A La protein requirement for efficient pre-tRNA folding

The La protein protects the 3' ends of many nascent small RNAs from exonucleases. Here we report that La is required for efficient folding of certain pre-tRNAs. A mutation in pre-tRNA(Arg)(CCG) causes yeast cells to be cold-sensitive and to require the La protein Lhp1p for efficient growth. When the mutant cells are grown at low temperature, or when Lhp1p is depleted, mature tRNA(Arg)(CCG) is not efficiently aminoacylated. The mutation causes the anticodon stem of pre-tRNA(Arg)(CCG) to misfold into an alternative helix in vitro. Intragenic suppressor mutations that disrupt the misfolded helix or strengthen the correct helix alleviate the requirement for Lhp1p, providing evidence that the anticodon stem misfolds in vivo. Chemical and enzymatic footprinting experiments suggest a model in which Lhp1p stabilizes the correctly folded stem. Lhp1p is also required for efficient aminoacylation of two wild-type tRNAs when yeast are grown at low temperature. These experiments reveal that pre-tRNAs can require protein assistance for efficient folding in vivo.     

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

Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 3' ends. The La protein binds the 3' ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4Delta cells requires Rex1p, these pre-tRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Delta cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4Delta cells. Our experiments reveal that one consequence of Rex1p-dependent 3' trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP.     

A Nuclear Import Pathway for a Protein Involved in tRNA Maturation

A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing.     

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

Previous analyses of eukaryotic pre-tRNAs processing have reported that 5'-cleavage by RNase P precedes 3'-maturation. Here we report that in contrast to all other yeast tRNAs analyzed to date, tRNA(Trp) undergoes 3'-maturation prior to 5'-cleavage. Despite its unusual processing pathway, pre-tRNA(Trp) resembles other pre-tRNAs, showing dependence on the essential Lsm proteins for normal processing and efficient association with the yeast La homolog, Lhp1p. tRNA(Trp) is also unusual in not requiring Lhp1p for 3' processing and stability. However, other Lhp1p-independent tRNAs, tRNA(2)(Lys) and tRNA(1)(Ile), follow the normal pathway of 5'-processing prior to 3-processing.     

Multiple functional interactions between components of the Lsm2-Lsm8 complex, U6 snRNA, and the yeast La protein

The U6 small nuclear ribonucleoprotein is a critical component of the eukaryotic spliceosome. The first protein that binds the U6 snRNA is the La protein, an abundant phosphoprotein that binds the 3' end of many nascent small RNAs. A complex of seven Sm-like proteins, Lsm2-Lsm8, also binds the 3' end of U6 snRNA. A mutation within the Sm motif of Lsm8p causes Saccharomyces cerevisiae cells to require the La protein Lhp1p to stabilize nascent U6 snRNA. Here we describe functional interactions between Lhp1p, the Lsm proteins, and U6 snRNA. LSM2 and LSM4, but not other LSM genes, act as allele-specific, low-copy suppressors of mutations in Lsm8p. Overexpression of LSM2 in the lsm8 mutant strain increases the levels of both Lsm8p and U6 snRNPs. In the presence of extra U6 snRNA genes, LSM8 becomes dispensable for growth, suggesting that the only essential function of LSM8 is in U6 RNA biogenesis or function. Furthermore, deletions of LSM5, LSM6, or LSM7 cause LHP1 to become required for growth. Our experiments are consistent with a model in which Lsm2p and Lsm4p contact Lsm8p in the Lsm2-Lsm8 ring and suggest that Lhp1p acts redundantly with the entire Lsm2-Lsm8 complex to stabilize nascent U6 snRNA.     

The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability

Although the La protein stabilizes nascent pre-tRNAs from nucleases, influences the pathway of pre-tRNA maturation, and assists correct folding of certain pre-tRNAs, it is dispensable for growth in both budding and fission yeast. Here we show that the Saccharomyces cerevisiae La shares functional redundancy with both tRNA modification enzymes and other proteins that contact tRNAs during their biogenesis. La is important for growth in the presence of mutations in either the arginyl tRNA synthetase or the tRNA modification enzyme Trm1p. In addition, two pseudouridine synthases, PUS3 and PUS4, are important for growth in strains carrying a mutation in tRNA(Arg)(CCG) and are essential when La is deleted in these strains. Depletion of Pus3p results in accumulation of the aminoacylated mutant tRNA(Arg)(CCG) in nuclei, while depletion of Pus4p results in decreased stability of the mutant tRNA. Interestingly, the degradation of mutant unstable forms of tRNA(Arg)(CCG) does not require the Trf4p poly(A) polymerase, suggesting that yeast cells possess multiple pathways for tRNA decay. These data demonstrate that La functions redundantly with both tRNA modifications and proteins that associate with tRNAs to achieve tRNA structural stability and efficient biogenesis.     

Precursors to the U3 small nucleolar RNA lack small nucleolar RNP proteins but are stabilized by La binding

Almost all small eukaryotic RNAs are processed from transiently stabilized 3'-extended forms. A key question is how and why such intermediates are stabilized and how they can then be processed to the mature RNA. Here we report that yeast U3 is also processed from a 3'-extended precursor. The major 3'-extended forms of U3 (U3-3'I and -II) lack the cap trimethylation present in mature U3 and are not associated with small nucleolar RNP (snoRNP) proteins that bind mature U3, i.e., Nop1p, Nop56p, and Nop58p. Depletion of Nop58p leads to the loss of mature U3 but increases the level of U3-3'I and -II, indicating a requirement for the snoRNP proteins for final maturation. Pre-U3 is cleaved by the endonuclease Rnt1p, but U3-3'I and -II do not extend to the Rnt1p cleavage sites. Rather, they terminate at poly(U) tracts, suggesting that they might be bound by Lhp1p (the yeast homologue of La). Immunoprecipitation of Lhp1p fused to Staphylococcus aureus protein A resulted in coprecipitation of both U3-3'I and -II. Deletion of LHP1, which is nonessential, led to the loss of U3-3'I and -II. We conclude that pre-U3 is cleaved by Rnt1p, followed by exonuclease digestion to U3-3'I and -II. These species are stabilized against continued degradation by binding of Lhp1p. Displacement of Lhp1p by binding of the snoRNP proteins allows final maturation, which involves the exosome complex of 3'-->5' exonucleases.     

Control of transfer RNA maturation by phosphorylation of the human La antigen on serine 366

Conversion of a nascent precursor tRNA to a mature functional species is a multipartite process that involves the sequential actions of several processing and modifying enzymes. La is the first protein to interact with pre-tRNAs in eukaryotes. An opal suppressor tRNA served as a functional probe to examine the activities of yeast and human (h)La proteins in this process in fission yeast. An RNA recognition motif and Walker motif in the metazoan-specific C-terminal domain (CTD) of hLa maintain pre-tRNA in an unprocessed state by blocking the 5'-processing site, impeding an early step in the pathway. Faithful phosphorylation of hLa on serine 366 reverses this block and promotes tRNA maturation. The results suggest that regulation of tRNA maturation at the level of RNase P cleavage may occur via phosphorylation of serine 366 of hLa.     

The fission yeast Schizosaccharomyces pombe has two distinct tRNase Z(L)s encoded by two different genes and differentially targeted to the nucleus and mitochondria

tRNase Z is the endonuclease that is involved in tRNA 3'-end maturation by removal of the 3'-trailer sequences from tRNA precursors. Most eukaryotes examined to date, including the budding yeast Saccharomyces cerevisiae and humans, have a single long form of tRNase Z (tRNase ZL). In contrast, the fission yeast Schizosaccharomyces pombe contains two candidate tRNase ZLs encoded by the essential genes sptrz1+ and sptrz2+. In the present study, we have expressed recombinant SpTrz1p and SpTrz2p in S. pombe. Both recombinant proteins possess precursor tRNA 3'-endonucleolytic activity in vitro. SpTrz1p localizes to the nucleus and has a simian virus 40 NLS (nuclear localization signal)-like NLS at its N-terminus, which contains four consecutive arginine and lysine residues between residues 208 and 211 that are critical for the NLS function. In contrast, SpTrz2p is a mitochondrial protein with an N-terminal MTS (mitochondrial-targeting signal). High-level overexpression of sptrz1+ has no detectable phenotypes. In contrast, strong overexpression of sptrz2+ is lethal in wild-type cells and results in morphological abnormalities, including swollen and round cells, demonstrating that the correct expression level of sptrz2+ is critical. The present study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein.