Depleting Components of the THO Complex Causes Increased Telomere Length by Reducing the Expression of the Telomere-Associated Protein Rif1p

Telomere length is regulated mostly by proteins directly associated with telomeres. However, genome-wide analysis of Saccharomyces cerevisiae mutants has revealed that deletion of Hpr1p, a component of the THO complex, also affects telomere length. The THO complex comprises four protein subunits, namely, Tho2p, Hpr1p, Mft1p, and Thp2p. These subunits interplay between transcription elongation and co-transcriptional assembly of export-competent mRNPs. Here we found that the deletion of tho2 or hpr1 caused telomere lengthening by ∼50–100 bps, whereas that of mft1 or thp2 did not affect telomere length. Since the THO complex functions in transcription elongation, we analyzed the expression of telomere-associated proteins in mutants depleted of complex components. We found that both the mRNA and protein levels of RIF1 were decreased in tho2 and hpr1 cells. RIF1 encodes a 1917-amino acid polypeptide that is involved in regulating telomere length and the formation of telomeric heterochromatin. Hpr1p and Tho2p appeared to affect telomeres through Rif1p, as increased Rif1p levels suppressed the telomere lengthening in tho2 and hpr1 cells. Moreover, yeast cells carrying rif1 tho2 or rif1 hpr1 double mutations showed telomere lengths and telomere silencing effects similar to those observed in the rif1 mutant. Thus, we conclude that mutations of components of the THO complex affect telomere functions by reducing the expression of a telomere-associated protein, Rif1p

Structural insights into the nucleic acid remodeling mechanisms of the yeast THO-Sub2 complex

The yeast THO complex is recruited to active genes and interacts with the RNA-dependent ATPase Sub2 to facilitate the formation of mature export-competent messenger ribonucleoprotein particles and to prevent the co-transcriptional formation of RNA:DNA-hybrid-containing structures. How THO-containing complexes function at the mechanistic level is unclear. Here, we elucidated a 3.4 angstrom resolution structure of Saccharomyces cerevisiae THO-Sub2 by cryoelectron microscopy. THO subunits Tho2 and Hpr1 intertwine to form a platform that is bound by Mft1, Thp2, and Text. The resulting complex homodimerizes in an asymmetric fashion, with a Sub2 molecule attached to each protomer. The homodimerization interfaces serve as a fulcrum for a seesaw-like movement concomitant with conformational changes of the Sub2 ATPase. The overall structural architecture and topology suggest the molecular mechanisms of nucleic acid remodeling during mRNA biogenesis.We thank Daniel Bollschweiler and Tillman Schäfer at the MPIB cryo-EM facility..

An hpr1 point mutation that impairs transcription and mRNP biogenesis without increasing recombination

THO/TREX, a conserved eukaryotic protein complex, is a key player at the interface between transcription and mRNP metabolism. The lack of a functional THO complex impairs transcription, leads to transcriptiondependent hyperrecombination, causes mRNA export defects and fast mRNA decay, and retards replication fork progression in a transcription-dependent manner. To get more insight into the interconnection between mRNP biogenesis and genomic instability, we searched for HPR1 mutations that differentially affect gene expression and recombination. We isolated mutants that were barely affected in gene expression but exhibited a hyperrecombination phenotype. In addition, we isolated a mutant, hpr1-101, with a strong defect in transcription, as observed for lacZ, and a general defect in mRNA export that did not display a relevant hyperrecombination phenotype. In THO single-null mutants, but not in the hpr1 point mutants studied, THO and its subunits were unstable. Interestingly, in contrast to hyperrecombinant null mutants, hpr1-101 did not cause retardation of replication fork progression. Transcription and mRNP biogenesis can therefore be impaired by THO/TREX dysfunction without increasing recombination, suggesting that it is possible to separate the mechanism(s) responsible for mRNA biogenesis defects from the further step of triggering transcriptiondependent recombination.Ministerio de Educación y Ciencia BMC2000-0409 SAF2003-00204Junta de Andalucía CVI10

Sumoylation of the THO complex regulates the biogenesis of a subset of mRNPs

International audienceAssembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, compromises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-dependent mRNP assembly is critical to allow a specific subset of mRNPs to escape degradation. This article thus provides the first example of a SUMO-dependent mRNP-assembly event allowing a refined tuning of gene expression, in particular under specific stress conditions

Genetic, Biochemical, and Electron Microscopic Analysis of Components Involved in Transcription Coupled mRNA Export

In the eukaryotic cell, the nuclear envelope separates the nucleoplasm from the cytoplasm. The nuclear pore complex (NPC) forms the conduit that regulates the exchange of macromolecules between these compartments. Import and export of protein and RNA through the NPCs are highly regulated and follow several different pathways. Messenger RNAs (mRNAs) are exported from the nucleus only after extensive processing and assembly into ribonucleoprotein particles (RNPs). Studies in yeast have identified many components involved in mRNA export. These include nuclear pore proteins, export receptors and components in the nucleus that couple formation of mRNPs with translocation through the pores. When I started my PhD work, I performed a synthetic lethal (sl) screen to investigate the role of Gle2 in mRNA export. Gle2 was a proposed to be an mRNA export factor. Thus, the aim has been to gain an understanding of the relationship between Gle2 and the Mex67-mediated mRNA export pathway. I could show that GLE2 is synthetic lethal with the mRNA export factors Sac3 and Mex67, with importins a and b, and with several nucleoporins, which are subunits of distinct subcomplexes of the NPC. This part of my studies indicated that the function of Gle2 is not restricted to nuclear export and suggested a more general role of Gle2 in bidirectional transport through the nuclear pore complexes. To investigate the Mex67-mediated mRNA export pathway, in the second part of my studies, I performed a synthetic lethal screen with SUB2, an intranuclear factor which in our lab was found to act in mRNA export. Initial work suggested that Sub2 was a splicing factor. The sl screen I performed revealed a genetic link between SUB2 and the THO complex, which is involved in transcription elongation. These data contributed to the identification of a novel conserved complex called TREX (transcription/export), formed by the export factors Sub2 and Yra1, a previously unknown factor, Tex1, and the THO complex. Thus, the TREX complex couples transcription elongation and mRNA export. To further characterize the TREX, I analyzed the genetic interactions of two components of the THO complex, THO2 and THP2. The import receptor MTR10 was found to be synthetic lethal with SUB2 and THP2. In addition, I found that Sub2-GFP and Thp2-GFP are mislocalized in MTR10 mutants, indicating a role of Mtr10 as import factor for components of the TREX complex. Furthermore, THO2 and THP2 are synthetic lethal with RRP6, a component of the exosome complex, which retains and eliminates improperly 3�-end processed mRNPs, suggesting a link between transcription elongation, and RNP quality control. Finally, I analyzed the TREX complex at the biochemical level. The TREX complex was purified using a variety of methods, including tandem affinity purification (TAP) and by gel filtration. Under stringent conditions, I could purify a stable core of the TREX complex, in which Sub2 and Yra1 were partly dissociated. In collaboration with the Böttcher lab (EMBL), I studied the morphology of this complex by electron microscopy (EM). The core of TREX shows a butterfly-like shape, with two-fold symmetry and a cleft in between the two winged arms. Under less stringent conditions, the TREX complex contains stoichiometric amounts of Sub2 and Yra1. Nevertheless, the complex mostly retains a butterfly-like morphology at the EM level. In conclusion, my studies identified new connections between mRNA export and protein import at the nuclear pore, and revealed that transcription, maturation and export of mRNAs are genetically and physically coupled

Comparative Genome-Wide Screening Identifies a Conserved Doxorubicin Repair Network That Is Diploid Specific in Saccharomyces cerevisiae

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Δ, ctf4Δ, ctk1Δ, hfi1Δ,nup133Δ, tho2Δ) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Δ and hfi1Δ) or deficient (tho2Δ) for recombination. Using these and other “THO2-like” hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Δ, thp1Δ, thp2Δ) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases

A new connection of mRNP biogenesis and export with transcription-coupled repair

Although DNA repair is faster in the transcribed strand of active genes, little is known about the possible contribution of mRNP biogenesis and export in transcription-coupled repair (TCR). Interestingly, mutants of THO, a transcription complex involved in maintenance of genome integrity, mRNP biogenesis and export, were recently found to be deficient in nucleotide excision repair. In this study we show by molecular DNA repair analysis, that Sub2-Yra1 and Thp1-Sac3, two main mRNA export complexes, are required for efficient TCR in yeast. Careful analysis revealed that THO mutants are also specifically affected in TCR. Ribozyme-mediated mRNA self-cleavage between two hot spots for UV damage showed that efficient TCR does not depend on the nascent mRNA, neither in wild-type nor in mutant cells. Along with severe UV damage-dependent loss in processivity, RNAPII was found binding to chromatin upon UV irradiation in THO mutants, suggesting that RNAPII remains stalled at DNA lesions. Furthermore, Def1, a factor responsible for the degradation of stalled RNAPII, appears essential for the viability of THO mutants subjected to DNA damage. Our results indicate that RNAPII is not proficient for TCR in mRNP biogenesis and export mutants, opening new perspectives on our knowledge of TCR in eukaryotic cells

R-loops do not accumulate in transcription-defective hpr1-101 mutants: implications for the functional role of THO/TREX

To get further insight into the effect that THO/TREX and R-loops have in transcription-associated recombination and transcription, we analyzed the ability to form R-loops of hpr1-101, a THO mutation that impairs transcription and mRNP biogenesis without triggering hyper-recombination. Human AID, a cytidine deaminase that acts on ssDNA displaced by RNA-DNA hybrids, strongly induced both hyper-recombination and hyper-mutation in hpr1-101, similar to hpr1Δ mutants. However, in contrast to hpr1Δ, AID-induced mutations in hpr1-101 occur at similar frequencies in both the transcribed and non-transcribed strands, implying that the enhanced AID action in these mutants is not caused by co-transcriptional R-loops. These results indicate for the first time that THO has a transcriptional function that is not mediated by R-loops, providing a new perspective for the understanding of the coupling of transcription with mRNP biogenesis and export