Export of discarded splicing intermediates requires mRNA export factors and the nuclear basket

Author:

Zeng Yi12ORCID,Staley Jonathan P.1

Affiliation:

1. Department of Molecular Genetics and Cell Biology, University of Chicago

2. Department of Genetics, Stanford University

Abstract

To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal splicing substrates after they have engaged the spliceosome. Although nuclear quality control mechanisms have been proposed to retain immature mRNPs, evidence indicates that discarded splicing substrates, including lariat intermediates, do export to the cytoplasm, as indicated by their translation and degradation by cytoplasmic nucleases. However, the mechanism for exporting these species has remained unknown. By single molecule (sm) RNA FISH in budding yeast, we have directly observed the nuclear export of lariat intermediates. Further, by crosslinking, export reporter assays, and smRNA FISH, we have demonstrated that the export of lariat intermediates requires the general mRNA export receptor Mex67p and three of its mRNA export adapter proteins, Nab2p, Yra1p, and Nlp3, establishing that mRNAs and lariat intermediates share the same export machinery. Unexpectedly, the export of lariat intermediates, but not mRNA, requires an interaction between Nab2p and Mlp1p, a nuclear basket component implicated in retaining immature mRNPs, including unspliced pre-mRNA, in the nucleus of budding yeast. Finally, the export of lariat intermediates, like mRNA, relies on the E3 ubiquitin ligase Tom1p and its target sites in Yra1p. Overall, our data indicate that the nuclear basket can promote, rather than antagonize, the export of an immature mRNP. Further, our data imply that the export of discarded lariat intermediates requires both Mlp1p-dependent docking onto the nuclear basket and subsequent Tom1p-mediated undocking, a mechanism our data suggests functions in the export of mRNA also but in a manner obscured by redundant pathways.

Publisher

eLife Sciences Publications, Ltd

Reference70 articles.

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