Many proteins interact with a newly synthesized RNA to mediate biogenesis and maturation. These temporally and spatially regulated interactions facilitate individual processing events and direct each RNA to a specific fate (e.g. nuclear export or decay). To further characterize mRNA biogenesis and export, we performed a fluorescent in situ hybridization (FISH) screen of >1000 essential gene mutants in Saccharomyces cerevisiae that resulted in the identification of 29 mutants that exhibited poly(A)-RNA accumulation in the nucleus. Gene ontology places the majority of the genes into three biological processes: RNA export from nucleus, nuclear RNA catabolic processes, and chromosome segregation.
It is known that defects in the processing of various non-coding RNAs (e.g. rRNA, snRNA, snoRNA, and tRNA) can lead to the stabilization of polyadenylated RNA species; therefore, mutants may accumulate poly(A)-RNA due to disruptions in RNA biogenesis that are independent of mRNA. To identify mutants that specifically alter mRNA biogenesis and export, single-molecule FISH (smFISH) assays were performed on all 29 mutants using probes against three different mRNAs (e.g. GFA1, ACT1 and CCW12). Based on transcript localization and abundance, we identified defects in mRNA processing and export in 26 of 29 mutants.
By employing smFISH and markers for various subdomains of the nucleus (e.g. Ndc1-GFP for the nuclear envelope, ITS1 for the nucleolus, and a LacO/LacI-GFP marked gene locus), we could further subdivide mutants into three classes based on the accumulation of poly(A)-RNA and mRNA within discrete regions of the nucleus. This included: (1) mRNAs and mRNA binding proteins becoming enriched in the nucleolus when nucleocytopalsmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, (2) the buildup of mRNAs near transcription sites in 3’ end processing and chromosome segregation mutants and (3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data demonstrate that alterations in a broad set of nuclear processes leads to the accumulation and possible retention of mRNAs at discrete locations within the nucleus. This may reflect common failure points in mRNA export and/or active mechanisms to protect the cell against cellular stress and dysfunction.