Nuclear precursor of mature messenger RNA (pre-mRNA) splicing is one of the most highly regulated processes in eukaryotic organisms. In addition to its role in the removal of constitutive or alternative introns present in the pre-mRNA, splicing is also highly integrated into other layers of gene expression. This study investigates the potential role of the nuclear branchpoint binding protein (BBP) outside of the pre-mRNA splicing cycle. More specifically, we were interested in the biological relevance of its association with two cytoplasmic proteins Smy2 and Syh1. Smy2 and Syh1 belong to the GYF family of poly-proline binding proteins, and their roles in cell biology have not been well elucidated. Here we report that Smy2 and Syh1 act redundantly in: (i) limiting pre-mRNA accumulation when yeast cultures reach high cell density, potentially through promoting pre-mRNA decay in the cytoplasm; (ii) restricting Ty1 retrotransposition, apparently by limiting the Ty1 transcript abundance; (iii) limiting the accumulation of BBP-associated yet intronless TDA1 mRNA. With the presence of UACUAAC motif and BBP association as common features of these Smy2/Syh1 sensitive substrates, we tested if BBP interaction is required for Smy2/Syh1 function in RNA metabolism. Interestingly, we found that deletion of BBP C-terminal region (bbp∆C), which largely reduces or abolishes its association with Smy2, does not lead to similar phenotypes as observed in smy2∆ syh1∆ deletion mutant cells. In addition, mutagenesis of the TACTAAC BBP-binding site within the TDA1 coding region does not seem to affect TDA1 mRNA abundance or its sensitivity to the smy2∆ syh1∆ deletions. Therefore, we concluded that while the two BBP-binding proteins Smy2 and Syh1 impact the levels of certain cellular RNAs, this phenomenon is not strictly dependent upon BBP-Smy2 interaction and may be independent of BBP contribution. A model is proposed for Smy2 and Syh1 function in RNA metabolism based on our observations and interactions between these proteins with other factors implicated in RNA stability or translation.

<p>Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from <i>Saccharomyces cerevisiae</i>, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner.</p><p>Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants.</p><p>We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.</p>

Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from Saccharomyces cerevisiae, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner. Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants. We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.