Nuclear mRNA surveillance in THO/sub2 mutants is triggered by inefficient polyadenylation.

The yeast THO complex and the associated RNA helicase Sub2p are important mRNP maturation factors. Transcripts produced in THO/sub2 mutants are subject to degradation by a surveillance mechanism that involves the nuclear RNA exosome. Here we show that inefficient polyadenylation forms the basis of this accelerated mRNA decay. A genetic screen reveals extensive interactions between deletions of THO subunits and mRNA 3' end processing mutants. Nuclear run-ons strengthen this link by showing premature transcription termination close to polyadenylation sites in THO/sub2 mutants in vivo. Moreover, in vitro, pre-mRNA substrates are poorly polyadenylated and consequently unstable in extracts from THO/sub2 mutant strains. Decreased polyadenylation correlates with a specific downregulation of the poly(A)-polymerase cofactor Fip1p by the ubiquitin/proteasome pathway. Both polyadenylation defects and Fip1p instability depend on the nuclear exosome component Rrp6p and its activator Trf4p. We suggest that removal of aberrant mRNA is facilitated by direct regulation of polyadenylation activity.

Formation of export-competent mRNP: escaping nuclear destruction.

In eukaryotic cells, primary transcripts are processed and bound by proteins before export to the cytoplasm. Nuclear production of export-competent messenger ribonucleoprotein particles (mRNPs) is a complicated process, and mRNP biogenic events that function sub-optimally are rapidly attacked by surveillance leading to degradation of the mRNA. Export of nuclear mRNAs is therefore constantly challenged by the opposing force of mRNA retention and decay. This balance ensures that only 'perfect' transcripts persist, and that non-functional and potentially deleterious transcripts are removed early in their biogenesis. Thus, eukaryotic systems of mRNP quality control can be viewed as simple Darwinian principles operating at the molecular level.

A link between transcription and mRNP quality in Saccharomyces cerevisiae.

The transcription machinery plays a direct role in the assembly of messenger ribonucleoprotein particles (mRNPs), contributing to the loading of proteins onto nascent transcripts. Such mRNP biogenesis is linked to the THO complex that operates at the boundary between transcription and nuclear export. Early mRNP assembly events are subject to surveillance by the nuclear exosome that retains, and degrades, aberrant mRNAs. A yeast strain that carries deletions of Hpr1p and Rrp6p of the THO complex and the nuclear exosome, respectively, grows slowly, possibly due to lack of Rrp6p-dependent mRNA quality control. We selected a number of spontaneous revertant strains from the slow growth phenotype. Interestingly, transcriptional activity was reduced in all revertants. These data corroborate earlier findings that transcriptional down regulation improves growth of cells containing a crippled mRNP formation/surveillance system and illustrates the impact transcriptional activity can have on early mRNP assembly.

Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants.

The nuclear exosome is involved in numerous RNA metabolic processes. Exosome degradation of rRNA, snoRNA, snRNA and tRNA in Saccharomyces cerevisiae is activated by TRAMP complexes, containing either the Trf4p or Trf5p poly(A) polymerase. These enzymes are presumed to facilitate exosome access by appending oligo(A)-tails onto structured substrates. Another role of the nuclear exosome is that of mRNA surveillance. In strains harboring a mutated THO/Sub2p system, involved in messenger ribonucleoprotein particle biogenesis and nuclear export, the exosome-associated 3' --> 5' exonuclease Rrp6p is required for both retention and degradation of nuclear restricted mRNAs. We show here that Trf4p, in the context of TRAMP, is an mRNA surveillance factor. However, unlike Rrp6p, Trf4p only partakes in RNA degradation and not in transcript retention. Surprisingly, a polyadenylation-defective Trf4p protein is fully active, suggesting polyadenylation-independent mRNA degradation. Transcription pulse-chase experiments show that HSP104 molecules undergoing quality control in THO/sub2 mutant strains fall into two distinct populations: One that is quickly degraded after transcription induction and another that escapes rapid decay and accumulates in foci associated with the HSP104 transcription site.

Modulation of transcription affects mRNP quality.

Cotranscriptional loading of proteins onto nascent transcripts contributes to the formation of messenger ribonucleoprotein particles (mRNPs) competent for nuclear export. The transcription machinery is believed to play a pivotal role in mRNP assembly, which is at least partially linked to the function of the THO/TREX complex and the mRNA termination/polyadenylation apparatus. Here we demonstrate a prominent role for the rate of transcription in the production of export-competent mRNPs. We show that a transcription-defective allele of the Rad3p helicase, a component of the TFIIH transcription initiation factor, suppresses several phenotypes associated with defective mRNA processing and export. Strikingly, the effects of compromised Rad3p activity can be phenocopied by a transcription elongation drug as well as by other mutations affecting transcription. Our results suggest that efficient mRNP assembly is under a kinetic control that is influenced by the rate of transcription.