Swt1, a Novel Yeast Protein, Functions in Transcription

Röther, Susanne; Clausing, Emanuel; Kieser, Anja; Sträßer, Katja

doi:10.1074/jbc.m607510200

Cited by 18 publications

(23 citation statements)

References 36 publications

(32 reference statements)

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“…RNAPI and RNAPIII were purified from S. cerevisiae by tandem affinity purification of Rpa190-TAP and Rpc160-TAP, respectively, according to Rother et al (2006) with the following modifications: All whole-cell protein extracts were treated with RNase and DNase prior to the purification. For the in vivo interaction assay of Nab2 with RNAPIII, the proteins/protein complexes were purified to the EGTA eluate.…”

Section: Protein Purificationmentioning

confidence: 99%

The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription

2015

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RNA polymerase III (RNAPIII) synthesizes most small RNAs, the most prominent being tRNAs. Although the basic mechanism of RNAPIII transcription is well understood, recent evidence suggests that additional proteins play a role in RNAPIII transcription. Here, we discovered by a genome-wide approach that Nab2, a poly(A)-binding protein important for correct poly(A) tail length and nuclear mRNA export, is present at all RNAPIII transcribed genes. The occupancy of Nab2 at RNAPIII transcribed genes is dependent on transcription. Using a novel temperature-sensitive allele of NAB2, nab2-34, we show that Nab2 is required for the occupancy of RNAPIII and TFIIIB at target genes. Furthermore, Nab2 interacts with RNAPIII, TFIIIB, and RNAPIII transcripts. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro. Taken together, we establish Nab2, an important mRNA biogenesis factor, as a novel player required for RNAPIII transcription by stabilizing TFIIIB and RNAPIII at promoters.[Keywords: RNA polymerase III; Nab2; tRNA; ncRNA; gene expression; TFIIIB] Supplemental material is available for this article. RNA polymerase III (RNAPIII) is responsible for the production of most noncoding RNAs (ncRNAs) such as tRNAs, the 5S rRNA (encoded by RDN5), the RNA of the signal recognition particle (SCR1), the spliceosomal U6 snRNA (SNR6), and the RNA component of RNase P (RPR1). Transcription by RNAPIII is initiated at three classes of promoters, which are highly diverse, have a relatively small number of cis-acting elements, and require comparably few transcription factors (TFs) (White 2011;Orioli et al. 2012;Acker et al. 2013 and references therein). Type 1 promoters are exclusively present at 5S rRNA-encoding genes (RN5S in mammals, and RDN5 in yeast). Type 1 promoters have three internal elements: the A box, which is recognized by TFIIIC, and the intermediate element (IE) and the C box, which are recognized by TFIIIA. The most common RNAPIII promoters are of type 2 and drive transcription of tRNA genes as well as, e.g., the SCR1 and RPR1 genes. Type 2 promoters consist of A-and B-box elements, which together recruit TFIIIC. TFIIIC recruits TFIIIB to the transcription start site at type 1 and type 2 promoters. Type 3 promoters are relatively rare, and, in contrast to type 1 and 2 promoters, all of their elements lie 5 ′ of the transcription start site: the distal sequence element (DSE), the proximal sequence element (PSE), and the TATA box. The DSE recruits the TFs Oct1 and STAF, whereas the PSE recruits SNAP c , which in turn binds TFIIIB and recruits it together with the TATA box to the promoter (Schramm and Hernandez 2002 and references therein). Type 3 promoters are only present in vertebrates; e.g., at the U6 snRNA gene. The promoter of the Saccharomyces cerevisiae U6 snRNA gene SNR6 is of type 2 and contains an upstream TATA box in addition to the canonical A and B boxes (Brow and Guthrie 1990;Eschenlauer et al. 1993). At all three types of promoters, TFIIIB recruits RNAPIII. TFIIIB i...

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Section: Protein Purificationmentioning

confidence: 99%

The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription

2015

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“…45 Like the exosome subunit Dis3/Rrp44, the evolutionarily conserved Swt1 contains a PIN domain which possesses RNA endonuclease activity in vitro. 12,13,45 First shown to genetically interact with transcription and mRNP assembly factors, suggesting a role in transcription, 46 Swt1 also genetically interacts with Mlp1, Esc1 and Nup60, suggesting a functional link between Swt1 and perinuclear QC components. 45 In addition, deletion of SWT1 causes robust nuclear accumulation of poly(A) RNA and enhances escape of the intron-containing premRNA reporter to the cytoplasm, suggesting Swt1 plays a role in the degradation of RNAs and unspliced mRNAs.…”

Section: Nuclear Mrna Quality Control: Retention At the Nuclear Porementioning

confidence: 99%

“…Swt1-cleaved mRNAs might then be subject to further degradation by 5'-3' exonucleases and the exosome. As Swt1 exhibits genetic interactions with transcription-associated components, such as the THO complex, 45,46 Swt1 may also participate in the degradation of aberrant mRNAs near the site of transcription, perhaps assisting the exosome.…”

Section: Nuclear Mrna Quality Control: Retention At the Nuclear Porementioning

confidence: 99%

Mechanisms of nuclear mRNA quality control

Fasken¹,

Corbett²

2009

RNA Biology

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“…We have been able to identify it by HyCCAPP and validate the interaction with ChIP, and the protein was also reported to bind to the GAL1 promoter in the previous study using the ChAP-MS approach [8]. Additionally we identified Swt1, a protein that interacts with the TREX complex during transcription [22], and Nhp6a, a high-mobility group protein involved in nucleosome remodeling [23]. While the previously reported functions of these proteins could potentially explain their binding to the ENO2 and GAL1 promoter regions, they have not been reported previously, highlighting the potential of the HyCCAPP approach to uncover novel DNA-binding proteins.…”

Section: Discussionmentioning

confidence: 92%

HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae

et al. 2016

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Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of S. cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges.

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Swt1, a Novel Yeast Protein, Functions in Transcription

Cited by 18 publications

References 36 publications

The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription

The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription

Mechanisms of nuclear mRNA quality control

HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae

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