Genome-Wide Mapping of Yeast RNA Polymerase II Termination

Schaughency, Paul; Merran, Jonathan; Corden, Jeffry L.

doi:10.1371/journal.pgen.1004632

Cited by 77 publications

(133 citation statements)

References 92 publications

(152 reference statements)

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“…Nuclear depletion of Nab3 results in readthrough transcription of snoRNA and CUTs as has been previously described for Nrd1 depletion (7,9,32,33,41). Figure 1A shows that snR47 and Nrd1 readthrough transcripts are slightly elevated in the FRB-tagged strain, and this level of readthrough is increased in the presence of rapamycin.…”

Section: Resultssupporting

confidence: 73%

“…To examine the effect on transcription of depleting Nab3 from the nucleus, we C-terminally tagged Nab3 with the FRB (FKBP12-rapamycin binding) domain in an anchor-away strain that contains an HTB (6His-TEVbiotin, where TEV is tobacco etch virus) tag on the RNA polymerase subunit Rpb2 (15,40,41). FRB-tagged Nab3 protein is normally expressed and renders this strain sensitive to rapamycin (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To control for effects of the tag, we carried out PAR-CLIP on the FRB-tagged strain with or without rapamycin and a control strain that does not contain an FRB tag but does include the downstream HIS3 marker that was used to introduce the tag. We also conducted PAR-CLIP on an NAB3-HTB strain to map Nab3 binding sites on nascent RNA transcripts (15,41,42). PAR-CLIP data sets were obtained from two biological replicates for each of these conditions.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to noncoding RNAs, the NNS complex has been shown to bind to a number of mRNAs (7,15,19,35,43,44). However, despite the prevalence of Nrd1-Nab3 binding to the 5= end of mRNAs, there are relatively few genes where regulation by premature termination has been confirmed (7,15,35,41,43,45). Nrd1 expression is autoregulated through attenuation and is also sensitive to mutations in Nab3 (9,36).…”

Section: Resultsmentioning

confidence: 99%

“…We along with others have previously used the anchor-away strategy (40) to create conditional mutants of Nrd1 that lead to nuclear depletion in the presence of rapamycin (7,41). This approach has led to the identification of NNS termination sites and has identified a limited set of mRNAs that are controlled through NNS.…”

mentioning

confidence: 99%

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

Merran

Corden

2017

Molecular and Cellular Biology

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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...

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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Merran

Corden

2017

Molecular and Cellular Biology

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Regulation of Gene Expression and Replication Initiation by Non‐Coding Transcription: A Model Based on Reshaping Nucleosome‐Depleted Regions

Soudet

Stutz

2019

BioEssays

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RNA polymerase II (RNAP II) non-coding transcription is now known to cover almost the entire eukaryotic genome, a phenomenon referred to as pervasive transcription. As a consequence, regions previously thought to be non-transcribed are subject to the passage of RNAP II and its associated proteins for histone modification. This is the case for the nucleosome-depleted regions (NDRs), which provide key sites of entry into the chromatin for proteins required for the initiation of coding gene transcription and DNA replication. In this review, recent data on the effects of pervasive transcription through NDRs are summarized and a model is proposed to explain how RNAP II-driven transcription is able to modify the nucleosomes flanking the NDRs, leading to nucleosome repositioning and NDR closure. Even though much of the mechanistic detail underlying these events remains to be elucidated, such a model provides a basis to explain how non-coding transcription through NDRs can regulate the initiation of coding gene expression and DNA replication.

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Development of a fluorescent three‐hybrid system for the identification of protein‐protein associators

et al. 2020

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The chemically‐induced dimerization of proteins is fundamental to many key regulatory pathways. A number of natural products exert their downstream effect through the stabilization of a protein complex, either by direct binding to two distinct protein partners or via an allosteric mechanism. Here, we report a bacterial three‐hybrid system, with dual life/death and fluorescent reporters, which detects protein association and is compatible with high‐throughput screening. We use rapamycin mediated mTOR‐FKBP12 (mammalian target of rapamycin—FK506 Binding Protein 12 kDa) dimerization to validate this platform; the addition of rapamycin results in the association of these target proteins, leading to the expression of two essential life/death reporter genes and a fluorescent signal. We further used this system to quantify the activity of rapamycin by utilizing the fluorescent readout, exemplifying its potential in screening and for ranking large in vivo libraries for compounds that upregulate the association of any two given proteins.

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Genome-Wide Mapping of Yeast RNA Polymerase II Termination

Cited by 77 publications

References 92 publications

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

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

Regulation of Gene Expression and Replication Initiation by Non‐Coding Transcription: A Model Based on Reshaping Nucleosome‐Depleted Regions

Development of a fluorescent three‐hybrid system for the identification of protein‐protein associators

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