Ana G. Rondón

TREX is a conserved complex coupling transcription with messenger RNA export.

The essential yeast proteins Yra1 and Sub2 are messenger RNA export factors that have conserved counterparts in metazoans, designated Aly and UAP56, respectively. These factors couple the machineries that function in splicing and export of mRNA. Here we show that both Yra1 and Sub2 are stoichiometrically associated with the heterotetrameric THO complex, which functions in transcription in yeast. We also show that Sub2 and Yra1 interact genetically with all four components of the THO complex (Tho2, Hpr1, Mft1 and Thp2). Moreover, these components operate in the export of bulk poly(A)+ RNA as well as of mRNA derived from intronless genes. Both Aly and UAP56 associate with human counterparts of the THO complex. Together, these data define a conserved complex, designated the TREX (‘transcription/export’) complex. The TREX complex is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II. Our data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export.

A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae

Transcription‐induced recombination has been reported in all organisms from bacteria to mammals. We have shown previously that the yeast genes HPR1 and THO2 may be keys to the understanding of transcription‐associated recombination, as they both affect transcription elongation and hyper‐recombination in a concerted manner. Using a yeast strain that has the wild‐type THO2 gene replaced by one encoding a His6‐HA‐tagged version, we have isolated an oligomeric complex containing four proteins: Tho2, Hpr1, Mft1 and a novel protein that we have named Thp2. We have reciprocally identified a complex containing Hpr1, Tho2 and Mft1 using anti‐Mft1 antibodies in immunoprecipitation experiments. The protein complex is mainly nuclear; therefore, Tho2 and Hpr1 are physically associated. Like hpr1Δ and tho2Δ cells, mft1Δ and thp2Δ cells show mitotic hyper‐ recombination and impaired transcription elongation, in particular, through the bacterial lacZ sequence. Hyper‐recombination conferred by mft1Δ and thp2Δ is only observed in DNA regions under transcription conditions. We propose that this protein complex acts as a functional unit connecting transcription elongation with the incidence of mitotic recombination.

The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability

The THO complex is a multimeric factor containing four polypeptides, Tho2, Hpr1, Mft1 and Thp2. Mutations in any of the genes encoding THO confer impairment of transcription and a transcription‐dependent hyper‐recombination phenotype, suggesting that THO has a functional role in gene expression. Using an in vivo assay developed to study expression of long and G+C‐rich DNA sequences, we have isolated SUB2, a gene involved in mRNA splicing and export, as a multicopy suppressor of the gene expression defect of hpr1Δ. Further investigation of a putative functional relationship between mRNA metabolism and THO revealed that mRNA export mutants sub2, yra1, mex67 and mtr2 have similar defective transcription and hyper‐recombination phenotypes as THO mutants. In addition, THO becomes essential in cells with a defective Mex67 mRNA export er. Finally, we have shown that THO has the ability to associate with RNA and DNA in vitro. These results indicate a functional link between the processes of elongation and metabolism of nascent mRNA mediated by THO and mRNA export proteins, which have important consequences for the maintenance of genome stability.

Yeast Sen1 Helicase Protects the Genome from Transcription-Associated Instability

Summary Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.

Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation.

THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1. Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects. To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes. With these assays, we demonstrate that hpr1Δ, tho2Δ, and mft1Δ mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation. The mRNA expression defect of hpr1Δ mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease. This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export.

Molecular evidence indicating that the yeast PAF complex is required for transcription elongation

PAF is a five‐subunit protein complex composed of Paf1, Cdc73, Leo1, Rtf1 and Ctr9, which was purified from yeast in association with RNA polymerase II and which is believed to function in transcription elongation. However, no direct proof exists for this yet. To assay whether PAF is required in elongation, we determined the in vitro transcription‐elongation efficiencies of mutant cell extracts using a DNA template containing two G‐less cassettes. paf1Δ or cdc73Δ cell extracts showed reduced transcription‐elongation efficiencies (16–18% of the wild‐type levels), whereas leo1Δ and rtf1Δ showed wild‐type levels. In vivo transcription efficiency was diminished in the four mutants analysed, as determined by their abilities to transcribe lacZ. Our work provides molecular evidence that PAF has a positive role in transcription elongation and is composed of at least two functionally different types of subunits (Paf1‐Cdc73 and Leo1‐Rtf1).

Fail-Safe Transcriptional Termination for Protein-Coding Genes in S. cerevisiae

Summary Transcription termination of RNA polymerase II (Pol II) on protein-coding genes in S. cerevisiae relies on pA site recognition by 3′ end processing factors. Here we demonstrate the existence of two alternative termination mechanisms that rescue polymerases failing to disengage from the template at pA sites. One of these fail-safe mechanisms is mediated by the NRD complex, similar to termination of short noncoding genes. The other termination mechanism is mediated by Rnt1 cleavage of the nascent transcript. Both fail-safe termination mechanisms trigger degradation of readthrough transcripts by the exosome. However, Rnt1-mediated termination can also enhance the usage of weak pA signals and thereby generate functional mRNA. We propose that these alternative Pol II termination pathways serve the dual function of avoiding transcription interference and promoting rapid removal of aberrant transcripts.

Molecular evidence for a positive role of Spt4 in transcription elongation.

We have previously shown that yeast mutants of the THO complex have a defect in gene expression, observed as an impairment of lacZ transcription. Here we analyze the ability of mutants of different transcription elongation factors to transcribe lacZ. We found that spt4Δ, like THO mutants, impaired transcription of lacZ and of long and GC‐rich DNA sequences fused to the GAL1 promoter. Using a newly developed in vitro transcription elongation assay, we show that Spt4 is required in elongation. There is a functional interaction between Spt4 and THO, detected by the lethality or strong gene expression defect and hyper‐recombination phenotypes of double mutants in the W303 genetic background. Our results indicate that Spt4–Spt5 has a positive role in transcription elongation and suggest that Spt4–Spt5 and THO act at different steps during mRNA biogenesis.

The interface between transcription and mRNP export: From THO to THSC/TREX-2

Eukaryotic gene expression is a multilayer process covering transcription to post-translational protein modifications. As the nascent pre-mRNA emerges from the RNA polymerase II (RNAPII), it is packed in a messenger ribonucleoparticle (mRNP) whose optimal configuration is critical for the normal pre-mRNA processing and mRNA export, mRNA integrity as well as for transcription elongation efficiency. The interplay between transcription and mRNP formation feeds forward and backward and involves a number of conserved factors, from THO to THSC/TREX-2, which in addition have a unique impact on transcription-dependent genome instability. Here we review our actual knowledge of the role that these factors play at the interface between transcription and mRNA export in the model organism Saccharomyces cerevisiae.

A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors

To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G‐less cassettes separated by either a long and GC‐rich or a short and GC‐poor DNA sequence (G‐less‐based run‐on (GLRO) assay). We demonstrate that PAF, THSC/TREX‐2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre‐mRNA processing and nuclear export with transcription elongation.