Project description:Nuclear export of mRNA is an essential process for eukaryotic gene expression. TREX complex couples the gene expression from transcription and splicing to mRNA export. Sub2, a core component of TREX complex in yeast is diversified to two closely related RNA helicases, UAP56 and URH49 in human.UAP56 and URH49 are required for bulk poly (A)+ RNA export but their target genes are quite different. In conclusion, UAP56 and URH49 have a different function in vivo despite the highly similarity. Cytoplasmic RNA was prepared in HeLa cells transfected with control-, UAP56- and URH49-siRNA and analyzed by gene expression array.
Project description:Nuclear export of mRNA is an essential process for eukaryotic gene expression. TREX complex couples the gene expression from transcription and splicing to mRNA export. Sub2, a core component of TREX complex in yeast is diversified to two closely related RNA helicases, UAP56 and URH49 in human.UAP56 and URH49 are required for bulk poly (A)+ RNA export but their target genes are quite different. In conclusion, UAP56 and URH49 have a different function in vivo despite the highly similarity.
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.
Project description:Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genomewide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes.
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
Project description:N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA. This modification has previously been shown to alter the export kinetics for mRNAs though the molecular details surrounding this phenomenon remain poorly understood. Here we show that the m6A complex (WTAP, KIAA1429, METTL3/14) drives recruitment of the TREX mRNA export complex onto m6A modified mRNAs and this process is essential for the efficient export of certain mRNAs. Depletion of the core m6A complex leads to loss of TREX from mRNAs which undergo the m6A modification. We show that TREX stimulates recruitment of the m6A reader protein YTHDC1 to the mRNP and the m6A complex influences the interaction of TREX with YTHDC1. We suggest that m6A acts as a surrogate for other TREX recruitment mechanisms such as splicing and 5’ capping, in long internal and final exons which may otherwise be devoid of this essential complex for mRNA export.
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.
Project description:mRNA export is central to gene regulation and expression. In trypanosomes, transcription is polycistronic, all mRNAs are processed by trans-splicing and export is mediated by non-canonical mechanisms. We have described several conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII, a core component of the exon junction complex (EJC). Few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes and examples of mechanistic divergence well known. To uncover additional components of the trypanosome mRNA maturation and export system we undertook an unbiased search for protein interactors of TcSub2 and TceIF4AIII. Significant overlap between TcSub2 and TceIF4AIII interacting protein cohorts suggests that both proteins associate with similar machinery. We confirmed several interactions with conserved core components of the EJC and multiple additional complexes, together with identification of proteins specific to trypanosomatids. The highly interactive super-interactome uncovered here, capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events, highlights kinetoplastid-specific and conserved components creating an amalgam to support the unique mRNA maturation mechanisms in trypanosomes.
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
Project description:Evidence indicates that transcription and mRNA export are linked processes. The molecular mechanisms of this coordination are not clear however. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator and TREX-2 that functions in mRNA biogenesis and export. Here we investigate the coordinated action of SAGA and TREX-2 that is required for gene expression. We demonstrate that the TREX-2/proteasomal subunit Sem1, influences Sus1 role in mRNA export and TREX-2 stability. Wide analyses of gene expression reveal that Sem1 and Sus1 have also overlapping functions in transcription. In the absence of Sem1, expression of some SAGA-dependent genes is compromised with a concomitant decrease of RNAP II recruitment to promoters. Notably, ChIP experiments revealed a distinct dependency for SAGA subunits recruitment on Sem1. While absence of Sem1 lowers Ada2 and Taf9 recruitment to GAL1 promoter upon activation, association of the deubiquitylation module remains intact. However, H2B deubiquitylation activity is dramatically decreased. These results unveil a new role for Sem1 in influencing activation of SAGA-dependent H2B deubiquitylation likely mediated by stabilization of TREX-2 complex and SAGA modular assembly. Our work gives insights in how modular architecture of SAGA is determinant for its function in gene expression.