Tho2 is critical for the recruitment of Rrp6 to chromatin in response to perturbed mRNP biogenesis.

The eukaryotic THO complex coordinates the assembly of so-called messenger RNA-ribonucleoprotein particles (mRNPs), a process that involves cotranscriptional coating of nascent mRNAs with proteins. Once formed, mRNPs undergo a quality control step that marks them either for active transport to the cytoplasm, or Rrp6/RNA exosome-mediated degradation in the nucleus. However, the mechanism behind the quality control of nascent mRNPs is still unclear. We investigated the cotranscriptional quality control of mRNPs in budding yeast by expressing the bacterial Rho helicase, which globally perturbs yeast mRNP formation. We examined the genome-wide binding profiles of the THO complex subunits Tho2, Thp2, Hpr1, and Mft1 upon perturbation of the mRNP biogenesis, and found that Tho2 plays two roles. In addition to its function as a subunit of the THO complex, upon perturbation of mRNP biogenesis Tho2 targets Rrp6 to chromatin via its carboxy-terminal domain. Interestingly, other THO subunits are not enriched on chromatin upon perturbation of mRNP biogenesis and are not necessary for localizing Rrp6 at its target loci. Our study highlights the potential role of Tho2 in cotranscriptional mRNP quality control, which is independent of other THO subunits. Considering that both the THO complex and the RNA exosome are evolutionarily highly conserved, our findings are likely relevant for mRNP surveillance in mammals.

Streamlining N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins.

Surface display co-opts yeast's innate ability to embellish its cell wall with mannoproteins, thus converting the yeast's outer surface into a growing and self-sustaining catalyst. However, the efficient toolbox for converting the enzyme of interest into its surface-displayed isoform is currently lacking, especially if the isoform needs to be anchored to the cell wall near the isoform's N-terminus, e.g., through a short GPI-independent protein anchor. Aiming to advance such N-terminally anchored surface display, we employed in silico and machine-learning strategies to study the 3D structure, function, genomic organisation, and evolution of the Pir protein family, whose members evolved to covalently attach themselves near their N-terminus to the ß-1,3-glucan of the cell wall. Through the newly-gained insights, we rationally engineered 14 S. cerevisiae Hsp150 (Pir2)-based fusion proteins. We quantified their performance, uncovering guidelines for efficient yeast surface display while developing a construct that promoted a 2.5-fold more efficient display of a reporter protein than the full-length Hsp150. Moreover, we developed a Pir-tag, i.e., a peptide spanning only 4.5 kDa but promoting as efficient surface display of a reporter protein as the full-length Hsp150. These constructs fortify the existing surface display toolbox, allowing for a prompt and routine refitting of intracellular proteins into their N-terminally anchored isoforms.

Nucleosome Remodeling at the Yeast PHO8 and PHO84 Promoters without the Putatively Essential SWI/SNF Remodeler.

Chromatin remodeling by ATP-dependent remodeling enzymes is crucial for all genomic processes, like transcription or replication. Eukaryotes harbor many remodeler types, and it is unclear why a given chromatin transition requires more or less stringently one or several remodelers. As a classical example, removal of budding yeast PHO8 and PHO84 promoter nucleosomes upon physiological gene induction by phosphate starvation essentially requires the SWI/SNF remodeling complex. This dependency on SWI/SNF may indicate specificity in remodeler recruitment, in recognition of nucleosomes as remodeling substrate or in remodeling outcome. By in vivo chromatin analyses of wild type and mutant yeast under various PHO regulon induction conditions, we found that overexpression of the remodeler-recruiting transactivator Pho4 allowed removal of PHO8 promoter nucleosomes without SWI/SNF. For PHO84 promoter nucleosome removal in the absence of SWI/SNF, an intranucleosomal Pho4 site, which likely altered the remodeling outcome via factor binding competition, was required in addition to such overexpression. Therefore, an essential remodeler requirement under physiological conditions need not reflect substrate specificity, but may reflect specific recruitment and/or remodeling outcomes.

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. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.

Interplay of the RNA Exosome Complex and RNA-Binding Protein Ssd1 in Maintaining Cell Wall Stability in Yeast.

Yeast cell wall stability is important for cell division and survival under stress conditions. The expression of cell-wall-related proteins is regulated by several pathways involving RNA-binding proteins and RNases. The multiprotein RNA exosome complex provides the 3'→5' exoribonuclease activity that is critical for maintaining the stability and integrity of the yeast cell wall under stress conditions such as high temperatures. In this work, we show that the temperature sensitivity of RNA exosome mutants is most pronounced in the W303 genetic background due to the nonfunctional ssd1-d allele. This gene encodes the RNA-binding protein Ssd1, which is involved in the posttranscriptional regulation of cell-wall-related genes. Expression of the functional SSD1-V allele from its native genomic locus or from a centromeric plasmid suppresses the growth defects and aberrant morphology of RNA exosome mutant cells at high temperatures or upon treatment with cell wall stressors. Moreover, combined inactivation of the RNA exosome catalytic subunit Rrp6 and Ssd1 results in a synthetically sick phenotype of cell wall instability, as these proteins may function in parallel pathways (i.e., via different mRNA targets) to maintain cell wall stability. IMPORTANCE Stressful conditions such as high temperatures can compromise cellular integrity and cause bursting. In microorganisms surrounded by a cell wall, such as yeast, the cell wall is the primary shield that protects cells from environmental stress. Therefore, remodeling its structure requires inputs from multiple signaling pathways and regulators. In this work, we identify the interplay of the RNA exosome complex and the RNA-binding protein Ssd1 as an important factor in the yeast cell wall stress response. These proteins operate in independent pathways to support yeast cell wall stability. This work highlights the contribution of RNA-binding proteins in the regulation of yeast cell wall structure, providing new insights into yeast physiology.

Regulation of the conserved 3'-5' exoribonuclease EXOSC10/Rrp6 during cell division, development and cancer.

The conserved 3'-5' exoribonuclease EXOSC10/Rrp6 processes and degrades RNA, regulates gene expression and participates in DNA double-strand break repair and control of telomere maintenance via degradation of the telomerase RNA component. EXOSC10/Rrp6 is part of the multimeric nuclear RNA exosome and interacts with numerous proteins. Previous clinical, genetic, biochemical and genomic studies revealed the protein's essential functions in cell division and differentiation, its RNA substrates and its relevance to autoimmune disorders and oncology. However, little is known about the regulatory mechanisms that control the transcription, translation and stability of EXOSC10/Rrp6 during cell growth, development and disease and how these mechanisms evolved from yeast to human. Herein, we provide an overview of the RNA- and protein expression profiles of EXOSC10/Rrp6 during cell division, development and nutritional stress, and we summarize interaction networks and post-translational modifications across species. Additionally, we discuss how known and predicted protein interactions and post-translational modifications influence the stability of EXOSC10/Rrp6. Finally, we explore the idea that different EXOSC10/Rrp6 alleles, which potentially alter cellular protein levels or affect protein function, might influence human development and disease progression. In this review we interpret information from the literature together with genomic data from knowledgebases to inspire future work on the regulation of this essential protein's stability in normal and malignant cells.

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation.

Nuclear RNA exosome is the main 3'→5' RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes.

Non-coding RNAs as cell wall regulators in Saccharomyces cerevisiae.

The cell wall of Saccharomyces cerevisiae is an extracellular organelle crucial for preserving its cellular integrity and detecting environmental cues. The cell wall is composed of mannoproteins attached to a polysaccharide network and is continuously remodelled as cells undergo cell division, mating, gametogenesis or adapt to stressors. This makes yeast an excellent model to study the regulation of genes important for cell wall formation and maintenance. Given that certain yeast strains are pathogenic, a better understanding of their life cycle is of clinical relevance. This is why transcriptional regulatory mechanisms governing genes involved in cell wall biogenesis or maintenance have been the focus of numerous studies. However, little is known about the roles of long non-coding RNAs (lncRNAs), a class of transcripts that are thought to possess little or no protein coding potential, in controlling the expression of cell wall-related genes. This review outlines currently known mechanisms of lncRNA-mediated regulation of gene expression in S. cerevisiae and describes examples of lncRNA-regulated genes encoding cell wall proteins. We suggest that the association of currently annotated lncRNAs with the coding sequences and/or promoters of cell wall-related genes highlights a potential role for lncRNAs as important regulators of the yeast cell wall structure.