identification of multiple

2017-05-19 | GSE99069 | GEO

Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis

Project description:Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells while we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.

2016-03-17 | GSE77989 | GEO

Expression data from Saccharomyces cerevisiae strains deleted for different subunits of the THO/TREX complex

Project description:THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis at the interface of transcription-RNA export with a key role in preventing transcription-associated genome instability. We used microarrays to analyze the impact of different THO/TREX mutations on gene expression and found that THO-Sub2 deletions have a high functional impact on highly expressed, long and G+C-rich genes regardless of gene function. S. cerevisiae strains were grown in YPAD liquid culture, total RNA was isolated and hybridized on Affymetrix microarrays.

2011-06-28 | E-GEOD-24802 | biostudies-arrayexpress

Expression data from Saccharomyces cerevisiae strains deleted for different subunits of the THO/TREX complex

2011-06-29 | GSE24802 | GEO

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