Cross Talk between tRNA and rRNA Synthesis in Saccharomyces cerevisiae

Abstract

Temperature-sensitive RNA polymerase III (rpc160-112 and rpc160-270) mutants were analyzed for the synthesis of tRNAs and rRNAs in vivo, using a double-isotopic-labeling technique in which cells are pulselabeled with [ 33 P]orthophosphate and coextracted with [ 3 H]uracil-labeled wild-type cells. Individual RNA species were monitored by Northern blot hybridization or amplified by reverse transcription. These mutants impaired the synthesis of RNA polymerase III transcripts with little or no influence on mRNA synthesis but also largely turned off the formation of the 25S, 18S, and 5.8S mature rRNA species derived from the common 35S transcript produced by RNA polymerase I. In the rpc160-270 mutant, this parallel inhibition of tRNA and rRNA synthesis also occurred at the permissive temperature (25°C) and correlated with an accumulation of 20S pre-rRNA. In the rpc160-112 mutant, inhibition of rRNA synthesis and the accumulation of 20S pre-rRNA were found only at 37°C. The steady-state rRNA/tRNA ratio of these mutants reflected their tRNA and rRNA synthesis pattern: the rpc160-112 mutant had the threefold shortage in tRNA expected from its preferential defect in tRNA synthesis at 25°C, whereas rpc160-270 cells completely adjusted their rRNA/tRNA ratio down to a wild-type level, consistent with the tight coupling of tRNA and rRNA synthesis in vivo. Finally, an RNA polymerase I (rpa190-2) mutant grown at the permissive temperature had an enhanced level of pre-tRNA, suggesting the existence of a physiological coupling between rRNA synthesis and pre-tRNA processing.The existence of three nuclear transcription systems is documented for all eukaryotes investigated so far. RNA polymerase I synthesizes the three largest rRNAs, RNA polymerase II produces mRNAs and many noncoding RNAs, and RNA polymerase III makes tRNAs and 5S rRNA, as well as a few small noncoding RNAs. Exceptions to this transcriptional specialization are rare and mostly concern noncoding RNA species that can be produced by either RNA polymerase II or III, depending on the phylum considered (reference 39 and references therein). Given its universality, the triplication of the transcriptional apparatus must provide a major selective advantage to the eukaryotic cell, probably by facilitating the separate control of mRNA, rRNA, and tRNA synthesis in response to changes in the environment or in the cell growth rate. On the other hand, RNA polymerases I and III deliver matching amounts of tRNAs and rRNAs to the protein synthesis machinery and may thus need to operate in a closely coordinated way (references 21, 38, and 39 and references therein). In fact, the extent to which the three nuclear RNA polymerases are coordinated relative to each other remains largely undetermined, although this is presumably a key aspect of the transcriptional regulation of growth.Yeast (Saccharomyces cerevisiae) is a particularly convenient model organism to study transcription and its regulation. Its three nuclear RNA polymerases are biochemically and genetically well characterized ...

Rsc4 Connects the Chromatin Remodeler RSC to RNA Polymerases

“…Total RNAs from a wild-type or rsc4-MYC strain were extracted, separated by gel electrophoresis, and blotted onto a membrane. The amounts of U6 snRNA, RPR1 RNA, RNase P RNA, and two different tRNAs (tRNA 3 Leu and tRNA 2 Lys ) and their short-lived precursors were quantified by Northern blot analysis and phosphorimaging ( Fig. 6 and data not shown).…”

Identification of factors that promote biogenesis of tRNACGASer

“…Transcription of tRNA genes as well as 5´-end processing of pre-tRNAs has been reported to localize to the nucleolus [47–49]. Moreover, dissociation of pre-tRNA from the nucleolus and defects in 5´ end processing have been observed in cells defective in Pol I transcription or rRNA processing [50,51]. As mutations in MOT1, RPA49 , and RRN3 affect nucleolar morphology [52,53], the mot1-190, rpa49-27 , and rrn3-32 alleles may influence biogenesis by disrupting the balance and/or order of pre-tRNA processing.…”

Subunits Common to RNA Polymerases

“…Two-hybrid analyses identified similar binding of Rpb8 to the Rpb1-like subunits of RNA pol I (Rpa190) and RNA pol III (Rpc160) [61]. In addition, mutational analysis of S. cerevisiae Rpb8 demonstrated a functional interaction with Rpb6 [67].…”

“…Rpb8 (also known as ABC14.5) is an essential subunit of 16.5 kDa conserved among eukaryotes and thought to be restricted to them [11,12,67]. However, recently, the Rpb8 archaeal orthologues, called G or Rpo8, has been identified in Sulfolobus acidocaldarius (18% identity) and other 15 of the 17 Crenarchaea.…”