Dataset Information

Phosphorylation controls the oligomeric state of She2 and mRNA localization in yeast.

ABSTRACT: Messenger RNA (mRNA) localization is an important mechanism controlling local protein synthesis. In budding yeast, asymmetric localization of transcripts such as ASH1 mRNA to the bud tip depends on the She2 RNA-binding protein. She2 assembles as a tetramer to bind RNA, but the regulation of this process as part of the mRNA locasome is still unclear. Here, we performed a phosphoproteomic analysis of She2 in vivo and identified new phosphosites, several of which are located at the dimerization or tetramerization interfaces of She2. Remarkably, phosphomimetic mutations at these residues disrupt the capacity of She2 to promote Ash1 asymmetric accumulation. A detailed analysis of one of these residues, T109, shows that a T109D mutation inhibits She2 oligomerization and its interaction with She3 and the importin-α Srp1. She2 proteins harboring the T109D mutation also display reduced expression. More importantly, this phosphomimetic mutation strongly impairs the capacity of She2 to bind RNA and disrupts ASH1 mRNA localization. These results demonstrate that the control of She2 oligomerization by phosphorylation constitutes an important regulatory step in the mRNA localization pathway.

SUBMITTER: Farajzadeh N

PROVIDER: S-EPMC10187671 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Phosphorylation controls the oligomeric state of She2 and mRNA localization in yeast.

Farajzadeh Nastaran N Shahbabian Karen K Bouaziz Yani Y Querido Emmanuelle E Chartrand Pascal P

RNA (New York, N.Y.) 20230315 6

Messenger RNA (mRNA) localization is an important mechanism controlling local protein synthesis. In budding yeast, asymmetric localization of transcripts such as <i>ASH1</i> mRNA to the bud tip depends on the She2 RNA-binding protein. She2 assembles as a tetramer to bind RNA, but the regulation of this process as part of the mRNA locasome is still unclear. Here, we performed a phosphoproteomic analysis of She2 in vivo and identified new phosphosites, several of which are located at the dimerizat ...[more]

PMID: 36921931

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Project description:In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro-interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.

Dataset Information

Phosphorylation controls the oligomeric state of She2 and mRNA localization in yeast.

Publications

Phosphorylation controls the oligomeric state of She2 and mRNA localization in yeast.

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