Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing

Kadener, Sebastián; Cramer, P.; Nogués, Guadalupe; Cazalla, Demián; Mata, Manuel de la; Fededa, Juan Pablo; Werbajh, Santiago; Srebrow, Anabella; Kornblihtt, Alberto R.

doi:10.1093/emboj/20.20.5759

Cited by 124 publications

(133 citation statements)

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“…These findings extend recent studies in which the effect of different promoters and activators on pre-mRNA processing was studied specifically in the context of alternative splicing regulation (24 -27). Several of these studies investigated effects on the inclusion level of the fibronectin EDI exon in the same reporter as we have examined in the present study (pSVEDaGal5HIV2) (24,25,27,35). Stronger promoters or activators resulted in the decreased inclusion of the EDI alternative exon (25,27).…”

Section: Discussionmentioning

confidence: 86%

“…This reporter, which also has an HIV2 promoter and five upstream Gal4 DNA binding sites, contains an alternative EDI exon and flanking constitutive exonic sequences from the human fibronectin gene downstream of the ␣ 1 -globin minigene (pSVEDaGal5HIV2, see Fig. 4A) (24,25) (see below). Consistent with the results observed with the dsx pre-mRNA reporters, when transcription was driven by the Gal4-VP16 and Gal-p53 activators, splicing of ␣ 1 -globin introns 1 and 2 was stimulated by approximately 8-and 4-fold, respectively, compared with when transcription was driven by the Gal4-SW6, -TAT, and -FBP activators (Fig.…”

Section: Figmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 86%

Section: Figmentioning

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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“…Balajee et al (1999) reported that RPII-speci®c transcription in WS B-lymphoblastoid cells transformed with SV40 is 40 ± 60% lower than that of cells from healthy individuals, and that RPII-dependent transcription is stimulated by adding puri®ed wild type, but not mutant WRN, in an in vitro assay using permeabilized cells. Recently, Kadener et al (2001) reported that SV40 large T-antigen activates template replication only twofold but transcription 25-fold. Therefore, it was not appropriate for the transformed WS B-lymphoblastoid cells to be used to compare native transcription activity among cells in the in vitro experiment.…”

Section: Resultsmentioning

confidence: 99%

WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex

et al. 2002

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Werner syndrome (WS) is a rare autosmomal recessive genetic disorder causing premature aging. The gene (WRN) responsible for WS encodes a protein homologous to the RecQ-type helicase. WRN has a nucleolar localization signal and shows intranuclear tra cking between the nucleolus and the nucleoplasm. WRN is recruited into the nucleolus when rRNA transcription is reactivated in quiescent cells. Inhibition of mRNA transcription with a-amanitin has no e ect on nucleolar localization of WRN whereas inhibition of rRNA transcription with actinomycin D releases WRN from nucleoli, suggesting that nucleolar WRN is closely related to rRNA transcription by RNA polymerase I (RPI). A possible function of WRN on rRNA transcription through interaction with RPI is supported by the results described here showing that WRN is coimmunoprecipitated with an RPI subunit, RPA40. Here we show that WS ®broblasts are characterized by a decreased level of rRNA transcription compared with wild-type cells, and that the decreased level of rRNA transcription in WS ®broblasts recovers when wild-type WRN is exogenously expressed. By contrast, exogenously expressed mutant-type WRN lacking an ability to migrate into the nucleolus fails to stimulate rRNA transcription. These results suggest that WRN promotes rRNA transcription as a component of an RPIassociated complex in the nucleolus.

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“…Changes in promoter structure and occupation as well as chromatin structure remodeling have been shown also to modify splicing patterns, indicating a coupling between transcription and alternative splicing (11,12,21). To study the involvement of cis and trans acting factors and the influence of promoter structure and/or chromatin organization on the described splicing regulation, we used a reporter system for EDI alternative splicing (11,12,21).…”

Section: Extracellular Regulation Of Fn Edi Alternative Splicing Is Amentioning

confidence: 99%

Mammary Epithelial-Mesenchymal Interaction Regulates Fibronectin Alternative Splicing via Phosphatidylinositol 3-Kinase

Blaustein

Pelisch

Coso

et al. 2004

Journal of Biological Chemistry

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The way alternative splicing is regulated within tissues is not understood. A relevant model of this process is provided by fibronectin, an important extracellular matrix protein that plays a key role in cell adhesion and migration and contains three alternatively spliced regions known as EDI, EDII, and IIICS. We used a cell culture system to simulate mammary epithelial-stromal communication, a process that is crucial for patterning and function of the mammary gland, and studied the effects of extracellular signals on the regulation of fibronectin pre-mRNA alternative splicing. We found that soluble factors from a mammary mesenchymal cell-conditioned medium, as well as the growth factors HGF/SF (hepatocyte growth factor/scatter factor), KGF (keratinocyte growth factor), and aFGF (acidic fibroblast growth factor), stimulate EDI and IIICS but not EDII inclusion into fibronectin mRNA in the mammary epithelial cell line SCp2, favoring fibronectin isoforms associated with proliferation, migration, and tissue remodeling. We explored the signaling pathways involved in this regulation and found that the mammary mesenchymal cell-conditioned medium and HGF/SF act through a phosphatidylinositol 3-kinase-dependent cascade to alter fibronectin alternative splicing. This splicing regulation is independent from promoter structure and de novo protein synthesis but does require two exonic elements within EDI. These results shed light on how extracellular stimuli are converted into changes in splicing patterns.Pre-mRNA alternative splicing is a widespread process that regulates gene expression and is the most important source of protein diversity in vertebrates (1). Fibronectin (FN), 1 the best characterized extracellular matrix (ECM) glycoprotein, plays a key role in cell adhesive and migratory behavior related to fundamental processes such as embryogenesis, wound healing, maintenance of tissue integrity, and malignancy. Different FN polypeptides arise through an intricate pattern of alternative splicing in three regions of the single primary transcript, (from 5Ј to 3Ј) extra domain II (EDII), extra domain I (EDI), and type III connecting segment (IIICS) (also called EDB or EIIIB, EDA or EIIIA, and V region, respectively), resulting in up to 12 variants in rodents. FN alternative splicing is modulated in a cell type-, development-, and age-specific manner and therefore constitutes a paradigm for studying the regulation of this complex process (2). EDI and EDII are cassette exons, either excluded from or included into the mature FN mRNA. The third site of alternative splicing, IIICS, is subject to total inclusion, partial inclusion, or total exclusion due to the presence of an internal 3Ј splice site within this exon.In vivo EDIϩ FN is poorly represented in the ECM of adult normal tissues. However, this variant is over-expressed in developing embryos, wound healing, liver fibrosis, ovary granulosa cell proliferation, and some tumors (2, 3). It has been proposed that EDI inclusion may mediate a conformational change in the whole m...

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Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing

Cited by 124 publications

References 46 publications

Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex

Mammary Epithelial-Mesenchymal Interaction Regulates Fibronectin Alternative Splicing via Phosphatidylinositol 3-Kinase

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