Requirement of the carboxy‐terminal domain of RNA polymerase II for the transcriptional activation of chromosomal <i>c‐fos</i> and <i>hsp</i>70A genes

Meininghaus, Mark; Eick, Dirk

doi:10.1016/s0014-5793(99)00184-2

Cited by 11 publications

(11 citation statements)

References 21 publications

(31 reference statements)

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“…Mapping of the 5Ј-end of RNAs demonstrated that pol II 1-22, pol II 31-52, and pol II 1-52 all initiated transcription at the expected position. As expected from previous reports (Gerber et al 1995;Okamoto et al 1996;Meininghaus and Eick 1999), pol II lacking a CTD (pol II ⌬) did not transcribe RNA from this minigene reporter (Fig. 2B, lane 1).…”

Section: Analysis Of the Ctd Requirement In Mrna Processingsupporting

confidence: 91%

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Analysis of the requirement for RNA polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 3′-end cleavage

Rosonina

Blencowe²

2004

RNA

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The carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) plays an important role in coupling transcription with precursor messenger RNA (pre-mRNA) processing. Efficient capping, splicing, and 3-end cleavage of pre-mRNA depend on the CTD. Moreover, specific processing factors are known to associate with this structure. The CTD is therefore thought to act as a platform that facilitates the assembly of complexes required for the processing of nascent transcripts. The mammalian CTD contains 52 tandemly repeated heptapeptides with the consensus sequence YSPTSPS. The C-terminal half of the mammalian CTD contains mostly repeats that diverge from this consensus sequence, whereas the N-terminal half contains mostly repeats that match the consensus sequence. Here, we demonstrate that 22 tandem repeats, from either the conserved or divergent halves of the CTD, are sufficient for approximate wild-type levels of transcription, splicing, and 3-end cleavage of two different pre-mRNAs, one containing a constitutively spliced intron, and the other containing an intron that depends on an exon enhancer for efficient splicing. In contrast, each block of 22 repeats is not sufficient for efficient inclusion of an alternatively spliced exon in another pre-mRNA. In this case, a longer CTD is important for counteracting the negative effect of a splicing silencer element located within the alternative exon. Our results indicate that the length, rather than the composition of CTD repeats, can be the major determinant in efficient processing of different pre-mRNA substrates. However, the extent of this length requirement depends on specific sequence features within the pre-mRNA substrate.

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Section: Analysis Of the Ctd Requirement In Mrna Processingsupporting

confidence: 91%

“…Truncation of the CTD in yeast and mammals reduces activator-dependent stimulation of transcription of some genes (Allison and Ingles 1989;Gerber et al 1995;Meininghaus and Eick 1999). Moreover, differential phosphorylation of the CTD correlates with steps in the transcription cycle.…”

Section: Introductionmentioning

confidence: 99%

Analysis of the requirement for RNA polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 3′-end cleavage

Rosonina

Blencowe²

2004

RNA

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“…Deletion of CTD Affects Initiation of pol II-We have shown previously that transiently expressed LS*wt but not LS*⌬5 is able to induce expression of the hsp70A and c-fos genes in 293 cells after appropriate stimuli (51). We confirmed this observation for Raji cells.…”

Section: Conditional Expression Of the Large Subunit Of Pol Ii-supporting

confidence: 80%

Conditional Expression of RNA Polymerase II in Mammalian Cells

Meininghaus

Chapman

Horndasch

et al. 2000

Journal of Biological Chemistry

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The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an ␣-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*⌬5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of ␣-amanitin. Moreover, pol II harboring LS*⌬5 failed to transcribe up to the promoterproximal pause sites in the hsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA.Eukaryotic mRNA synthesis is catalyzed by the multisubunit RNA polymerase II (pol II).1 The large subunit of pol II (LS) is highly conserved among eukaryotic RNA polymerases and also shows striking homology to the large subunit of Escherichia coli RNA polymerase (1). The LS has evolved a particularly structured carboxyl-terminal domain (CTD) that is not present in other RNA polymerases (2). This CTD comprises multiple copies of a heptapeptide repeat with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats varies from 26/27 in yeast to 52 in mouse and human cells (3). Deletion of more than half of the repeats in yeast and mouse interferes with cell viability (4 -6). Mice homozygous for a deletion of 13 repeats are smaller than wild-type littermates and have a high rate of neonatal lethality (7), suggesting that CTD is important in regulating growth during mammalian development. In cells, two forms of pol II are detectable containing either a hypophosphorylated (pol IIA) or hyperphosphorylated CTD (pol II0). Although pol IIA is consistently found in the initiation complex, pol II0 is associated with elongating complexes.An increasing number of genes have been shown to be regulated by promoter-proximal pausing of pol II. These genes include Drosophila hsp70 and hsp26 genes, as well as the mammalian c-myc, c-fos, and immunoglobulin genes (8 -15). The passage of the paused pol II into a processive mode coincides in vivo with hyperphosphorylation of the CTD (11, 16).Recent studies suggest that the hyperphosphorylated CTD functions as a platform for the assembly of complexes that cap, splice, cleave, and polyadenylate pre-mRNA (2, 17, 18). Capping of mRNA occurs shortly after transcription initiation (19), preceding other mRNA processing events such as mRNA splicing and polyadenylation. The capping enzyme is not stably associated with basal transcription factors or the RNA pol II holoenzyme but is directly and specifically recruited to the hyperphosphorylated form of CTD (20 -22, 24). Similarly, several components of the splicing machinery (25, 26) and related factors such as SR proteins and SR-like proteins (27-29) are recruited to pol II by the hyperphosphorylated CT...

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“…In this model, gene activity is primarily controlled by chromatin state and PIC assembly. Promoter-proximal pausing in eukaryotes was characterized at Drosophila major heat shock genes 19,20,112 , and similar analyses revealed human c-myc and c-fos genes appeared to have a similar promoter-proximal pausing [113][114][115] . This mode of regulation was initially considered by the field to be a regulatory step that was specific to a few highly-inducible genes; however, studies tracking nascent RNA synthesis, together with other methods, including genome-wide ChIP-and PIP-seq 67 , demonstrated that promoter-proximal pausing is widespread throughout metazoan genomes 17,18,22,23,44,46,51,55,67,[116][117][118] (reviewed in Refs.…”

Section: Gene Activation Is Defined By Initiation and Promoter-proximal Pausingmentioning

confidence: 99%

Nascent RNA analyses: tracking transcription and its regulation

et al. 2019

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The genome encodes information to program an organism's development and maintenance, and its decoding begins with regulated transcription of genomic DNA into RNA. Transcription and its control can be tracked indirectly by measuring stable RNAs, or directly by measuring nascent RNAs. The latter reveals the immediate regulatory changes in response to developmental, environmental, disease, and metabolic signals. Multiple complementary methods have been developed to quantitatively track nascent transcription genome-wide at nucleotide-resolution, providing novel insights to mechanisms of gene regulation and transcription-coupled RNA processing. Here, we critically evaluate the array of strategies used for investigating nascent transcription and discuss recent conceptual advances they provide.

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Requirement of the carboxy‐terminal domain of RNA polymerase II for the transcriptional activation of chromosomal c‐fos and hsp70A genes

Cited by 11 publications

References 21 publications

Analysis of the requirement for RNA polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 3′-end cleavage

Analysis of the requirement for RNA polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 3′-end cleavage

Conditional Expression of RNA Polymerase II in Mammalian Cells

Nascent RNA analyses: tracking transcription and its regulation

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