In vivo pre-tRNA processing in Saccharomyces cerevisiae.

Abstract

We have surveyed intron-containing RNAs of the yeast Saccharomyces cerevisiae by filter hybridization with pre-tRNA intron-specific oligonucleotide probes. We have classified various RNAs as pre-tRNAs, splicing intermediates, or excised intron products according to apparent size and structure. Linear, excised intron products were detected, and one example was isolated and sequenced directly. Additional probes designed to detect other precursor sequences were used to verify the identification of several intermediates. Pre-tRNA species with both 5' leader and 3' extension, with 3' extension only, and with mature ends were distinguished. From these results, we conclude that the processing reactions used to remove the 5' leader and 3' extension from the transcript are ordered 5' end trimming before 3' end trimming. Splicing intermediates containing the 5' exon plus the intron were detected. The splice site cleavage reactions are probably ordered 3' splice site cleavage before 5' splice site cleavage. Surprisingly, we also detected a splicing intermediate with the 5' leader and a spliced product with both 5' leader and 3' extension. Evidently, splicing and end trimming are not ordered relative to each other, splicing occurring either before or after end trimming.

Repression of Ribosome and tRNA Synthesis in Secretion-Defective Cells Is Signaled by a Novel Branch of the Cell Integrity Pathway

“…Total RNA was extracted by glass-bead disruption in the presence of hot phenol (23). For Northern analysis of tRNA precursors, RNA (20 g) was resolved on 10% polyacrylamide-8.3 M urea gels in Tris-borate-EDTA (TBE) buffer.…”

“…At various times before and after the temperature shift, 1 ml of cells was removed to tubes containing 150 Ci of carrier-free [ 32 P]orthophosphate for a further 5-min incubation. Cells were harvested rapidly and frozen prior to the preparation of total RNA (23). RNA (5 g) was resolved on 10% denaturing polyacrylamide gels (see above) which were fixed, dried, and exposed to phosphor storage plates.…”

Dual function of the tRNA(m5U54)methyltransferase in tRNA maturation

“…Eukaryotic transfer RNA genes are transcribed by RNA polymerase III generating precursor tRNAs that have to be further processed to generate functional tRNAs (for review, see Wolin & Matera, 1999)+ One of the first proteins that bind the newly synthesized pre-tRNA is the La protein (Lhp1p in yeast), which binds to the 39 end of the transcript and protects this end from exonucleolytic digestion (Yoo & Wolin, 1997;Fan et al+, 1998)+ This is followed by removal of the 59 leader by RNase P (for review, see Frank & Pace, 1998), which precedes an endonucleolytic removal of the 39 trailer+ Furthermore, eukaryotic tRNAs have to undergo posttranscriptional addition of CCA to their 39 termini catalyzed by the tRNA nucleotidyltransferase (for review, see Deutscher, 1990)+ Some tRNA genes contain an intron and splicing of the pre-tRNA occurs before or after end maturation+ However, in Saccharomyces cerevisiae, end maturation normally precedes splicing (O'Connor & Peebles, 1991)+ During the maturation of tRNA, a variety of different nucleoside modifications occurs that allows the mature tRNA to function with high efficiency and flexibility+ Modified nucleosides can be found in all phylogenetic domains and also in identical positions of the tRNA, suggesting a conserved function of some tRNA modifications (Björk, 1986;Cermakian & Cedergren, 1998;Björk et al+, 2001)+ A 5-methyluridine residue at position 54 (m 5 U 54 ) is a highly conserved feature of eukaryotic and bacterial tRNAs+ The presence of this modification influences, in vitro, the fidelity and rate of protein synthesis as well as the stability of tRNA tertiary structure (Davanloo et al+, 1979;Kersten et al+, 1981)+ The in vivo role has been more difficult to elucidate+ An Escherichia coli strain with a mutation in the gene (trmA) encoding the tRNA(m 5 U 54 )methyltransferase and that lacks the m 5 U 54 nucleoside show a slight reduction in growth rate (Björk, 1986)+ However, a truncation of the trmA gene was shown to be lethal, indicative of an additional and essential function of the protein (Persson et al+, 1992)+ In contrast, an S. cerevisiae strain with either a mutation in or a deletion of the TRM2 gene, encoding the yeast tRNA(m 5 U 54 )methyltransferase, lacks the m 5 U 54 nucleoside but is viable and exhibits no apparent phenotype (Hopper et al+, 1982;Nordlund et al+, 2000)+ The methylation of U 54 is an early event in tRNA maturation in S. cerevisiae, as intron-containing pre-tRNAs have been shown to contain the m 5 U nucleoside (Knapp et al+, 1978;Etcheverry et al+, 1979)+ In this study, we report that yeast strains with each of four different mutant alleles of the single copy and essential sup61 ϩ gene, encoding tRNA CGA Ser , have a requirement for the TRM2 gene for growth at 30 8C+ The absence of the Trm2 protein ...…”

Have tRNA, will travel

“…Thus, steady-state analysis of tRNA processing in Saccharomyces cerevisiae demonstrated the existence of at least one species of tRNA that was spliced but not end-processed (17). Similarly, disruption of a nuclear retention element of Schizosaccharomyces pombe La protein, a protein required for tRNA 3Ј end processing (18), led to the accumulation of spliced tRNA precursors that had not been processed at their 5Ј and 3Ј ends (19).…”