Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

Abstract

The co-transcriptional biogenesis of export-competent messenger ribonucleoprotein particles (mRNPs) in yeast is under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from inappropriate or inefficient processing and packaging reactions are detected by the QC system and retained in the nucleus with ensuing elimination of their mRNA component by a mechanism that requires the catalytic activity of Rrp6p, a 3 0 -5 0 exonuclease associated with the RNA exosome. In previous studies, we implemented a new experimental approach in which the production of aberrant mRNPs is massively increased upon perturbation of mRNP biogenesis by the RNA-dependent helicase/translocase activity of the bacterial Rho factor expressed in S. cerevisiae. The analyses of a subset of transcripts such as PMA1 led us to substantiate the essential role of Rrp6p in the nuclear mRNP QC and to reveal a functional coordination of the process by Nrd1p. Here, we extended those results by showing that, in contrast to PMA1, Rho-induced aberrant HXK1 mRNPs are targeted for destruction by an Nrd1p-and Rrp6p-independent alternative QC pathway that relies on the 5 0 -3 0 exonuclease activity of Rat1p. We show that the degradation of aberrant HXK1 mRNPs by Rat1p occurs cotranscriptionally following decapping by Dcp2p and leads to premature transcription termination. We discuss the possibility that this alternative QC pathway might be linked to the well-known specific features of the HXK1 gene transcription such as its localization at the nuclear periphery and gene loop formation.

Perturbation of mRNP biogenesis reveals a dynamic landscape of the Rrp6-dependent surveillance machinery trafficking along the yeast genome

“…meta-transcript profiles obtained with the pool of nonrescued mRNAs reveal an averaged higher expression of the transcripts with a clear absence of rescue in the rrp47Δ strain. We infer that the absence of recovery in the rrp47Δ strain for this mRNA group is likely an indication that the corresponding Rho-induced defective mRNPs are targeted by the alternative Rat1-dependent QC pathway, as we reported previously for HXK1 mRNP [18]. An in-depth comparative analysis between the two mRNA populations and their specific processing by the two QC pathways (Rrp6-dependent and Rat1-dependent) will be published elsewhere (Moreau et al manuscript in preparation).…”

“…The importance of the Nrd1-Nab3 association in this early step of the targeting process is also inferred from our previous studies with model genes. In effect, we showed that the targeting of aberrant mRNPs as well as the growth defect phenotype induced by Rho were suppressed when the Nrd1 and Nab3 coupling was flawed by a specific mutation in the Nab3 RNA-binding domain (nab3-11 allele in [11]) or a deletion of the Nab3association domain within Nrd1 (nrd1ΔNID in [18] and in Figure S8). Thus, the formation of the Nrd1-Nab3 dimer and its stabilization by RNA binding play a key role in the recognition and targeting of aberrant mRNPs.…”

“…All the S. cerevisiae yeast strains used in this study are derived from the wild-type strain BMA41 (MATa or MATα ade2-1 ura3-1 leu2-3,112 his3-11,15 trp1Δ can1-100) [48]. They have been previously described [11,13,18] except NRD1-MYC and NAB3-MYC which were newly created. To construct these new strains expressing C-terminally MYC-tagged Nrd1 or Nab3 proteins, PCR products were amplified with the forward and reverse primers (listed in Table S1) on plasmid template pYM19 [49] and integrated into the NRD1 locus or the NAB3 locus by homologous recombination.…”

Mec1/ATR, the Program Manager of Nucleic Acids Inc.

“…For instance, an aberrant mRNP (messenger ribonucleoprotein) particle may be obstructing the damaged site. This hypothesis is substantiated by recent findings that Rrp6 plays an important role in the quality control of specific mRNPs [ 51 , 52 ]. Moreover, a previous proteomic analysis revealed a multitude of interactions between RPA and the chromatin remodeling proteins, including Ino80, Isw1, Isw2, Swic, Rsc2, and SWI/SNF [ 53 ].…”

Tho2 is critical for the recruitment of Rrp6 to chromatin in response to perturbed mRNP biogenesis

“…The mean values and standard deviations (SDs) were calculated from three independent experiments. For RT-qPCR, RNAs were first purified with the NucleoSpin RNA II kit (Macherey Nagel) and then reverse transcribed with the cDNA Synthesis kit from Thermo Scientific [the kit includes random hexamers and oligo(dT)20] as described in Mosrin-Huaman et al (2016) . Real-time PCR quantifications for ChIP were then performed as described; see above.…”