Eukaryotic cells have evolved an mRNP quality control system by which aberrant transcripts with processing and assembly defects are recognized as export-incompetent triggering their degradation by the nuclear exosome. Previous studies have suggested the interconnection of this surveillance process with transcription elongation and mRNP biogenesis. However, the molecular mechanisms by which a surveillance apparatus recognizes aberrancies at each step of mRNP formation and targets the defective molecules for destruction remained unknown. To address this question, we took advantage of a new assay in which the heterologous expression of a bacterial RNA-dependent helicase/translocase (transcription termination factor Rho) in yeast induces the production of full-length yet aberrant transcripts that are targeted and degraded by the nuclear exosome, causing a growth defect phenotype (44
). In vitro
, Rho functions as a powerful molecular motor that tracks along the RNA chain and can melt nucleic acid base pairs or disrupt RNA–protein complexes present on its path (49–51
). We have postulated that Rho loading and subsequent translocation along nascent transcripts in yeast cells could antagonize the normal deposition of processing and packaging factors, yielding stripped mRNPs that are recognized as defective. This assumption was supported by the identification of several mRNP processing and packaging factors in a screen for dosage suppressors of the Rho-induced defect, suggesting that an overload of these RNA-interacting proteins can counteract the denuding effect of the bacterial helicase or neutralize the detection of the aberrant transcripts by the surveillance mechanisms. Additional support to our assumption is provided by a work to be published elsewhere in which decreased occupancy of some mRNP assembly proteins upon Rho action was revealed by ChIP experiments (R. H., C. M., N. C. and A. R. R., manuscript in preparation).
Among the dosage suppressors found in the Rho-based screen, Pcf11 is an essential factor for transcription termination of poly(A)-containing RNAs which is known to interact with the nascent transcript upon co-transcriptional recruitment by the CTD of RNAP II (28–32
). These functional features incited us to use the Pcf11 suppressing ability as a starting point to investigate the mechanisms by which Rho-induced aberrant transcripts are recognized and targeted for destruction. A systematic deletion analysis of Pcf11 indicates that the rescue of Rho-induced aberrant transcripts and the accompanying relief of growth defect by overproduction of the protein stems from a physical interaction of its CID domain with the transcription complex. The interaction requires both CTD and RNA contacts and is more efficient with isolated CID fragment than the full-length protein. Similar rescue of Rho-induced aberrant transcripts and relief of growth defect were obtained when endogenous Nrd1, the other CID-containing protein involved in transcription termination of small non-coding RNAs, was altered in its CTD and RNA binding determinants by deletion of its CID or mutations within its RRM. Thus, overproduced Pcf11 or its CID-containing fragments exert their suppressing effect by outcompeting Nrd1 for recruitment by the transcription complex. Consistent with this conclusion, a similar competition-mediated suppression of Rho-induced defect is achieved by overproduction of functionally-deficient CID-containing Nrd1 fragments. Together, these results show that the recognition and targeting for destruction of Rho-induced aberrant transcripts is mediated by Nrd1 association with the transcription complex, a fact that was substantiated with the direct evaluation of Nrd1 occupancy by ChIP experiments. Indeed, Nrd1 recruitment across two representative genes, PMA1
( and Supplementary Figure S2
), is shown to increase dramatically in the presence of Rho expression and this trend is alleviated by overproduction of competing suppressors.
What makes the transcription complex so attractive for Nrd1 association in the presence of Rho helicase/translocase activity? Co-transcriptional recruitment of Nrd1 was shown to be predominant at promoter-proximal regions matching the high level of CTD Ser5 phosphorylation. This was taken to explain the preferential involvement of Nrd1 complex in termination of short transcripts such as non-coding RNAs and CUTs (39
). However, as detected with our ChIP experiments in the absence of Rho, recent data indicate that a basal level of Nrd1 association with transcribing RNAP II persists throughout elongation, apparently due to the persistence of a certain level of phosphorylated Ser5 residues (42
). Moreover, this basal level of Nrd1 recruitment was shown to increase substantially and provoke termination at a site located >1.5
kb from a promoter when poly(A)-dependent termination is impaired (43
). These results along with others suggest that the choice between the two termination modes is governed by a continuous competition between the two termination machineries for recruitment by the transcription complex, with the CID-containing proteins Nrd1 and Pcf11 being the prominent components of the two machineries (39
We propose that a similar competition process, but in a biased way, is taking place for the recognition and targeting of Rho-induced aberrant transcripts. In effect, stripping of nascent transcripts from processing and packaging factors by Rho action should uncover specific RNA-binding sites promoting preferential recruitment of Nrd1 (). For instance, this is well illustrated by our results with the CID-defective pcf11–13
strain in which the Rho-induced defect is aggravated, reflecting the unbalance in the competition between mutant Pcf11–13 and Nrd1. Conversely, the competition is counterbalanced at the expense of Nrd1 in the case of Pcf11 overproduction or in mutant strains with defective Nrd1 (nrd1Δ1–151
alleles). Work in progress in our laboratory indicates that a similar competition process is obtained by overexpression of other CTD-interacting proteins. In addition to Pcf11, Rna14 which is known to interact with the CTD was found as a dosage suppressor of the Rho-induced defect in our previous screen (44
). In contrast, overexpression of the two other members of the CF1A complex, Rna15 and Clp1, for which CTD binding has not been found in two-hybrid experiments (31
), did not confer any suppressing phenotype. A similar correlation between known CTD binding activity and dosage suppression of the Rho-induced defect is also found for some components of the CPF complex.
Figure 9. Model illustrating the Nrd1-mediated recognition and degradation of Rho-induced aberrant transcripts. (A) Normal conditions of mRNP biogenesis. Nrd1 complex is recruited to the transcription complex at early elongation then is outcompeted by the deposition (more ...)
In this biased competition model, Nrd1 might be weakly anchored to the transcription complex through interaction of its CID with the CTD, then the interaction is further stabilized by binding to specific RNA segments uncovered by Rho action. In this regard, our results suggest that Nab3 association with Nrd1 plays a key role in the stabilization process. First, a rescue of Rho-induced aberrant transcripts with accompanying relief of growth defect is observed in a strain with a defect in Nab3 RNA-binding domain (nab3–11
allele). Second, the dominant-negative effect induced by overexpression of Nrd1 deletion mutants is more robust when the protein fragments include the Nab3-interacting domain in addition to the CID. We note that the transcripts derived from the two representative genes studied in this work (PMA1
) contain much more Nab3 (UCUU) than Nrd1 (GUAA/G) binding sites (27 and 15 compared to 2 and 5, respectively) which should expend the opportunity for RNA stabilizing interactions and thus the recognition of a wide range of aberrant transcripts. These considerations are consistent with previous reports in which the formation of Nrd1–Nab3 heterodimer was shown to be the primary specificity determinant for transcription termination of small non-coding RNAs and CUTs. For instance, a CUT terminator containing only Nab3 binding sites functions efficiently in wild-type cells but does not function in the nab3–11
mutant strain, indicating that the Nab3 interaction with the transcript is sufficient to stabilize the recruitment of the heterodimer (36
Previous co-immunoprecipitation studies have detected physical interactions between the exosome–TRAMP complex and Nrd1 (22
). This was taken to suggest that co-transcriptional recruitment of Nrd1 could help to tether the exosome-dependent degradation machinery to the transcription complex. Our ChIP results provide a direct evidence for such suggestion by showing a correlation between the Rho-induced large increase of Nrd1 recruitment and the enrichment of nuclear-specific exosome component Rrp6 across the PMA1
gene. A general belief for nuclear mRNA surveillance is that Rrp6 exoribonuclease activity acts in concert with the core exosome through physical interactions. However, our ChIP experiments using tagged Rrp4 and Rrp41 did not reveal any association of these core exosomal subunits with the tethered surveillance machinery. This could be due to the fact that the core exosome does not have close contacts with RNA and DNA within the transcription complex and thus is less efficiently cross-linked to chromatin. A second possibility could be that the interactions of the core exosome with Nrd1 and Rrp6 are rather weak, which may explain the fact that in the Nrd1 co-immunoprecipitates, the core exosome components were significantly underrepresented as compared to Nab3, Rrp6 or the TRAMP component Air2 (22
). A third possibility, which we favor, could be that co-transcriptional recruitment and RNA binding of Nrd1 followed by Rrp6 tethering constitutes the first step at which a defect is detected and the transcript is labeled as aberrant. Subsequently, the labeled transcript would be prevented from export then directed for destruction by facilitating a close coupling of Rrp6 with the core exosome scaffold (). This second step is probably achieved upon transcription completion since exoribonuclease degradation by the exosome–Rrp6 complex requires exposed RNA 3′-end. However, an alternative scenario of this last model could be that Rrp6 exoribonuclease activity acts independently from core exosome to degrade aberrant transcripts. This scenario is supported by recent findings in which Rrp6 was found to carry out some of its exoribonuclease functions without physical association with the core exosome and to be stimulated by the TRAMP complex (52
). Whatever the precise mechanism, this Nrd1–Rrp6-tagging model could explain how a general quality control system recognizes various aberrancies arising at different steps of mRNP biogenesis such as splicing, termination and 3′-end formation or assembly and export, then directs the defective molecules to the same outcome, destruction. In this regard, although our experimental approach uses an artificial system in which mRNPs are actively depleted from processing and packaging factors by Rho action, it gives a hint of what is likely to happen under naturally occurring circumstances that result in defective mRNP formation. However, it should be emphasized that the existence of alternative pathways for the mRNA surveillance mechanism cannot be excluded, especially in the case of transcripts in which Nrd1 and Nab3 binding sites are poorly represented or absent.