A novel connection between [PSI+] prion formation and RNA splicing in S. cerevisiae

Abstract

Relatively new studies on prions in Saccharomyces cerevisiae show that some protein prion conformations may be beneficial to cell survival in response to environmental stress. Sup35, a translation termination release factor, forms the prion [PSI+] . When [PSI+] forms, translation termination efficiency decreases, which has been shown to result in the alteration of the abundance of cellular proteins, including splicing factor abundance. Therefore, we are testing the hypothesis that [PSI+] prion formation alters splicing efficiency in yeast. First, we employed quantitative RT-PCR and revealed that splicing efficiency improves upon [PSI+] prion formation. The proposed mechanisms for the effect of [PSI+] on protein abundance include nonsense suppression and non-stop decay. Non-stop decay would lead to degradation of the mRNA encoding a splicing protein and therefore would reduce splicing factor protein levels. Nonsense suppression, a result of translation readthrough, generates a C-terminally extended protein, which could alter the function of splicing factors, or alter protein levels. We are initiating RT-qPCR and Western Blot analyses to test the contribution of the two mechanisms on splicing factor abundance. To further test the relationship between RNA splicing and [PSI+] prion formation, we used knockout PCR to create splicing factor deletion mutations in a [psi-] strain, as well as a weak [PSI+]WK strain or a strong [PSI+]STR strain. We found that the [PSI+]WK conformation can suppress the growth defect in a yeast strain with a deletion of the gene encoding SNU66, a component of the tri-snRNP. Similarly, the [PSI+]WK conformation can suppress the growth defect in a yeast strain with a deletion of the MUD2 gene, which encodes a gene important for commitment complex formation. In addition, we found that [PSI+]WK can rescue the splicing defect observed in a mud2Δ strain, consistent with the suppressive growth phenotype. Together, the results show that [PSI+] formation impacts RNA splicing in S. cerevisiae and our future work will focus on determining the mechanisms that underlie this effect.