Nuclear transit of the RNA‐binding protein She2 is required for translational control of localized ASH1 mRNA

Abstract

Cytoplasmic localization and localized translation of messenger RNAs contribute to asymmetrical protein distribution. Recognition of localized mRNAs by RNA-binding proteins can occur in the cytoplasm or, alternatively, co-or post-transcriptionally in the nucleus. In budding yeast, mRNAs destined for localization are bound by the She2 protein before their nuclear export. Here, we show that a specific transcript, known as ASH1 mRNA, and She2 localize specifically to the nucleolus when their nuclear export is blocked. Nucleolar She2 localization is enhanced in a She2 mutant that cannot bind to RNA. A fusion protein of the amino terminus of She3 and She2 (She3N-She2) fails to enter the nucleus, but does not impair ASH1 mRNA localization. Instead, these cells fail to distribute Ash1 protein asymmetrically, which is caused by a defective translational control of ASH1 mRNA. Our results indicate that the nucleolar transit of RNA-binding proteins such as She2 is necessary for the correct assembly of translationally silenced localizing messenger ribonucleoproteins.

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

“…A role of nuclear She2p in translational control is indeed supported by recent data from the Jansen lab, which showed that nuclear exclusion of She2p accelerates Ash1p synthesis (Du et al, 2008). Intriguingly, Du et al did not report any defect in ASH1 mRNA localization when She2p was excluded from the nucleus, unlike what we observed (see Figure 7).…”

“…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). However, the specific role of She2p in the nucleus is still unclear.…”

“…Interacts Directly with the Importin ␣ Srp1p in Order to Enter the Nucleus Previous work has shown that She2p transits through the nucleus (Kruse et al, 2002;Du et al, 2008). Because of its size (26 kDa), which is below the nuclear pore diffusion limit (40 kDa in yeast), a She2p monomer may enter passively through the nuclear pores.…”

“…One possibility is that, being in the nucleolus, Puf6p and Loc1p are spatially restricted from polyA ϩ mRNAs. Because She2p has been recently shown to transit through the nucleolus (Du et al, 2008), it may either bring the ASH1 mRNA in the nucleolus, where Puf6p and Loc1p can bind this transcript, or She2p may recruit these two factors in the nucleolus and bring them to the ASH1 mRNA in the nucleoplasm.…”

The cytoplasmic fate of an mRNP is determined cotranscriptionally: exception or rule?: Figure 1.

“…Other components, like She1p-5p, are essential for proper localization of ASH1 mRNA and Ash1p sorting (Long et al 1997). Interestingly, when the She2p NLS is mutated so that it can no longer enter the nucleus, it can still bind to the target mRNA on its own in the cytoplasm (Du et al 2008), but with a diffuse RNA phenotype rather than bud tip-anchored mRNA localization (Shen et al 2009). Furthermore, when the N terminus of She3p, an adapter of She2 interaction with Myo4p, was fused with She2p to prevent its nuclear import, ASH1 mRNAs could localize to the bud tip as in wild-type cells, but, remarkably, failed to redistribute Ash1p asymmetrically (Du et al 2008).…”