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Abstract:
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 RNAbinding
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.
