A new regulatory pathway of mRNA export by an F-box protein, Mdm30

Durairaj, Geetha; Lahudkar, Shweta; Bhaumik, Sukesh R.

doi:10.1261/rna.042325.113

Cited by 20 publications

(61 citation statements)

References 59 publications

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“…To this end, we introduced a plasmid expressing hexahistidine-tagged ubiquitin under the control of the CUP1 promoter (pUB221) in the wild-type strain expressing Myc-tagged Spt16. Using this strain, we performed the ubiquitylation assay as described previously (22,25). Ubiquitin and ubiquitylated proteins were precipitated from the WCE using nickel-nitrilotriacetic acid (Ni 2ϩ -NTA)-agarose beads, which bound to the hexahistidine tag attached to ubiquitin.…”

Section: Resultsmentioning

confidence: 99%

“…Total RNA was prepared from yeast cell culture as done previously (22); the method is briefly described in the supplemental material.…”

Section: Wce Preparation and Western Blot Analysismentioning

confidence: 99%

“…Reverse transcription-PCR (RT-PCR) analysis was performed as done previously (22)(23)(24) and as briefly described in the supplemental material. The primer pairs used for PCR analysis of cDNAs are also described in the supplemental material.…”

Section: Wce Preparation and Western Blot Analysismentioning

confidence: 99%

“…The ubiquitylation assay was performed as done previously (22,25) and is briefly described in the supplemental material.…”

Section: Wce Preparation and Western Blot Analysismentioning

confidence: 99%

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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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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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Section: Resultsmentioning

confidence: 99%

“…Total RNA was prepared from yeast cell culture as done previously (22); the method is briefly described in the supplemental material.…”

Section: Wce Preparation and Western Blot Analysismentioning

confidence: 99%

Section: Wce Preparation and Western Blot Analysismentioning

confidence: 99%

“…The ubiquitylation assay was performed as done previously (22,25) and is briefly described in the supplemental material.…”

Section: Wce Preparation and Western Blot Analysismentioning

confidence: 99%

See 2 more Smart Citations

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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“…Total RNA was prepared from yeast cell culture as described previously (54,(65)(66)(67). Briefly, 10 ml yeast culture was harvested and suspended in 100 l RNA preparation buffer (500 mM NaCl, 200 mM Tris-HCl, 100 mM Na 2 -EDTA, and 1% SDS), along with 100 l phenol-chloroform-isoamyl alcohol and a 100-l volume equivalent of glass beads (acid washed; Sigma).…”

Section: Plasmidsmentioning

confidence: 99%

Eaf1p Is Required for Recruitment of NuA4 in Targeting TFIID to the Promoters of the Ribosomal Protein Genes for Transcriptional Initiation In Vivo

Uprety

Sen

Bhaumik

2015

Molecular and Cellular Biology

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NuA4 (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a ⌬eaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions.H istone H4 acetylation plays important roles in the regulation of eukaryotic transcription and other biological processes (1-3). In Saccharomyces cerevisiae, NuA4 (nucleosome acetyltransferase of H4) acetylates histone H4. In addition, NuA4 is involved in acetylation of histones H2A and H2A.Z (4-7). NuA4 is a multisubunit protein complex and is conserved from yeast to humans (Tip60 is the human homologue of yeast NuA4) (8). Like other histone lysine (K) acetyltransferases (KATs), NuA4 is involved in various cellular events, such as transcription, DNA repair, and cell cycle progression (9-27). In addition, NuA4 is proposed to regulate cellular aging and autophagy via acetylation of nonhistone proteins (28-30). Likewise, Tip60 has numerous nonhistone targets involved in various cellular activities (31, 32). In addition, Tip60 has been found to be involved in performing critical functions in DNA repair and stem cell regulation (33-36). Therefore, NuA4 and its human homologue are multifunctional in maintaining normal cellular functions.Esa1p is the catalytic subunit of NuA4 with KAT activity (37, 38). In addition, NuA4 has 12 other subunits (39, 40). These subunits include Tra1p (ATM-related factor), Epl1p (enhancer of polycomb homologue), Arp4p (actin-related protein), Yaf9p (leukemogenic factor ENL/AF9 homologue), Act1p, and 7 Esa1p-associated factors, Eaf1p to Eaf7p. Eaf2p and Eaf4p are also known as Swc4p and Yng2p, respectively. Although Esa1p is the catalytic subunit of NuA4, it cannot acetylate nucleosomal histones on its own but can acetylate naked/f...

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