Nuclear RNA Surveillance in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e: Trf4p-dependent Polyadenylation of Nascent Hypomethylated tRNA and an Aberrant Form of 5S rRNA

1-Methyladenosine modification at position 58 of tRNA is catalyzed by a two-subunit methyltransferase composed of Trm6p and Trm61p in Saccharomyces cerevisiae. Initiator tRNA (tRNAiMet) lacking m1A58 (hypomethylated) is rendered unstable through the cooperative function of the poly(A) polymerases, Trf4p/Trf5p, and the nuclear exosome. We provide evidence that a catalytically active Trf4p poly(A) polymerase is required for polyadenylation of hypomethylated tRNAiMet in vivo. DNA sequence analysis of tRNAiMet cDNAs and Northern hybridizations of poly(A)+ RNA provide evidence that nascent pre-tRNAiMet transcripts are targeted for polyadenylation and degradation. We determined that a mutant U6 snRNA and an aberrant form of 5S rRNA are stabilized in the absence of Trf4p, supporting that Trf4p facilitated RNA surveillance is a global process that stretches beyond hypomethylated tRNAiMet. We conclude that an array of RNA polymerase III transcripts are targeted for Trf4p/ Trf5p-dependent polyadenylation and turnover to eliminate mutant and variant forms of normally stable RNAs

Substrate specificity of the TRAMP nuclear surveillance complexes

During nuclear surveillance in yeast and human cells, the RNA exosome functions together with the TRAMP complexes. Here, the authors defined the protein composition of the TRAMP complexes and identified specific RNA binding sites for the different TRAMP components

Identifying Subunit Organization and Function of the Nuclear RNA Exosome Machinery

IDENTIFYING SUBUNIT ORGANIZATION AND FUNCTION OF THE NUCLEAR RNA EXOSOME MACHINERY Jillian Strother Losh, A.S., B.S. The eukaryotic RNA exosome processes and degrades many classes of RNA. It is present in the nucleus and the cytoplasm, highly evolutionarily conserved, and essential for viability. Since the RNA exosome is such a significant component of the RNA degradation machinery, it is unsurprising that even single point mutations in a few of its subunits have been linked to human disease. For example, at least eight point mutations in a single subunit of the RNA exosome have been linked to pontocerebellar hypoplasia subtype 1b (PCH1b). My work has included the development of a laboratory model system to assess the specific effects of these mutations on the structure and function of the RNA exosome. My collaborators and I have employed the common model organism Saccharomyces cerevisiae for this work since both the RNA exosome and other components of RNA degradation machinery are conserved throughout eukaryotes. Our research has shown that at least one PCH1b-associated mutation negatively affects the stability of the RNA exosome, although it remains functional. The effect of this mutation is conserved between yeast and mouse cells. The RNA exosome requires various cofactors in both the nucleus and the cytoplasm for substrate delivery. The other half of my work focuses on a nuclear cofactor of the RNA exosome, the TRAMP complex. This complex is comprised of an RNA helicase and a poly(A) polymerase, as well as an RNA-binding subunit. However, it is currently unclear how the TRAMP complex is specifically assembled and moreover, if it is essential for life. The poly(A) polymerase subunit consists of a catalytic domain, as well as disordered regions that are required for protein interactions. My work has shown that the catalytic core of the TRAMP complex is necessary and sufficient for its essential functions, although a specific interaction between the two enzymatic subunits is required for snoRNA biogenesis and possibly other cellular functions. These and future studies will help define the role of the TRAMP complex in the RNA degradation process and determine its importance for cellular viability

Contribution of Trf4/5 and the Nuclear Exosome to Genome Stability Through Regulation of Histone mRNA Levels in Saccharomyces cerevisiae

Balanced levels of histones are crucial for chromosome stability, and one major component of this control regulates histone mRNA amounts. The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in a quality control mechanism that mediates polyadenylation and consequent degradation of various RNA species by the nuclear exosome. None of the known RNA targets, however, explains the fact that trf mutants have specific cell cycle defects consistent with a role in maintaining genome stability. Here, we investigate the role of Trf4/5 in regulation of histone mRNA levels. We show that loss of Trf4 and Trf5, or of Rrp6, a component of the nuclear exosome, results in elevated levels of transcripts encoding DNA replication-dependent histones. Suggesting that increased histone levels account for the phenotypes of trf mutants, we find that TRF4 shows synthetic genetic interactions with genes that negatively regulate histone levels, including RAD53. Moreover, synthetic lethality of trf4Δ rad53Δ is rescued by reducing histone levels whereas overproduction of histones is deleterious to trf's and rrp6Δ mutants. These results identify TRF4, TRF5, and RRP6 as new players in the regulation of histone mRNA levels in yeast. To our knowledge, the histone transcripts are the first mRNAs that are upregulated in Trf mutants

Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control

Trf4 is the poly(A) polymerase component of TRAMP4, which stimulates nuclear RNA degradation by the exosome. We report that in Saccharomyces cerevisiae strains lacking Trf4, cryptic transcripts are detected from regions of repressed chromatin at telomeres and the rDNA intergenic spacer region (IGS1-R), and at CEN3. Degradation of the IGS1-R transcript was reduced in strains lacking TRAMP components, the core exosome protein Mtr3 or the nuclear-specific exosome component Rrp6. IGS1-R has potential binding sites for the RNA-binding proteins Nrd1/Nab3, and was stabilized by mutation of Nrd1. IGS1-R passes through the replication fork barrier, a region required for rDNA copy number control. Strains lacking Trf4 showed sporadic changes in rDNA copy number, whereas loss of both Trf4 and either the histone deacetylase Sir2 or the topoisomerase Top1 caused dramatic loss of rDNA repeats. Chromatin immunoprecipitation analyses showed that Trf4 is co-transcriptionally recruited to IGS1-R, consistent with a direct role in rDNA stability. Co-transcriptional RNA binding by Trf4 may link RNA and DNA metabolism and direct immediate IGS1-R degradation by the exosome following transcription termination

Distinct Roles of Non-Canonical Poly(A) Polymerases in RNA Metabolism

Trf4p and Trf5p are non-canonical poly(A) polymerases and are part of the heteromeric protein complexes TRAMP4 and TRAMP5 that promote the degradation of aberrant and short-lived RNA substrates by interacting with the nuclear exosome. To assess the level of functional redundancy between the paralogous Trf4 and Trf5 proteins and to investigate the role of the Trf4-dependent polyadenylation in vivo, we used DNA microarrays to compare gene expression of the wild-type yeast strain of S. cerevisiae with either that of trf4Δ or trf5Δ mutant strains or the trf4Δ mutant expressing the polyadenylation-defective Trf4(DADA) protein. We found little overlap between the sets of transcripts with altered expression in the trf4Δ or the trf5Δ mutants, suggesting that Trf4p and Trf5p target distinct groups of RNAs for degradation. Surprisingly, most RNAs the expression of which was altered by the trf4 deletion were restored to wild-type levels by overexpression of TRF4(DADA), showing that the polyadenylation activity of Trf4p is dispensable in vivo. Apart from previously reported Trf4p and Trf5p target RNAs, this analysis along with in vivo cross-linking and RNA immunopurification-chip experiments revealed that both the TRAMP4 and the TRAMP5 complexes stimulate the degradation of spliced-out introns via a mechanism that is independent of the polyadenylation activity of Trf4p. In addition, we show that disruption of trf4 causes severe shortening of telomeres suggesting that TRF4 functions in the maintenance of telomere length. Finally, our study demonstrates that TRF4, the exosome, and TRF5 participate in antisense RNA–mediated regulation of genes involved in phosphate metabolism. In conclusion, our results suggest that paralogous TRAMP complexes have distinct RNA selectivities with functional implications in RNA surveillance as well as other RNA–related processes. This indicates widespread and integrative functions of TRAMP complexes for the coordination of different gene expression regulatory processes

Current perspectives on the role of TRAMP in nuclear RNA surveillance and quality control

The TRAMP complex assists the nuclear exosome to degrade a broad range of ribonucleic acid (RNA) substrates by increasing both exoribonucleolytic activity and substrate specificity. However, how the interactions between the TRAMP subunits and the components of the nuclear exosome regulate their functions in RNA degradation and substrate specificity remain unclear. This review aims to provide a summary of the recent findings on the role of the TRAMP complex in nuclear RNA degradation. The new insights from recent structural biological studies are discussed.published_or_final_versio

New liver cell mutants defective in the endocytic pathway

AbstractTo isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2–Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 α″ (CK2α″) which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of 125I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%–60% in Trf2–Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on 125I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2–Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa488-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein–protein or protein–lipid interactions that orchestrate specific steps in membrane protein trafficking

tRNA Fragments: Expression and Function in Ovarian Cancer

University of Minnesota Ph.D. dissertation.September 2017. Major: Biochemistry, Molecular Bio, and Biophysics. Advisors: Lester Drewes, Lynne Bemis. 1 computer file (PDF); xi, 127 pages.Deep sequencing studies of noncoding RNA in liquid biopsies are revealing a vast repertoire of potential biomarkers. Ovarian cancer is a difficult-to-diagnose disease, urgently requiring novel and readily accessible biomarkers. We hypothesized that urine, one source of liquid biopsy samples, may contain novel noncoding RNAs (ncRNAs) that could serve as biomarkers for ovarian cancer. We proceeded to deep sequence RNA extracted from urine collected from ovarian cancer patients to better understand the repertoire of small RNAs in this type of liquid biopsy sample. The ncRNAs identified in these urine samples were predominantly microRNAs (miRNAs), ribosomal RNA (rRNA) fragments and tRNA fragments (tRFs). tRFs are a group of ncRNAs, which have been found across the biological kingdom and are increasingly being studied for their role in cancer biology. Several tRFs have been studied in cancer, although not previously in ovarian cancer. We have studied the expression of one specific tRF, 5’ fragment of tRNA-Glu-CTC (tRF5-Glu), in five different ovarian cancer cell lines. Several variants of tRF5-Glu were identified and we have now confirmed the expression of tRF5-Glu in ovarian cancer cells by quantitative real-time PCR (qRT-PCR), Northern analysis and ligation PCR. Additionally, we determined that angiogenin (ANG) plays a role in the biogenesis of tRF5-Glu. Furthermore, we have shown that tRF5-Glu targets the mRNA of the Breast Cancer Anti-estrogen Resistance 3 (BCAR3). While BCAR3 is known to regulate cancer cell migration and contributes to anti-estrogen resistance in breast cancer cells, it has not previously been studied in ovarian cancer or shown to be targeted by a tRF. Using synthetic mimics of tRF5-Glu and siRNAs targeting BCAR3, we were able to show that tRF5-Glu expression and the knock down of BCAR3 expression inhibits proliferation in ovarian cancer cells. These studies demonstrate that tRF5-Glu contributes to the regulation of BCAR3 and provides a novel mechanism of the regulation of proliferation in ovarian cancer cell lines