RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals

Scafe, Charles; Chao, David M.; Lopes, John M.; Hirsch, Jeanne P.; Henry, Susan A.; Young, Richard A.

doi:10.1038/347491a0

Cited by 161 publications

(148 citation statements)

References 30 publications

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“…Pol II at the stage of transcription initiation is associated with a large holoenzyme complex, which is recruited to promoters by sequence-specific transcriptional activators (6 -10). In some cases, stimulation of transcription by activators requires the CTD (11)(12)(13). Recent work has demonstrated that the CTD is also important for pre-mRNA processing.…”

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confidence: 99%

Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

Rosonina

Bakowski

McCracken

et al. 2003

Journal of Biological Chemistry

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We have investigated whether transcriptional activators influence the efficiency of constitutive splicing and 3 -end formation, in addition to transcription levels. Remarkably, strong activators result in higher levels of splicing and 3 -cleavage than weak activators and can control the efficiency of these steps in pre-mRNA processing separately. The pre-mRNA processing stimulatory property of activators is dependent on their binding to promoters, but is not an indirect consequence of the levels of transcripts produced. Moreover, stimulation of splicing and cleavage by a strong activator operates by a mechanism that requires the carboxyl-terminal domain of RNA polymerase II. The splicing stimulatory property of activators was observed for unrelated transcripts and for separate introns within a transcript, indicating a possible general role for strong activators in facilitating pre-mRNA processing levels. The results suggest that the efficiency of constitutive splicing and 3 -end cleavage is closely coordinated with transcription levels by promoter-bound activators.The generation of mature mRNA involves the addition of an m 7 G cap at the 5Ј-end, splicing, and cleavage and polyadenylation at the 3Ј-end of precursor (pre-)mRNAs. Although these steps can occur in isolation of each other, the majority of pre-mRNA processing occurs as RNA polymerase II (pol II) 1 is transcribing. This is evident from numerous microscopy studies, in which the removal of most introns is observed to coincide with nascent transcript synthesis, as well as from more recent studies that demonstrate that transcription and pre-mRNA processing steps are physically coupled and can influence one another (1-3).The largest subunit of pol II contains a carboxyl-terminal domain (CTD), which in mammals consists of 52 repeats of the heptapeptide consensus sequence YSPTSPS (4, 5). Pol II at the stage of transcription initiation is associated with a large holoenzyme complex, which is recruited to promoters by sequence-specific transcriptional activators (6 -10). In some cases, stimulation of transcription by activators requires the CTD (11-13). Recent work has demonstrated that the CTD is also important for pre-mRNA processing. Truncation of the CTD in vivo reduces the efficiency of capping, splicing, and 3Ј-end formation (14 -16). Moreover, purified pol II, or the CTD itself, can stimulate splicing as well as the 3Ј-end processing of transcripts in vitro (17)(18)(19). Consistent with these results, factors involved in capping, splicing, and 3Ј-end formation have been found to interact with the CTD (1, 3, 20).It is not known at which stage of transcription different pre-mRNA processing components are recruited to transcription complexes, nor how CTD-associated components influence pre-mRNA processing. The 3Ј-end cleavage polyadenylation stimulatory factor (CPSF) interacts with the general transcription factor TFIID during transcription initiation (21), as well as with the CTD (15), indicating that it is recruited to transcription initiation complexes pr...

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confidence: 99%

Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

Rosonina

Bakowski

McCracken

et al. 2003

Journal of Biological Chemistry

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“…For example, Pol II carrying altered CTDs responds differently to certain wildtype and mutant transcriptional regulators in vivo (Allison et al 1988;Scafe et al 1990;Peterson et al 1991), suggesting that the CTD may be involved in receiving regulatory signals at certain promoters. In vivo and in vitro experiments suggest that these responses might be mediated through the TATA-binding protein (TBP) component of TFIID (Koleske et al 1992;Usheva et al 1992; Thompson et al 1993).…”

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confidence: 99%

“…The CTD has been shown to carry out essential in vivo roles in yeast (Nonet et al 1987), Drosophila (Zehring et al 1988), and mammalian cells (Bartolomei et al 1988), but precisely what those roles are is not well understood. Suggested roles for the CTD include interacting with transcription initiation factors, serving as a molecular "cowcatcher" to facilitate movement of polymerase on chromatin templates, providing a link between transcription and RNA processing, and localizing polymerase to specific nuclear compartments (Corden 1990 (Allison et al 1988;Scafe et al 1990;Peterson et al 1991), suggesting that the CTD may be involved in receiving regulatory signals at certain promoters. In vivo and in vitro experiments suggest that these responses might be mediated through the TATA-binding protein (TBP) component of TFIID (Koleske et al 1992;Usheva et al 1992;Thompson et al 1993).…”

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confidence: 99%

Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing.

Weeks¹,

Hardin²,

Shen³

et al. 1993

Genes & Development

172

146

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To investigate functional differences between RNA polymerases IIA and II0 (Pol IIA and Pol II0), with hypoand hyperphosphorylated carboxy-terminal repeat domains (CTDs), respectively, we have visualized the in vivo distributions of the differentially phosphorylated forms of Pol II on Drosophila polytene chromosomes.Using phosphorylation state-sensitive antibodies and immunofluorescence microscopy with digital imaging, we find Pol IIA and Pol II0 arrayed in markedly different, locus-and condition-specific patterns. Major ecdysone-induced puffs, for example, stain exclusively for Pol II0, indicating that hyperphosphorylated Pol II is the transcriptionally active form of the enzyme on these genes. In striking contrast, induced heat shock puffs stain strongly for both Pol IIA and Pol II0, suggesting that heat shock genes are transcribed by a mixture of hypo-and hyperphosphorylated forms of Pol II. At the insertion sites of a transposon carrying a hybrid hsp70-lacZ transgene, we observe only Pol IIA before heat shock induction, consistent with the idea that Pol II arrested on the hsp70 gene is form IIA. After a 90-sec heat shock, we detect heat shock factor (HSF) at the transposon insertion sites; and after a 5-min shock its spatial distribution on the induced transgene puffs is clearly resolved from that of Pol II. Finally, using antibodies to hnRNP proteins and splicing components, we have discerned an apparent overall correlation between the presence and processing of nascent transcripts and the presence of Pol II0.[Key Words: RNA polymerase IIA/II0; CTD phosphorylation; transcription factors; in situ localization; immunofluorescence microscopy; digital imaging] Received July 28, 1993; revised version accepted September 29, 1993.The carboxy-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is an unusual entity composed of multiple repeats of the 7-amino-acid consensus sequence YSPTSPS (for review, see Corden 1990;Young 1991). The number of repeats ranges from 26 in yeast, to 42 in Drosophila, to 52 in mammals. The CTD has been shown to carry out essential in vivo roles in yeast (Nonet et al. 1987), Drosophila (Zehring et al. 1988), and mammalian cells (Bartolomei et al. 1988), but precisely what those roles are is not well understood. Suggested roles for the CTD include interacting with transcription initiation factors, serving as a molecular "cowcatcher" to facilitate movement of polymerase on chromatin templates, providing a link between transcription and RNA processing, and localizing polymerase to specific nuclear compartments (Corden 1990 (Allison et al. 1988;Scafe et al. 1990;Peterson et al. 1991), suggesting that the CTD may be involved in receiving regulatory signals at certain promoters. In vivo and in vitro experiments suggest that these responses might be mediated through the TATA-binding protein (TBP) component of TFIID (Koleske et al. 1992;Usheva et al. 1992;Thompson et al. 1993). On the other hand, the precise nature of the CTD involvement in initiation is not known, ...

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“…The viral transactivating protein HBx interacts with RPB5 (46,47). Successive truncation of the C-terminal domain of RPB1 in yeast results in the decreased transcriptional induction from multiple genes and decreased response to specific activators (26,27), probably because of effects on the Pol II holoenzyme. An RPB5 point mutant similarly has decreased transcriptional induction of multiple genes, and RPB5 and C-terminal domain truncation double mutants are synthetically lethal, suggesting an overlap in function for the two subunits (28).…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of individual Pol II subunits in yeast has revealed a role for a number of Pol II subunits in polymerase assembly (16)(17)(18)(19)(20), elongation (21,22), and accurate initiation of transcription (23)(24)(25). However, only the C-terminal domain of RPB1 and the full-length polypeptides of RPB5 and RPB7 have been shown to function in the stimulation of transcription from specific promoters (26)(27)(28)(29)). …”

Section: R Egulation Of Class II Gene Expression In Eukaryotes Involvesmentioning

confidence: 99%

BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10alpha in activated transcription

Schlegel

Green²,

Ladias³

et al. 2000

Proceedings of the National Academy of Sciences

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The functions of most of the 12 subunits of the RNA polymerase II (Pol II) enzyme are unknown. In this study, we demonstrate that two of the subunits, hRPB2 and hRPB10␣, mediate the regulated stimulation of transcription. We find that the transcriptional coactivator BRCA1 interacts directly with the core Pol II complex in vitro. We tested whether single subunits from Pol II would compete with the intact Pol II complex to inhibit transcription stimulated by BRCA1. Excess purified Pol II subunits hRPB2 or hRPB10␣ blocked BRCA1-and VP16-dependent transcriptional activation in vitro with minimal effect on basal transcription. No other Pol II subunits tested inhibited activated transcription in these assays. Furthermore, hRPB10␣, but not hRPB2, blocked Sp1-dependent activation.R egulation of class II gene expression in eukaryotes involves a myriad of transcription factors and protein complexes that ultimately exert control over the enzymatic activity of the multisubunit RNA polymerase II enzyme (Pol II). Pol II and the general transcription factors bind to template DNA in a sequential fashion before the initiation of transcription to form a preinitiation complex (1-3). The process is more complex because Pol II also exists in a preformed complex known as the Pol II holoenzyme, which contains general transcription factors, mediator and SRB proteins, chromatin remodeling factors, and coactivators such as BRCA1 and CBP (4). Further, regulated transcription requires promoter-specific DNA-binding factors to target stimulation of specific genes and multiple other factors that function in the gene activation process (5, 6).Although hundreds of polypeptides orchestrate gene expression in the nucleus, the mass of a protein required to catalyze a DNA-dependent RNA synthesis is not excessive. Indeed, a single 99-kDa polypeptide, such as the bacteriophage T7 RNA polymerase, is sufficient to catalyze promoter-dependent RNA polymerization (7). Bacterial RNA polymerase consists of an enzymatic core of three subunits (␣ 2 ␤␤Ј) and a variable specificity factor ( ) that selects for various classes of promoters. Promoter-specific activators primarily interact with the ␣ and subunits, although all four subunits can be targets (8-12). By analogy, some of the many subunits of the eukaryotic RNA polymerase would also be targets for activators.Human Pol II consists of 12 subunits (see Table 1 and ref. 14). Several subunits are homologous to prokaryotic subunits, and presumably function similarly. The two largest subunits, hRPB1 and hRPB2, are homologues of the prokaryotic ␤Ј and ␤ subunits that bind DNA and nucleotide triphosphate substrate, respectively (15). hRPB3 contains some sequence similarity to ␣, and likewise contributes to core Pol II assembly. Analysis of individual Pol II subunits in yeast has revealed a role for a number of Pol II subunits in polymerase assembly (16)(17)(18)(19)(20), elongation (21,22), and accurate initiation of transcription (23-25). However, only the C-terminal domain of RPB1 and the full-length polypeptide...

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RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals

Cited by 161 publications

References 30 publications

Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing.

BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10alpha in activated transcription

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