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

Abstract

Eukaryotic cells have evolved a nuclear quality control (QC) system to monitor the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export-competent ribonucleoprotein particles (mRNPs). Aberrant mRNPs that fail to pass the QC steps are retained in the nucleus and eliminated by the exonuclease activity of Rrp6. It is still unclear how the surveillance system is precisely coordinated both physically and functionally with the transcription machinery to detect the faulty events that may arise at each step of transcript elongation and mRNP formation. To dissect the QC mechanism, we previously implemented a powerful assay based on global perturbation of mRNP biogenesis in yeast by the bacterial Rho helicase. By monitoring model genes, we have shown that the QC process is coordinated by Nrd1, a component of the NNS complex (Nrd1-Nab3-Sen1) involved in termination, processing and decay of ncRNAs which is recruited by the CTD of RNAP II. Here, we have extended our investigations by analyzing the QC behaviour over the whole yeast genome. We performed high-throughput RNA sequencing (RNA-seq) to survey a large collection of mRNPs whose biogenesis is affected by Rho action and which can be rescued upon Rrp6 depletion. This genome-wide perspective was extended by generating high-resolution binding landscapes (ChIP-seq) of QC components along the yeast chromosomes before and after perturbation of mRNP biogenesis. Our results show that perturbation of mRNP biogenesis redistributes the QC components over the genome with a significant hijacking of Nrd1 and Nab3 from genomic loci producing ncRNAs to Rhoaffected protein-coding genes, triggering termination and processing defects of ncRNAs.

Degradation of Non-coding RNAs Promotes Recycling of Termination Factors at Sites of Transcription

“…Defective termination due to the inability of a crippled exosome to negotiate timely degradation of ncRNAs reveals that the Nrd1-Nab3 heterodimer can become limiting in yeast cells. Consistent with that notion, a recent study proposed that the two proteins have reduced binding to CUTs when the bacterial terminator Rho is expressed in yeast, presumably because Nab3 and Nrd1 recruitment is diverted to a set of Rho-sensitive mRNA coding genes (Moreau et al, 2019). This limiting pool of the Nrd1-Nab3 module could reflect a requirement for an optimal cellular concentration of these factors and surveillance components to allow control of pervasive transcription events, still avoiding the inappropriate triggering of transcription termination at mRNA transcription units, which are largely populated by the Cell Reports 32, 107942, July 21, 2020 11 Article ll NNS (Creamer et al, 2011;Wlotzka et al, 2011;Webb et al, 2014;this study).…”

Nrd1p identifies aberrant and natural exosomal target messages during the nuclear mRNA surveillance inSaccharomyces cerevisiae

“…Moreover, despite being theoretically hypothesized [4,19], the existence of 'Exosome Specificity Factor' (ESF), and its universality and functionality remained elusive. Only recently, Nrd1p was demonstrated to facilitate the exosomal degradation of Rho-induced transcription-elongation processing defective [47] mRNPs in Saccharomyces cerevisiae [20,21]. Although these study unraveled a key role of Nrd1p in the degradation of Rho-induced faulty messages, Nrd1p/NNS was neither demonstrated as the primary recruiter of Rrp6 nor its contribution in the degradation of other kinds of aberrant nuclear mRNAs was addressed.…”

“…Notably, the decay of Rho-induced aberrant transcripts involved the Nrd1p-dependent co-ordinated recruitment of Rrp6p after being recruited by the RNAP II [20]. In an extension of this investigation, genome-wide high-resolution landscapes of Rrp6p, Trf4p, and Nrd1p/Nab3p were utilized to show that Nrd1p/Nab3p redistribute from the genomic loci producing non-coding RNAs to Rho-affected protein-coding genes, thereby triggering their decay and elimination [21]. Despite this study assigns a functional role of Nrd1p/Nab3p in the quality control of anomalous transcripts lacking mRNA processing factors, the role of Nrd1p complex in the surveillance of entire spectrum of aberrant mRNAs was never addressed.…”

“…The RNA binding protein Nrd1p, complexed with its partners, Nab3p (another RNA binding protein) and Sen1p (a putative helicase), dubbed NNS (Nrd1p-Nab3p-Sen1p) complex, participates in transcription termination of pre-snRNAs, pre-snoRNAs, and Cryptic Unstable Transcripts (CUTs) [17], thereby leading either to their maturation or degradation by the exosome and TRAMP [18,19]. This complex also degrades (i) antisense mRNA/transcripts, (ii) bacterial Rho helicase-induced aberrant mRNAs [20,21], and (iii) a few normal mRNAs [22]. Notably, the decay of Rho-induced aberrant transcripts involved the Nrd1p-dependent co-ordinated recruitment of Rrp6p after being recruited by the RNAP II [20].…”

Degradation of non-coding RNAs promotes recycling of termination factors at sites of transcription

“…This could reflect a requirement for an optimal cellular concentration of these factors and surveillance components to allow control of pervasive transcription events, still avoiding the inappropriate triggering of transcription termination at mRNA transcription units, which are largely populated by the NNS (Creamer et al, 2011;Wlotzka et al, 2011;Webb et al, 2014; this study). Consistent with the notion that the Nrd1-Nab3 module might be limiting, a recent study proposed that the two proteins have reduced binding to CUTs when the bacterial terminator Rho is expressed in yeast, presumably because Nab3 and Nrd1 recruitment is diverted to a set of Rho-sensitive mRNA coding genes (Moreau et al, 2019).…”