Isolation of a Yeast Gene Involved in Species-Specific Pre-tRNA Processing

Abstract

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.

Conformational transition required for efficient splicing of transcripts from hybrid .lambda.-promoter yeast tRNA gene fusion

“…(1) the detection of distinct sets of intermediates among whole cell RNA from wild-type yeast strains and of end-mature IVS-containing precursors accumulated in strains with splicing defects (Hopper & Kurjan, 1981;Willis et al, 1984; Wang & Hopper, 1988;O'Connor & Peebles, 1991), (2) analysis of the time course for appearance of intermediates in yeast transcription and processing extracts (Pearson et al, 1985;Engelke et al, 1985; our unpublished observations), and (3) the demonstration that certain base modifications may take place only prior to splicing (Johnson & Abelson, 1983; Strobel & Abelson, 1986). Thus, pre-tRNA substrates for the splicing enzymes in vivo may normally include base modifications.…”

“…A candidate for this latter category of factors is the STP1 gene product identified recently by Wang and Hopper (1988).…”

STP1, a Gene Involved in Pre-tRNA Processing, Encodes a Nuclear Protein Containing Zinc Finger Motifs

“…Strains, media, and genetic procedure. The following yeast strains were used: M54b (MATa SUP4(G37) ade2-1 met4-1 lys2-1 trp5-2 canl-100 leul-12 ura3-1 [69]), M54b-17 (MATa SUP4(G37) ade2-1 met4-1 lys2-1 trp5-2 canl-100 leul-12 ura3-1 stpl::URA3 [69]), M54b(pC6) (same as M54b, but contains pC6, a multicopy plasmid encoding STPI [69]), SW2001 (A 4Ta ade2-1 ura3-1 leu2-3, 112 trpl lys2-1 or lysl-1] hisS-2 [or his5-2 [or his5-419] stpl::URA3), and MDlBxMD14a (MATa/4MATot ade2-1/ade2-1 leu2-3,112/ leu2-3, 112 ura3/ura3 his5-2 [or hisS-419]/his5-2 [or his5-419] lys2-1/lys2-1 [or lysl-lIlysl-1 or lys2-1Ilys2-1 lysl-llysI -1] trpl/trp1 [this study]). Cells were grown in YEPD or synthetic media as described by Sherman (60).…”

“…STPI was identified by searching for wild-type genes that when overexpressed would suppress the defects caused by a mutation at the 5' splice junction of a tRNATYr (69). STPI is unessential, but a mutation of this gene causes defects in the removal of IVS from a subset of pre-tRNAs.…”

“…The STPI-dependent and STPJ-independent tRNA families have structurally distinct IVS. To explain these results, we proposed that the STPJ gene product interacts with pre-tRNAs to generate structures efficiently recognized by the splicing machinery (69). Those pre-tRNAs whose processing is independent of this gene product might utilize an analogous protein for this function or might assume an optimal structure in the absence of a protein product.…”