Abstract: The yeast [PSI+] prion is an epigenetic modifier of translation termination fidelity that causes nonsense suppression. The prion [PSI+] forms when the translation termination factor Sup35p adopts a self-propagating conformation. The presence of the [PSI+] prion modulates survivability in a variety of growth conditions. Nonsense suppression is essential for many [PSI+]-mediated phenotypes, but many do not appear to be due to read-through of a single stop codon, but instead are multigenic traits. We hypothesized that other global mechanisms act in concert with [PSI+] to influence [PSI+]-mediated phenotypes. We have identified one such global regulator, the Paf1 complex (Paf1C). Paf1C is conserved in eukaryotes and has been implicated in several aspects of transcriptional and posttranscriptional regulation. Mutations in Ctr9p and other Paf1C components reduced [PSI+]-mediated nonsense suppression. The CTR9 deletion also alters nonsense suppression afforded by other genetic mutations but not always to the same extent as the effects on [PSI+]-mediated read-through. Our data suggest that the Paf1 complex influences mRNA translatability but not solely through changes in transcript stability or abundance. Finally, we demonstrate that the CTR9 deletion alters several [PSI+]-dependent phenotypes. This provides one example of how [PSI+] and genetic modifiers can interact to uncover and regulate phenotypic variability.

Abstract: The ciliate Tetrahymena thermophila, having both germ line micronuclei and somatic macronuclei, must possess a specialized nucleocytoplasmic transport system to import proteins into the correct nucleus. To understand how Tetrahymena can target proteins to distinct nuclei, we first characterized FG repeat-containing nucleoporins and found that micro- and macronuclei utilize unique subsets of these proteins. This finding implicates these proteins in the differential permeability of the two nuclei and implies that nuclear pores with discrete specificities are assembled within a single cell. To identify the import machineries that interact with these different pores, we characterized the large families of karyopherin homologs encoded within the genome. Localization studies of 13 putative importin (imp) alpha- and 11 imp beta-like proteins revealed that imp alpha-like proteins are nucleus specific--nine localized to the germ line micronucleus--but that most imp beta-like proteins localized to both types of nuclei. These data suggest that micronucleus-specific proteins are transported by specific imp alpha adapters. The different imp alpha proteins exhibit substantial sequence divergence and do not appear to be simply redundant in function. Disruption of the IMA10 gene encoding an imp alpha-like protein that accumulates in dividing micronuclei results in nuclear division defects and lethality. Thus, nucleus-specific protein import and nuclear function in Tetrahymena are regulated by diverse, specialized karyopherins.