Nuclear surveillance and degradation of hypomodified initiator tRNA^Met in S. cerevisiae

Abstract

The tRNA m 1 A58 methyltransferase is composed of two subunits encoded by the essential genes TRM6 and TRM61 (formerly GCD10 and GCD14). The trm6-504 mutation results in a defective m 1 A methyltransferase (Mtase) and a temperature-sensitive growth phenotype that is attributable to the absence of m 1 A58 and consequential tRNA i Met instability. We used a genetic approach to identify the genes responsible for tRNA i Met degradation in trm6 cells. Three recessive extragenic mutations that suppress trm6-504 mutant phenotypes and restore hypomodified tRNA i Met to near normal levels were identified. The wild-type allele of one suppressor, DIS3/RRP44, encodes a 3-5 exoribonuclease and a member of the multisubunit exosome complex. We provide evidence that a functional nuclear exosome is required for the degradation of tRNA i The relatively unstable nature of messenger RNAs fueled the discovery of pathways that control the degradation of normal and abnormal mRNAs in the nucleus and cytoplasm (Hilleren and Parker 1999;Mitchell and Tollervey 2001;Wilusz et al. 2001;Maquat 2002;Moore 2002;Long and McNally 2003). Two general pathways of mRNA decay have been characterized in the yeast Saccharomyces cerevisiae, and homologs of most of the yeast proteins involved in mRNA turnover have been identified in metazoans. The first pathway initially requires shortening of the mRNA polyadenylate tail, followed by removal of the 5Ј cap structure (Wilusz et al. 2001), which leaves the body of the mRNA susceptible to 5Ј-3Ј exonucleolytic degradation by Xrn1p. The second pathway involves deadenylation of mRNAs and the 3Ј-5Ј degradation of the body of the mRNA by the exosome (Jacobs et al. 1998;Burkard and Butler 2000;van Hoof et al. 2000b;van Hoof and Parker 2002;Mitchell and Tollervey 2003).The exosome is a multisubunit complex of proteins with multiple functions in the processing, degradation, and retention of stable and unstable RNAs in the nucleus and cytoplasm. The cytoplasmic exosome directly interacts with Ski7p (Araki et al. 2001) and recruits the Ski2p, Ski3p, and Ski8p complex to the 3Ј end of a deadenylated mRNA (Brown et al. 2000) or an mRNA that is stalled on the ribosome because it lacks a stop codon (Jacobs et al. 1998;van Hoof et al. 2000b), and in turn each is degraded in a 3Ј-to-5Ј direction. In the nucleus, the exosome has been implicated in elimination of by-products of rRNA processing (ETS sequence). The nuclear exosome possesses an exonuclease, Rrp6p, not found in the cytoplasmic form (Allmang et al. 1999b). A specialized function of Rrp6p and the nuclear exosome appears to be in retaining mRNAs incorrectly processed at their 3Ј ends at the site of transcription to prevent their release into the cytoplasm (Hilleren et al. 2001;Libri et al. 2002). Thus far, the exosome has not been implicated in the destruction of stable RNAs that are rendered unstable due to mutations or defects in processing.

tRNA modifications: Necessary for correct tRNA‐derived fragments during the recovery from stress?

“…The first pathway is triggered by a loss of essential tRNA modifications and is mediated by the nuclear TRAMP complex. Poly-adenylation of hypo-modified tRNAs by TRAMP is followed by tRNA degradation in the nuclear exosome [14]. The second pathway occurs both in the cytoplasm and in the nucleus and involves the loss of non-essentíal tRNA modifications.…”

Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae

“…Recently, a decay complex now referred to as TRAMP was described (13)(14)(15)(16). Trf4p is a component of this complex that acts by adding 10-40 adenines onto the 3Ј end of RNAs before their decay by the exosome (13)(14)(15)(16).…”

“…Trf4p is a component of this complex that acts by adding 10-40 adenines onto the 3Ј end of RNAs before their decay by the exosome (13)(14)(15)(16). To test whether loss of Trf4p could cause accumulation of the RNAs we identified, we blotted RNA from a trf4⌬ strain and probed with several genomic regions.…”

“…The other class of RNA was unexpected and consists of transcripts that appear to originate from promoter regions of standard protein-encoding genes. In wild-type cells, these transcripts are decayed through a Trf4p-Rrp6p pathway (13)(14)(15)(16). We propose that these transcripts are produced at mRNA promoters across the genome as a consequence of RNA polymerase II activity that normally does not lead to functional mRNA.…”

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“…Ainsi, la voie majeure de la surveillance nucléaire, la voie dite TRAMP-exosome, a initialement été mise en évi-dence sur un précurseur d'ARNt (ARNt MET initiateur) défectueux structuralement en raison de l'absence d'une seule de ses modifications [10,15,16] (Figure 2A). Dans cette voie, l'addition de courtes queues de poly(A) et/ou la liaison du complexe TRAMP à l'ARN condamnent véritablement les molécules aberrantes à la dégradation, à la fois en discriminant les ARN normaux des ARN anormaux et en stimulant l'activité de l'exosome à ARN [2].…”