A mechanism for co-transcriptional recruitment of mRNA localization factor on nascent mRNAs in budding yeast

Cytoplasmic transport and localization of messenger RNAs allows temporal and spatial expression of specific factors involved in cell fate determination, synaptic plasticity, cellular polarity or asymmetric cell division. In S. cerevisiae, over thirty transcripts are actively transported and localized to the bud tip of budding yeast.  One of them, the ASH1 mRNA (for Asymmetric Synthesis of HO), is localized at the bud tip in late anaphase cells.  This allows Ash1p, a transcriptional repressor of the HO endonuclease, to be sorted exclusively to the daughter cell nucleus, where it prevents mating type switching.  Proper ASH1 mRNA localization and Ash1p asymmetric expression involve localization factors, which are part of the She-proteins (She1p/Myo4p, She2p and She3p), and translational repressors (the proteins Puf6, Loc1 and Khd1).  The nucleo-cytoplasmic shuttling protein She2p binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the She3p-Myo4p complex.  Translational repressors regulate the translation of ASH1 mRNA and avoid ectopic expression of the Ash1 protein during the transport of its transcript. While the cytoplasmic role of She2p in mRNA localization is known, its nuclear function is still unclear.   We now show that She2p contains a non-classical nuclear localization signal sequence (NLS) which is essential for its nuclear import via the importin  Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS disrupt the binding of Loc1p and Puf6p to the ASH1 mRNA, leading to defective mRNA localization and Ash1p sorting. To further investigate the assembly of the mRNA localization machinery in the nucleus, we used chromatin immunoprecipitation (ChIP) to follow the recruitment of localization factors and translational repressors on nascent localized mRNAs.  We found that She2p is recruited on the ASH1 gene during transcription, via its interaction with the nascent ASH1 mRNA. Puf6p is also recruited on the ASH1 gene, but in a She2p-dependent manner.  Interestingly, we detected an interaction between She2p and Rpb1p, the largest subunit of RNA polymerase II in vivo.  This interaction is independent of the RNA-binding properties of She2p, and involves the elongating form of the RNA polymerase II. We also found that She2p interacts with both members of the elongation factors Spt4p /Spt5p; Deletion of SPT4 or Ts spt5 mutants at restrictive temperature disrupted the interaction between She2p and Rpb1p, and then reduced the recruitment of She2p on the ASH1 gene, resulting in ASH1 mRNA delocalization and defective Ash1p sorting.  Altogether, our results show that factors involved in cytoplasmic mRNA localization and translational control are recruited co-transcriptionally on nascent mRNAs via interation with the transcription machinery, pointing toward a role of the transcription machinery in the mRNA localization process.

Keyphrases: She2p, recrutement co-transcriptionnel, complexe Spt4-Spt5p, mRNA localization, ASH1 mRNA, nuclear localization signal (NLS), She2p, Co-transcriptional recruitment, RNA polymerase II, Spt4-Spt5 complex, yeast