A new yeast PUF family protein, Puf6p, represses ASH1 mRNA translation and is required for its localization

Abstract

In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression. The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs. To study the mechanism regulating ASH1 mRNA translation, we isolated the ASH1 locasome and characterized the associated proteins by MALDI-TOF. One of these proteins was Puf6p, a new member of the PUF family of highly conserved RNA-binding proteins such as Pumilio in Drosophila, responsible for translational repression, usually to effect asymmetric expression. Puf6p-bound PUF consensus sequences in the 3 UTR of ASH1 mRNA and repressed the translation of ASH1 mRNA both in vivo and in vitro. In the puf6⌬ strain, asymmetric localization of both Ash1p and ASH1 mRNA were significantly reduced. We propose that Puf6p is a protein that functions in the translational control of ASH1 mRNA, and this translational inhibition is necessary before localization can proceed.

Pumilio Puf domain RNA-binding proteins in Arabidopsis

“…Nucleolar localized Puf proteins have also been reported in Saccharomyces cerevisae (ScPuf6 and ScNop9), 10,11,30 and Trypanosoma brucei (TbPUF7). 29 Most of the nucleolar Puf proteins are associated with rRNA processing and ribosome biogenesis.…”

“…8,9 Similarly, yeast Puf6p represses the translation of Ash1 mRNA during localization to the distal tip of the budding cell. 10,11 Translational repression of Ash1 mRNA by Puf6p is inhibited by eIF-5B, 11 which is responsible for the conversion of 48S to the 60S ribosomal subunit. 12 This suggests that RNA transport is tightly linked to ribosome maturation and assembly.…”

Nuclear Shuttling of She2p Couples ASH1 mRNA Localization to its Translational Repression by Recruiting Loc1p and Puf6p

“…She2p also interacts with the transport machinery, constituted of the type V myosin Myo4p, via the bridging protein She3p (Bohl et al, 2000;Long et al, 2000;Takizawa and Vale, 2000). During its transport to the bud tip, the ASH1 mRNA is translationally repressed by the RNA-binding proteins Khd1p (Irie et al, 2002) and Puf6p (Gu et al, 2004), and phosphorylation of these factors at the bud tip promotes the local synthesis of the Ash1 protein (Paquin et al, 2007;Deng et al, 2008).…”

“…Puf6p and Loc1p are predominantly nucleolar RNA-binding proteins that bind directly the 3Ј untranslated region (UTR) of ASH1 mRNA in vitro and are involved in the translational repression of this transcript Gu et al, 2004;Komili et al, 2007). Knockouts of these genes disrupt ASH1 mRNA localization and Ash1p sorting to the daughter cell Gu et al, 2004). She2p shuttles between the cytoplasm and the nucleus (Kruse et al, 2002), and recent evidence suggests that the nuclear transit of She2p is involved in the translational regulation of ASH1 mRNA (Du et al, 2008).…”

“…Puf6p and Loc1p are predominantly nucleolar RNA-binding proteins that bind directly the 3Ј untranslated region (UTR) of ASH1 mRNA in vitro and are involved in the translational repression of this transcript Gu et al, 2004;Komili et al, 2007). Knockouts of these genes disrupt ASH1 mRNA localization and Ash1p sorting to the daughter cell Gu et al, 2004).…”