The mRNA cap-binding complex stimulates the formation of pre-initiation complex at the promoter via its interaction with Mot1p in vivo

FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone involved in transcription and other DNA transactions, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine-tuned for normal cellular functions. Here, we show that the FACT subunit Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, we found that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similarly to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. Taken together, our results demonstrate for the first time a fine-tuning of FACT by a ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation, thus revealing novel regulatory mechanisms of gene expression. In eukaryotes, DNA is packaged into nucleosomes to form chromatin. Each nucleosome within chromatin consists of ϳ147 bp of DNA wrapped around a histone octamer containing one histone H3-H4 tetramer and two histone H2A-H2B dimers (1). Thus, chromatin structure plays crucial functions in regulation of DNA transactions such as transcription, replication, and DNA repair (2-4). A variety of factors are involved in altering chromatin structure and, hence, DNA transactions. These factors are generally classified as ATP-independent histone modifying enzymes, ATP-dependent chromatin remodelers, and histone chaperones. Histone modifying enzymes function through addition or removal of specific chemical groups (e.g., acetyl, methyl, ubiquitin, phospho, and SUMO), while ATP-dependent chromatin remodelers alter DNA-histone interactions or the composition of the nucleosome in an ATP-dependent manner. On the other hand, histone chaperones function by binding with nucleosomes or histones to facilitate assembly and/or disassembly of nucleosomes in an ATP-independent fashion. The histone chaperone that was first found to alter chromatin structure during transcription is FACT (facilitates chromatin transcription), which is evolutionarily conserved among eukaryotes (5, 6). In budding yeast (Saccharomyces cerevisiae), FACT is composed of Spt16 (suppressor of Ty) and Pob3 and physically interacts with nucleosomes with the assistance of the HMG (high mobility group) protein Nhp6. Likewise, FACT is also a heterodimer of Spt16 and SSRP1 (structurespecific recognition protein 1) in humans. SSRP1 contains HMG domain, while HMG domain is p...

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“…The coimmunoprecipitation (co-IP) assay was performed as done previously (26,27) and is briefly described in the supplemental material.…”

Section: Wce Preparation and Western Blot Analysismentioning

confidence: 99%

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation

Sen

Ferdoush

Kaja

et al. 2016

Molecular and Cellular Biology

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“…In this direction, we first analyzed the role of CBC in association with RNA polymerase II with ADH1. We have recently demonstrated that both Cbp20p and Cbp80p are recruited to ADH1 (Lahudkar et al 2011). The deletion of CBP80 impairs the recruitment of Cbp20p (Lahudkar et al 2011), but does not alter Cbp20p's stability (Lahudkar et al 2011).…”

Section: Resultsmentioning

confidence: 98%

“…We have recently demonstrated that both Cbp20p and Cbp80p are recruited to ADH1 (Lahudkar et al 2011). The deletion of CBP80 impairs the recruitment of Cbp20p (Lahudkar et al 2011), but does not alter Cbp20p's stability (Lahudkar et al 2011). Therefore, we have used the Dcbp80 mutant for analysis of the effect of CBC on the association of RNA polymerase II with ADH1.…”

Section: Resultsmentioning

confidence: 99%

“…This study ZDY4 MATa his3-11,15 trp1-1 leu2-3,112 ura3-1 ade2-1 can1-100 RPB1-MYC (KAN) Lahudkar et al (2011) Regulation of RNA Polymerase II by Cet1p Primer pairs used for PCR analysis (A, B, C, D, etc. represent regions amplified; Figure 1A and Figure 3A) were as follows:…”

Section: Buy36mentioning

confidence: 99%

“…The cap structure recruits cap-binding complex (CBC) that plays crucial roles in maintaining the stability of mRNA and its splicing, export, and translation (Izaurralde et al 1994;Colot et al 1996;Izaurralde et al 1995;Flaherty et al 1997;Schwer et al 1998;Shatkin and Manley 2000;Shen et al 2000;Ishigaki et al 2001;McKendrick et al 2001;Maquat 2004;Gornemann et al 2005;Hosoda et al 2005;Svitkin et al 2005;Balatsos et al 2006;Nojima et al 2007;Wong et al 2007;Kuhn et al 2008;Laubinger et al 2008). Further, CBC has been recently implicated in controlling transcriptional initiation and elongation, histone H2B ubiquitylation, histone H3K36 (lysine 36) methylation, RNA interference, cell proliferation, and processing of primary microRNAs (Kim et al 2008;Gruber et al 2009;Hossain et al 2009Hossain et al , 2013Nielsen et al 2009;Sabin et al 2009;Lahudkar et al 2011;Lidschreiber et al 2013). Thus, the 59 end capping of mRNA has critical functions in eukaryotic gene expression.…”

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confidence: 99%

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A Novel Role for Cet1p mRNA 5′-Triphosphatase in Promoter Proximal Accumulation of RNA Polymerase II in Saccharomyces cerevisiase

et al. 2014

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Yeast mRNA 59-triphosphatase, Cet1p, recognizes phosphorylated-RNA polymerase II as a component of capping machinery via Ceg1p for cotranscriptional formation of mRNA cap structure that recruits cap-binding complex (CBC) and protects mRNA from exonucleases. Here, we show that the accumulation of RNA polymerase II at the promoter proximal site of ADH1 is significantly enhanced in the absence of Cet1p. Similar results are also found at other genes. Cet1p is recruited to the 59 end of the coding sequence, and its absence impairs mRNA capping, and hence CBC recruitment. However, such an impaired recruitment of CBC does not enhance promoter proximal accumulation of RNA polymerase II. Thus, Cet1p specifically lowers the accumulation of RNA polymerase II at the promoter proximal site independently of mRNA cap structure or CBC. Further, we show that Cet1p's N-terminal domain, which is not involved in mRNA capping, decreases promoter proximal accumulation of RNA polymerase II. An accumulation of RNA polymerase II at the promoter proximal site in the absence of Cet1p's N-terminal domain is correlated with reduced transcription. Collectively, our results demonstrate a novel role of Cet1p in regulation of promoter proximal accumulation of RNA polymerase II independently of mRNA capping activity, and hence transcription in vivo.

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C ap ‐b inding complex (CBC)

2015

The Dictionary of Genomics, Transcriptomics and Proteomics

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The 7mG (7-methylguanosine cap) formed on mRNA is fundamental to eukaryotic gene expression. Protein complexes recruited to 7mG mediate key processing events throughout the lifetime of the transcript. One of the most important mediators of 7mG functions is CBC (cap-binding complex). CBC has a key role in several gene expression mechanisms, including transcription, splicing, transcript export and translation. Gene expression can be regulated by signalling pathways which influence CBC function. The aim of the present review is to discuss the mechanisms by which CBC mediates and co-ordinates multiple gene expression events.

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The mRNA cap-binding complex stimulates the formation of pre-initiation complex at the promoter via its interaction with Mot1p in vivo

Cited by 32 publications

References 116 publications

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation

A Novel Role for Cet1p mRNA 5′-Triphosphatase in Promoter Proximal Accumulation of RNA Polymerase II in Saccharomyces cerevisiase

C ap ‐b inding complex (CBC)

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