The cullin Rtt101 promotes ubiquitin-dependent DNA-protein crosslink repair across the cell cycle.

DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1.

Cdc48/p97 segregase: Spotlight on DNA-protein crosslinks.

The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including the removal of DNA-protein crosslinks (DPCs) from the DNA. Growing evidence demonstrates in the last years that Cdc48/p97 is pivotal in targeting ubiquitinated and SUMOylated substrates on chromatin, thereby supporting the DNA damage response. Along with its cofactors, notably Ufd1-Npl4, Cdc48/p97 has emerged as a central player in the unfolding and processing of DPCs.

SUMO in the regulation of DNA repair and transcription at nuclear pores.

Two related post-translational modifications, the covalent linkage of Ubiquitin and the Small Ubiquitin-related MOdifier (SUMO) to lysine residues, play key roles in the regulation of both DNA repair pathway choice and transcription. Whereas ubiquitination is generally associated with proteasome-mediated protein degradation, the impact of sumoylation has been more mysterious. In the cell nucleus, sumoylation effects are largely mediated by the relocalization of the modified targets, particularly in response to DNA damage.

Ubx5-Cdc48 assists the protease Wss1 at DNA-protein crosslink sites in yeast.

DNA-protein crosslinks (DPCs) pose a serious threat to genome stability. The yeast proteases Wss1, 26S proteasome, and Ddi1 are safeguards of genome integrity by acting on a plethora of DNA-bound proteins in different cellular contexts. The AAA ATPase Cdc48/p97 is known to assist Wss1/SPRTN in clearing DNA-bound complexes; however, its contribution to DPC proteolysis remains unclear.

Antisense non-coding transcription represses the PHO5 model gene at the level of promoter chromatin structure.

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system.

Antisense-mediated repression of SAGA-dependent genes involves the HIR histone chaperone.

Eukaryotic genomes are pervasively transcribed by RNA polymerase II (RNAPII), and transcription of long non-coding RNAs often overlaps with coding gene promoters. This might lead to coding gene repression in a process named Transcription Interference (TI). In Saccharomyces cerevisiae, TI is mainly driven by antisense non-coding transcription and occurs through re-shaping of promoter Nucleosome-Depleted Regions (NDRs).

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae.

Fine Chromatin-Driven Mechanism of Transcription Interference by Antisense Noncoding Transcription.

Eukaryotic genomes are almost entirely transcribed by RNA polymerase II. Consequently, the transcription of long noncoding RNAs often overlaps with coding gene promoters, triggering potential gene repression through a poorly characterized mechanism of transcription interference. Here, we propose a comprehensive model of chromatin-based transcription interference in Saccharomyces cerevisiae (S.

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast.

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not repaired in a timely manner. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including stabilized topoisomerase-1 cleavage complexes (Top1ccs). Here, we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae.

Regulation of Gene Expression and Replication Initiation by Non-Coding Transcription: A Model Based on Reshaping Nucleosome-Depleted Regions: Influence of Pervasive Transcription on Chromatin Structure.

RNA polymerase II (RNAP II) non-coding transcription is now known to cover almost the entire eukaryotic genome, a phenomenon referred to as pervasive transcription. As a consequence, regions previously thought to be non-transcribed are subject to the passage of RNAP II and its associated proteins for histone modification. This is the case for the nucleosome-depleted regions (NDRs), which provide key sites of entry into the chromatin for proteins required for the initiation of coding gene transcription and DNA replication.

The functional complexity of the RNA-binding protein Yra1: mRNA biogenesis, genome stability and DSB repair.

The mRNA export adaptor Yra1 is essential in S. cerevisiae, and conserved from yeast to human (ALY/REF). It is well characterized for its function during transcription elongation, 3' processing and mRNA export.

The mRNA export adaptor Yra1 contributes to DNA double-strand break repair through its C-box domain.

Yra1 is an mRNA export adaptor involved in mRNA biogenesis and export in S. cerevisiae. Yra1 overexpression was recently shown to promote accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), cell senescence and telomere shortening, via an unknown mechanism.

Noncoding transcription influences the replication initiation program through chromatin regulation.

In eukaryotic organisms, replication initiation follows a temporal program. Among the parameters that regulate this program in , chromatin structure has been at the center of attention without considering the contribution of transcription. Here, we revisit the replication initiation program in the light of widespread genomic noncoding transcription.

Translesion synthesis DNA polymerase η exhibits a specific RNA extension activity and a transcription-associated function.

Polymerase eta (Polη) is a low fidelity translesion synthesis DNA polymerase that rescues damage-stalled replication by inserting deoxy-ribonucleotides opposite DNA damage sites resulting in error-free or mutagenic damage bypass. In this study we identify a new specific RNA extension activity of Polη of Saccharomyces cerevisiae. We show that Polη is able to extend RNA primers in the presence of ribonucleotides (rNTPs), and that these reactions are an order of magnitude more efficient than the misinsertion of rNTPs into DNA.

Role of chromatin, environmental changes and single cell heterogeneity in non-coding transcription and gene regulation.

The number and variety of factors underlying control of gene expression have been frequently underestimated. Non-coding RNAs generated through pervasive transcription have recently been implicated in shaping the transcriptional landscape in different organisms from bacteria to higher eukaryotes, adding a previously unexpected layer of complexity to the process of gene regulation. In this review, we highlight non-coding transcription-dependent regulatory mechanisms linked to chromatin organization and environmental changes, and particular emphasis is given to single-cell approaches, which have been crucial in dissecting cell-to-cell variability.

Sumoylation and transcription regulation at nuclear pores.

Increasing evidence indicates that besides promoters, enhancers, and epigenetic modifications, nuclear organization is another parameter contributing to optimal control of gene expression. Although differences between species exist, the influence of gene positioning on expression seems to be a conserved feature from yeast to Drosophila and mammals. The nuclear periphery is one of the nuclear compartments implicated in gene regulation.

Role of histone modifications and early termination in pervasive transcription and antisense-mediated gene silencing in yeast.

Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays.

The nuclear pore regulates GAL1 gene transcription by controlling the localization of the SUMO protease Ulp1.

Transcription activation of some yeast genes correlates with their repositioning to the nuclear pore complex (NPC). The NPC-bound Mlp1 and Mlp2 proteins have been shown to associate with the GAL1 gene promoter and to maintain Ulp1, a key SUMO protease, at the NPC. Here, we show that the release of Ulp1 from the NPC increases the kinetics of GAL1 derepression, whereas artificial NPC anchoring of Ulp1 in the Δmlp1/2 strain restores normal GAL1 regulation.

Bimodal expression of PHO84 is modulated by early termination of antisense transcription.

Many Saccharomyces cerevisiae genes encode antisense transcripts, some of which are unstable and degraded by the exosome component Rrp6. Loss of Rrp6 results in the accumulation of long PHO84 antisense (AS) RNAs and repression of sense transcription through PHO84 promoter deacetylation. We used single-molecule resolution fluorescent in situ hybridization (smFISH) to investigate antisense-mediated transcription regulation.

Gene loops and HDACs to promote transcription directionality.

Gene loops have been described in different organisms from yeast to human and form through interaction between components of the transcription pre-initiation complex and Ssu72, a member of the 3' end cleavage and polyadenylation complex. A recent study by Tan-Wong et al. reports a new role for gene loops in promoting ORF transcription directionality from otherwise bidirectional promoters.

Structural basis for polyadenosine-RNA binding by Nab2 Zn fingers and its function in mRNA nuclear export.

Polyadenylation regulation and efficient nuclear export of mature mRNPs both require the polyadenosine-RNA-binding protein, Nab2, which contains seven CCCH Zn fingers. We describe here the solution structure of fingers 5-7, which are necessary and sufficient for high-affinity polyadenosine-RNA binding, and identify key residues involved. These Zn fingers form a single structural unit.

Ubiquitin and assembly of export competent mRNP.

The production of mature and export competent mRNP (mRNA ribonucleoprotein) complexes depends on a series of highly coordinated processing reactions. RNA polymerase II (RNAPII) plays a central role in this process by mediating the sequential recruitment of mRNA maturation and export factors to transcribing genes, thereby establishing a strong functional link between transcription and export through nuclear pore complexes (NPC). Growing evidence indicates that post-translational modifications participate in the dynamic association of processing and export factors with mRNAs ensuring that the transitions and rearrangements undergone by the mRNP occur at the right time and place.

Keeping mRNPs in check during assembly and nuclear export.

The cell nucleus is an intricate organelle that coordinates multiple activities that are associated with DNA replication and gene expression. In all eukaryotes, it stores the genetic information and the machineries that control the production of mature and export-competent messenger ribonucleoproteins (mRNPs), a multistep process that is regulated in a spatial and temporal manner. Recent studies suggest that post-translational modifications play a part in coordinating the co-transcriptional assembly, remodelling and export of mRNP complexes through nuclear pores, adding a new level of regulation to the process of gene expression.

Ubiquitin-mediated mRNP dynamics and surveillance prior to budding yeast mRNA export.

The evolutionarily conserved mRNA export receptor Mex67/NXF1 associates with mRNAs through its adaptor, Yra1/REF, allowing mRNA ribonucleoprotein (mRNP) exit through nuclear pores. However, alternate adaptors should exist, since Yra1 is dispensable for mRNA export in Drosophila and Caenorhabditis elegans. Here we report that Mex67 interacts directly with Nab2, an essential shuttling mRNA-binding protein required for export.