Yeast RNA-Binding Protein Nab3 Regulates Genes Involved in Nitrogen Metabolism

Abstract

Termination of Saccharomyces cerevisiae RNA polymerase II (Pol II) transcripts occurs through two alternative pathways. Termination of mRNAs is coupled to cleavage and polyadenylation while noncoding transcripts are terminated through the Nrd1-Nab3-Sen1 (NNS) pathway in a process that is linked to RNA degradation by the nuclear exosome. Some mRNA transcripts are also attenuated through premature termination directed by the NNS complex. In this paper we present the results of nuclear depletion of the NNS component Nab3. As expected, many noncoding RNAs fail to terminate properly. In addition, we observe that nitrogen catabolite-repressed genes are upregulated by Nab3 depletion.KEYWORDS nitrogen metabolism, noncoding RNA, termination, transcription S accharomyces cerevisiae RNA polymerase II (Pol II) synthesizes both mRNAs and noncoding RNAs (ncRNAs), including snRNAs, snoRNAs, and a large number of RNAs with unknown functions (1, 2). The latter class includes cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs) (3, 4). Both coding and noncoding transcripts originate from promoters located in nucleosome-free regions in a process that requires both general transcription factors and gene-specific factors (5-7), but termination of these different classes of Pol II transcripts takes place through two different processes. Stable transcripts like mRNA and SUTs terminate through a process linked to cleavage and polyadenylation while snoRNAs and CUTs terminate through the Nrd1-Nab3-Sen1 (NNS) pathway (8)(9)(10)(11)(12)(13)(14).The NNS complex contains two RNA-binding proteins, Nrd1 and Nab3, which recognize specific sequences in nascent transcripts and direct termination in a process that requires the RNA helicase Sen1 (8,9,(15)(16)(17)(18)(19)(20). NNS interacts with Pol II in part through binding of Nrd1 to phosphorylated Ser5 on the C-terminal domain (CTD) (21-23), a pattern of CTD phosphorylation most prominent in the early stages of the transcription cycle, limiting NNS termination to promoter-proximal transcripts (24-29). The NNS complex also interacts with the TRAMP (Trf4/Trf5-Air1/Air2-Mtr4 polyadenylation) complex to couple termination to processing by the nuclear exosome (30)(31)(32)(33).While the two yeast Pol II termination pathways generally operate on distinct sets of transcripts, there are some genes that can use either termination pathway (11). In some cases NNS acts downstream of genes as a fail-safe termination mechanism to ensure that transcripts that fail to terminate through the cleavage/polyadenylation mechanism do not read through into downstream genes (34,35). In other cases NNS functions upstream to terminate transcripts before the poly(A) site is reached. For example, Nrd1 autoregulates its own transcription by binding, along with Nab3, to sites in the 5= end of its nascent pre-mRNA (9, 36). The result of this binding leads to premature termination of the majority of Nrd1 transcripts close to the 5= end. Nrd1 transcripts that escape the NNS pathway go on to t...

RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination

“…The DEF1 attenuator exhibits Sen1-dependence but little-to-no dependence on Nrd1 or Nab3 despite sequence similarity to consensus Nrd1 (GUAA, GUAG) and Nab3 RNA-binding sites (UCUU) ( Steinmetz and Brow 1998 ; Carroll et al 2004 ; Creamer et al 2011 ; Porrua et al 2012 ). The very limited role for Nrd1 and Nab3 at the DEF1 attenuator is a contrast to what has been observed for most other attenuators, including NRD1 , IMD2 , URA2 , FKS2 , CLN3 , GPH1 , and GLT1 , which exhibit strong dependence on Nrd1, Nab3, or both ( Arigo et al 2006 ; Jenks et al 2008 ; Kuehner and Brow 2008 ; Thiebaut et al 2008 ; Kim and Levin 2011 ; Darby et al 2012 ; Chen et al 2017 ; Merran and Corden 2017 ). The lack of Nrd1/Nab3 involvement that we observe is consistent with DEF1 expression levels not increasing upon Nrd1/Nab3 depletion and Nrd1/Nab3 failing to crosslink to DEF1 during in-vivo crosslinking studies ( Jamonnak et al 2011 ; Merran and Corden 2017 ).…”

“…Attenuator recognition and bypass has been linked to changes in cell metabolism and stress response genes, but in most cases the signaling mechanism is unknown. Examples of mRNA gene attenuation targets include IMD2 and URA2 (nucleotide biosynthesis), FKS2 (cell wall damage), CLN3 (glucose starvation), GPH1 (glycogen metabolism), and GLT1 (nitrogen metabolism) ( Jenks et al 2008 ; Kuehner and Brow 2008 ; Thiebaut et al 2008 ; Kwapisz et al 2008 ; Kim and Levin 2011 ; Darby et al 2012 ; Chen et al 2017 ; Merran and Corden 2017 ).…”

Transcription-dependent spreading of the Dal80 yeast GATA factor across the body of highly expressed genes

“…The addition of nitrogen to nitrogen-limited cells rapidly results in the transient overproduction of transcripts required for protein translation (stimulated growth) whereas accelerated mRNA degradation favours rapid clearing of the most abundant transcripts, like those involved in high affinity permease production, that are highly expressed NCR-sensitive genes, for example [ 64 ]. The involvement of the Nrd1-Nab3-Sen1 (NNS) and TRAMP complexes in these regulatory responses has been envisioned very recently [ 81 , 82 ]; deadenylation, decapping and exonuclease mutants display impaired GAP1 mRNA clearance upon nitrogen upshift [ 83 ]. Thus, a possible role of Dal80 (and possibly of the other GATA factors) binding along highly expressed genes could be to transmit nutritional signals to elongation-related processes, like histone modification, chromatin remodelling [ 84 , 85 ], mRNA export/processing [ 86 ] or roadblock termination [ 87 ].…”

RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination

“…Two-hundred and seventy-six transcripts showed upregulation of more than 1.5-fold, many of which were ORF regions ( Fig 2C). The most significantly reduced transcript was IMD2, whose expression is regulated by GTP levels as well as an NNS terminator (Fig 2A, labeled in green, [46,47,[78][79][80]). We also confirmed that Imd2 protein levels are significantly decreased in rtr1Δ cells using global proteomics analysis (S2 Fig, S4 Table).…”