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 tRNATYr. 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, STPI, 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 STPI rtesulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stpl-disruption. At least five of the nine families of pre-tRNAs were affected.Two other species, pre-tRNA"' and pre-tRNALj", were not. We propose that STPI encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STPI product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.In eucaryotes, tRNA species originate as precursor transcripts that contain extra nucleotides at both the 5' and 3' termini. These tRNA precursors may also contain intervening sequences (IVS). The removal of these extra sequences from tRNA precursors is a multistep process that occurs in the nucleus. The processing of 5' and 3' ends is thought to precede splicing of the IVS (7,14,25). In the yeast Saccharomyces cerevisiae, nine families of tRNA have been found to be derived from intron-containing tRNA precursors (27). The rnal-I mutant accumulates IVS-containing precursor tRNA molecules at the restrictive temperature (15,19) and has considerably facilitated the study of the pre-tRNA splicing reaction (19,29). This reaction is separable into two distinct steps: (i) an endonucleolytic step that excises the IVS and produces half-size tRNA molecules bearing 5' hydroxyl and 2',3'-cyclic phosphodiester termini, and (ii) an ATP-requiring ligation step that joins the tRNA halves and generates mature tRNA sequences. The endonuclease has been partially purified. It behaves as an integral membrane protein and has similar specific activities for all nine tRNA precursors that contain IVS (28). The ligase is a soluble single polypeptide located at the inner surface of the nuclear membrane (4). Three activities are associated with the ligation reaction: polynucleotide kinase, cyclic phosphodiesterase, and RNA ligase. All three of these activities have been shown to copurify (10, 30). The gene encoding ligase has been cloned (30). New evidence has suggested that the yeast endonuclease and ligase form a tRNA splicing complex in vitro (9).In addition to the rnal-I mutation, which defines the RNA] gene, temperature-sensitive and deletion mutations of the LOS] locus that affect pre-tRNA splicing have been identifie...

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.

STP1 is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STP1 might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STP1 gene and its products. The STP1 locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STP1 open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STP1 ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STP1 ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing.