Endonucleolytic cleavage of a long 3'-trailer sequence in a nuclear yeast suppressor tRNA

Abstract

Transcripts of Saccharomyces cerevisiae nuclear tRNA genes are normally terminated within a few nucleotides of the tRNA coding region, in contrast to mitochondrially encoded tRNAs, which are contained within polycistronic transcripts and thus require 3'-processing by mitochondrial endonucleases. We show that 3'-processing activities capable of removing artificially extended 3'-trailer sequences from some tRNA substrates are also present in the yeast nucleus. Correct 3'-processing in vivo resulted in the formation of functional suppressor tRNA. The 3'-processing activities were also identified in vitro through analysis of transcription-processing products in cell-free yeast S-100 extracts. Comparison of several pre-tRNA substrates showed that the tRNA structure played a major role in determining the processability of a substrate but that the nature of the 3'-trailer sequence also modulated the rate of 3'-processing. Pre-tRNA containing mitochondrial tRNA(Val) sequence was a good substrate for in vitro processing, independent of its 3'-trailer. A 200-nt-long pre-tRNA, encoding the nuclear SUP4 tRNA gene and a mitochondrial 3'-trailer, was processed in yeast S-100 extract in a multistep pathway into mature-sized tRNA(Tyr). Part of the 3'-processing was due to an endonuclease which cleaved near or precisely at the 3'-end of the coding region of the tRNA. A short sequence around this endonucleolytic 3'-cleavage site was crucial for the formation of active suppressor tRNA in vivo. A 9-nt-long sequence motif derived from the mitochondrial 3'-trailer allowed processing, while sequences derived from lacZ or pBR322 DNA were processed neither in vitro nor in vivo.

Competition between the Rex1 exonuclease and the La protein affects both Trf4p-mediated RNA quality control and pre-tRNA maturation

“…Our findings, together with previous studies (Evans and Engelke 1990;Furter et al 1992;Yoo and Wolin 1997;van Hoof et al 2000), indicate that exonucleases and at least one endonuclease contribute to pre-tRNA maturation. In prokaryotes, exonucleolytic maturation is largely confined to tRNAs in which the CCA is encoded, while RNase Z carries out maturation of most pre-tRNAs that lack CCA.…”

3′-processing of yeast tRNATrp precedes 5′-processing

“…All previous analyses of tRNA processing in yeast have concluded that 5Ј-end maturation obligatorily precedes 3Ј-maturation (Engelke et al 1985;Lee et al 1991;O'Connor and Peebles 1991;Furter et al 1992;Lygerou et al 1994). In contrast to other pre-tRNAs, 5Ј-unprocessed, 3Ј-processed forms of pretRNA Trp were readily detected.…”

“…In both prokaryotes and eukaryotes, maturation of the 5Ј-end is performed by endonuclease RNase P (for review, see Altman et al 1995;Frank and Pace 1998). Processing of the 3Ј terminus of bacterial tRNAs is exonucleolytic (Li and Deutscher 1996), whereas in eukaryotes, the major pathway involves an endonucleolytic cleavage (Garber and Altman 1979;Hagenbüchle et al 1979;Castaño et al 1985;Frendewey et al 1985;Manam and Van Tuyle 1987;Chen and Martin 1988;Furter et al 1992;Oommen et al 1992;Han and Kang 1997;Nashimoto 1997;Kunzmann et al 1998;Mayer et al 2000;Schiffer et al 2002), with exonucleolytic trimming as an alternative pathway (Garber and Altman 1979;Solari and Deutscher 1983;Engelke et al 1985).…”

“…In the budding yeast Saccharomyces cerevisiae, in vivo analysis of pre-tRNA processing revealed endonucleolytic 3Ј-processing only after 5Ј-processing (Engelke et al 1985;O'Connor and Peebles 1991;Furter et al 1992). This was confirmed by analysis of mutants defective in RNase P. Mutations in either the RNA or protein components of RNase P inhibited both 5Ј-and 3Ј-processing of pre-tRNA in vivo, even though the enzyme has no known direct role in 3Ј-processing (Lee et al 1991;Lygerou et al 1994).…”

Transcription Termination by RNA Polymerase III in Fission Yeast

“…Preliminary data suggested that constructs bearing inefficient terminators nonetheless produced functional tRNA, presumably because readthrough transcripts were correctly processed at their 3Ј-ends (47-49) (not shown). tRNA structure appears to be a major determinant of recognition by 3Ј-processing enzymes (50,51), and although certain artificial sequences placed downstream of an inefficient terminator can interfere with 3Ј-processing, others support processing (47,49). To ensure that tRNA expression would be dependent on accurate termination, we inserted a sequence downstream of the test terminator to interfere with tRNA formation if pol III was to read through the test terminator.…”