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.

SCFCdc4 acts antagonistically to the PGC-1alpha transcriptional coactivator by targeting it for ubiquitin-mediated proteolysis.

Peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) is a highly regulated transcriptional coactivator that coordinates energy metabolism in mammals. Misregulation of PGC-1alpha has been implicated in the pathogenesis of several human diseases, including diabetes, obesity, and neurological disorders. We identified SCF(Cdc4) as an E3 ubiquitin ligase that regulates PGC-1alpha through ubiquitin-mediated proteolysis. PGC-1alpha contains two Cdc4 phosphodegrons that bind Cdc4 when phosphorylated by Glycogen Synthase Kinase 3beta (GSK3beta) and p38 MAPK, leading to SCF(Cdc4)-dependent ubiquitylation and proteasomal degradation of PGC-1alpha. Furthermore, SCF(Cdc4) negatively regulates PGC-1alpha-dependent transcription. We demonstrate that RNAi-mediated reduction of Cdc4 in primary neurons results in an increase of endogenous PGC-1alpha protein, while ectopic expression of Cdc4 leads to a reduction of endogenous PGC-1alpha protein. Finally, under conditions of oxidative stress in neurons, Cdc4 levels are decreased, leading to an increase in PGC-1alpha protein and PGC-1alpha-dependent transcription. These results suggest that attenuation of SCF(Cdc4)-dependent proteasomal degradation of PGC-1alpha has a role in mediating the PGC-1alpha-dependent transcriptional response to oxidative stress.

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.