Effects of the Paf1 Complex and Histone Modifications on snoRNA 3′-End Formation Reveal Broad and Locus-Specific Regulation

Tomson, Brett N.; Crisucci, Elia M.; Heisler, Lawrence E.; Gebbia, Marinella; Nislow, Corey; Arndt, Karen M.

doi:10.1128/mcb.01233-12

Cited by 24 publications

(31 citation statements)

References 83 publications

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“…The PAF1C also plays important roles in transcription elongation through chromatin, as well as on naked DNA (13)(14)(15)(16). Furthermore, the PAF1C is involved in transcription termination and 3= processing of polyadenylated and nonpolyadenylated Pol II transcripts (17)(18)(19)(20)(21)(22)(23)(24)(25).…”

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confidence: 99%

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

Cao

Yamamoto

Isobe

et al. 2015

Molecular and Cellular Biology

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bRestores TBP function 1 (Rtf1) is generally considered to be a subunit of the Paf1 complex (PAF1C), a multifunctional protein complex involved in histone modification and transcriptional or posttranscriptional regulation. Rtf1, however, is not stably associated with the PAF1C in most species except Saccharomyces cerevisiae, and its biochemical functions are not well understood. Here, we show that human Rtf1 is a transcription elongation factor that may function independently of the PAF1C. Rtf1 requires "Rtf1 coactivator" activity, which is most likely unrelated to the PAF1C or DSIF, for transcriptional activation in vitro. A mutational study revealed that the Plus3 domain of human Rtf1 is critical for its coactivator-dependent function. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation studies in HeLa cells showed that Rtf1 and the PAF1C play distinct roles in regulating the expression of a subset of genes. Moreover, contrary to the finding in S. cerevisiae, the PAF1C was apparently recruited to the genes examined in an Rtf1-independent manner. The present study establishes a role for human Rtf1 as a transcription elongation factor and highlights the similarities and differences between the S. cerevisiae and human Rtf1 proteins. Restores TBP (TATA box-binding protein) function 1 (Rtf1) was identified as a suppressor of a TBP mutant in Saccharomyces cerevisiae (1). Subsequent genetic and biochemical studies in yeast have shown that Rtf1 functions as a component of the polymerase-associated factor 1 (Paf1) complex (PAF1C) containing Paf1, Ctr9, Leo1, and Cdc73 (2-5). The PAF1C is a multifunctional protein complex whose primary role is to facilitate histone modifications, such as H2B monoubiquitination at K123 and H3 methylation at K4 and K79 (6-12). The PAF1C also plays important roles in transcription elongation through chromatin, as well as on naked DNA (13-16). Furthermore, the PAF1C is involved in transcription termination and 3= processing of polyadenylated and nonpolyadenylated .Paf1, Ctr9, Leo1, Cdc73, and Rtf1 are highly conserved in eukaryotes; however, the subunit compositions of the PAF1C vary among species. Purification of the PAF1C from human HeLa cells yielded a five-subunit complex lacking Rtf1 but containing Ski8 as an additional subunit (22,26,27). Similarly, coimmunoprecipitation studies in zebrafish, Drosophila, and Schizosaccharomyces pombe showed that PAF1C homologs in these species lack a stably associated Rtf1 subunit (28-30). At the functional level, however, Rtf1 and other PAF1C subunits have a number of similarities. For example, knockout or knockdown of Rtf1 and other PAF1C subunits results in similar defects in histone modifications in all the species examined (27,28,31). At the organismal level, inhibition of Rtf1 and other PAF1C subunits causes similar defects in epidermal morphogenesis in Caenorhabditis elegans and in somitogenesis and cardiomyocyte development in zebrafish (29,32,33). Rtf1 and Paf1 colocalize with each other and with RNA polymerase II (RNAPII) in...

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confidence: 99%

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

Cao

Yamamoto

Isobe

et al. 2015

Molecular and Cellular Biology

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“…Whereas many formed chimeric transcripts that used the polyA signals of downstream protein-coding genes to terminate transcription (e.g., SNR33) (Fig. 1C) (34)(35)(36), others were read through and terminated even in the absence of a downstream gene (e.g., SNR48) (SI Appendix, Fig. S3).…”

Section: Resultsmentioning

confidence: 99%

“…We quantified readthrough at each snoRNA, as previously described (35) (Fig. 2B), and identified 37 snoRNAs with a 3ʹ extension ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner

Nemec

Yang

Gilmore

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptapeptide repeats of the CTD engage specific "readers." Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phosphoSer2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engages two distinct CTD modifications that are differentially enriched at noncoding genes, we relied on NMR analysis of Rtt103 in complex with phospho-Thr4-or phospho-Ser2-bearing CTD peptides. The structural data reveal that Rtt103 interacts with phospho-Thr4 in a manner analogous to its interaction with phospho-Ser2-modified CTD. The same set of hydrogen bonds involving either the oxygen on phospho-Thr4 and the hydroxyl on Ser2, or the phosphate on Ser2 and the Thr4 hydroxyl, can be formed by rotation of an arginine side chain, leaving the intermolecular interface otherwise unperturbed. This economy of design enables Rtt103 to engage Pol II at distinct sets of genes with differentially enriched CTD marks.ach stage of transcription relies on ordered recruitment and exchange of specific protein complexes that act on RNA polymerase II, its nascent transcripts, and the underlying chromatin. This dynamic process is orchestrated via patterned posttranslational modifications of the carboxyl-terminal domain (CTD). This unusual and essential domain of Rpb1, the largest component of the 12-subunit polymerase, consists of repeating Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptapeptides (26 repeats in budding yeast and 52 in humans) (1). The mechanistic consequences of phosphorylating Ser5 and Ser2 have been well documented (2-11). However, the role of Thr4 phosphorylation (pThr4), and even the necessity of Thr4 for cellular survival, appears to differ among closely related species and between growth conditions within a given species (12-15). Recent mass spectrometric analysis of an extensively engineered CTD revealed a paucity of pThr4, raising questions about the importance of this mark (16). In contrast, similar studies found pThr4 marks at levels comparable to or greater than the ubiquitously placed pSer2 mark in both yeast and human cells (17). Thus, much remains to be understood about the natural abundance and functional role of pThr4 marks on the endogenous, unmodified CTD.Previous studies suggest that pThr4 has roles in transcriptional elongation, 3ʹ-...

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“…Paf1C also facilitates H3 K36 trimethylation on the bodies of active genes (15). Additional functions of Paf1C include the Rtf1-mediated recruitment of the Chd1 chromatin remodeling protein (16), the stimulation of serine 2 phosphorylation on the C-terminal domain (CTD) of RNA Pol II (17,18), the maintenance of nucleosome occupancy on highly expressed regions of the genome (19), and the recruitment of factors involved in transcription termination and RNA processing (17,18,20,21). Either individually or collectively, the multiple functions of Paf1C can influence the rate of transcription elongation in vitro and in vivo (22)(23)(24).…”

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confidence: 99%

“…Either individually or collectively, the multiple functions of Paf1C can influence the rate of transcription elongation in vitro and in vivo (22)(23)(24). In addition to its roles in elongation, Paf1C has also been shown to affect the initiation and termination stages of the transcription cycle (17,18,20,21,(25)(26)(27).…”

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confidence: 99%

The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

Mayekar

Gardner

Arndt

2013

Molecular and Cellular Biology

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cTranscription elongation factors associate with RNA polymerase II and aid its translocation through chromatin. One such factor is the conserved Paf1 complex (Paf1C), which regulates gene expression through several mechanisms, including the stimulation of cotranscriptional histone modifications. Previous studies revealed a prominent role for the Rtf1 subunit in tethering Paf1C to the RNA polymerase II elongation machinery. Here, we investigated the mechanism by which Rtf1 couples Paf1C to active chromatin. We show that a highly conserved domain of Rtf1 is necessary and sufficient for mediating a physical interaction between Rtf1 and the essential transcription elongation factor Spt5. Mutations that alter this Rtf1 domain or delete the Spt5 C-terminal repeat domain (CTR) disrupt the interaction between Rtf1 and Spt5 and release Paf1C from chromatin. When expressed in cells as the only source of Rtf1, the Spt5-interacting domain of Rtf1 can associate independently with active genes in a pattern similar to that of full-length Rtf1 and in a manner dependent on the Spt5 CTR. In vitro experiments indicate that the interaction between the Rtf1 Spt5-interacting domain and the Spt5 CTR is direct. Collectively, our results provide molecular insight into a key attachment point between Paf1C and the RNA polymerase II elongation machinery.

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Effects of the Paf1 Complex and Histone Modifications on snoRNA 3′-End Formation Reveal Broad and Locus-Specific Regulation

Cited by 24 publications

References 83 publications

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner

The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

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