Cloning and Characterization of LOS1, a Saccharomyces cerevisiae Gene That Affects tRNA Splicing

Abstract

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.

Three tRNA nuclear exporters in S. cerevisiae: parallel pathways, preferences, and precision

“…However, there is considerable evidence that there are additional tRNA nuclear exporters in most eukaryotes. First, in all tested organisms, including haploid human cancer cells, LOS1 and its homologs are unessential ( 21 , 27–31 ). Second, Drosophila melanogaster lacks a Los1 homologue ( 32 ).…”

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

“…Diploid yeast strains were selected either by prototroph selection or by physical isolation with a micromanipulator. Diploids were sporulated on medium as previously described (17).…”

“…Gamma-delta (-y) mutagenesis was performed by a modification of the procedure of Guyer (11) as described by Hurt et al (17). Plasmids were extracted and restriction mapped to determine the position of the transposon.…”

“…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 identified (16,17). At the nonpermissive temperature, both the rnal-I and losi cells accumulate tRNA precursors that have mature termini but contain IVS.…”

“…This suggests that the RNA] product may have an indirect effect on pre-tRNA splicing by modifying the RNA processing activities or affecting nuclear structure. The LOS] gene may encode a product in an alternative tRNA splicing pathway or may enhance interactions between the pre-tRNA and splicing enzymes (17). On the other hand, the LOS] product, like the RNA] product, may affect pre-tRNA splicing in an indirect manner.…”