The yeast splicing factor Prp40p contains functional leucine-rich nuclear export signals that are essential for splicing.

To investigate the function of the essential U1 snRNP protein Prp40p, we performed a synthetic lethal screen in Saccharomyces cerevisiae. Using an allele of PRP40 that deletes 47 internal residues and causes only a slight growth defect, we identified aphenotypic mutations in three distinct complementation groups that conferred synthetic lethality. The synthetic phenotypes caused by these mutations were suppressed by wild-type copies of CRM1 (XPO1), YNL187w, and SME1, respectively. The strains whose synthetic phenotypes were suppressed by CRM1 contained no mutations in the CRM1 coding sequence or promoter. This indicates that overexpression of CRM1 confers dosage suppression of the synthetic lethality. Interestingly, PRP40 and YNL187w encode proteins with putative leucine-rich nuclear export signal (NES) sequences that fit the consensus sequence recognized by Crm1p. One of Prp40p's two NESs lies within the internal deletion. We demonstrate here that the NES sequences of Prp40p are functional for nuclear export in a leptomycin B-sensitive manner. Furthermore, mutation of these NES sequences confers temperature-sensitive growth and a pre-mRNA splicing defect. Although we do not expect that yeast snRNPs undergo compartmentalized biogenesis like their metazoan counterparts, our results suggest that Prp40p and Ynl187wp contain redundant NESs that aid in an important, Crm1p-mediated nuclear export event.

Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain.

Aminoacyl-tRNA synthetases are multidomain proteins responsible for the attachment of specific amino acids to their tRNA substrates. Prolyl-tRNA synthetases (ProRSs) are notable due to their particularly diverse architectures through evolution. For example, Saccharomyces cerevisiae ProRS possesses an N-terminal extension with weak homology to a bacterial-specific domain typically present as an insertion (INS) within the aminoacylation active site. The INS domain has been shown to contain a "posttransfer" editing active site responsible for cleaving the aminoacyl-ester bond of misacylated Ala-tRNA(Pro) species. However, wild-type S. cerevisiae ProRS does not perform posttransfer editing in vitro. Here, we show that replacement of the N-terminal domain of S. cerevisiae ProRS with the Escherichia coli INS domain confers posttransfer editing function to this chimeric enzyme, with specificity for yeast Ala-tRNA(Pro). In contrast, the isolated INS domain displays only weak editing activity and lacks tRNA sequence specificity. These results emphasize the modular nature of synthetase editing active sites and demonstrate how in evolution, a weak editing activity can be converted to a more robust state through fusion to the body of a synthetase. In this manner, a single editing module can be distributed to different synthetases, and simultaneously acquire specificity and enhanced activity.

Pre-transfer editing by class II prolyl-tRNA synthetase: role of aminoacylation active site in "selective release" of noncognate amino acids.

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids to specific tRNA molecules. To prevent potential errors in protein synthesis caused by misactivation of noncognate amino acids, some synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). In the case of post-transfer editing, synthetases employ a separate editing domain that is distinct from the site of amino acid activation, and the mechanism is believed to involve shuttling of the flexible CCA-3' end of the tRNA from the synthetic active site to the site of hydrolysis. The mechanism of pre-transfer editing is less well understood, and in most cases, the exact site of pre-transfer editing has not been conclusively identified. Here, we probe the pre-transfer editing activity of class II prolyl-tRNA synthetases from five species representing all three kingdoms of life. To locate the site of pre-transfer editing, truncation mutants were constructed by deleting the insertion domain characteristic of bacterial prolyl-tRNA synthetase species, which is the site of post-transfer editing, or the N- or C-terminal extension domains of eukaryotic and archaeal enzymes. In addition, the pre-transfer editing mechanism of Escherichia coli prolyl-tRNA synthetase was probed in detail. These studies show that a separate editing domain is not required for pre-transfer editing by prolyl-tRNA synthetase. The aminoacylation active site plays a significant role in preserving the fidelity of translation by acting as a filter that selectively releases non-cognate adenylates into solution, while protecting the cognate adenylate from hydrolysis.

A diverse set of nuclear RNAs transfer between nuclei of yeast heterokaryons.

Small nuclear RNAs and small nucleolar RNAs function in the nucleus of eukaryotic cells during pre-mRNA splicing and ribosomal RNA processing, respectively. In metazoan cells, the small nuclear RNAs shuttle between the nucleus and the cytoplasm during ribonucleoprotein particle assembly. Nuclear export of these small RNAs in yeast, however, has not been demonstrated. Therefore, we have attempted to visualize internuclear RNA movements by in situ hybridization in heterokaryon yeast cells. Using the kar1Delta15 mutation to block karyogamy, we mated two strains, each expressing a unique allele of U1 snRNA. In these heterokaryons, we observed a time-dependent transfer of U1 snRNA from one nucleus to the other. This transfer was reduced two-fold by the addition of the Crm1p-inhibitor leptomycin B. Interestingly, however, we observed identical transfer of the U2 and U6 snRNAs and SNR4, SNR8, SNR9 and SNR11 snoRNAs. Remarkably, when the U2, U6 or SNR4 RNAs were observed in the same heterokaryon as the U1 snRNA, both RNAs always transferred simultaneously. These data suggest a global leaking or transport of material between nuclei of yeast heterokaryons. Our results suggest that caution must be taken when testing nuclear envelope shuttling in yeast heterokaryons.