Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

Rosonina, Emanuel; Bakowski, Malina A.; McCracken, Susan; Blencowe, Benjamin J.

doi:10.1074/jbc.m307289200

Cited by 62 publications

(59 citation statements)

References 45 publications

(50 reference statements)

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“…Recruitment of several different transcriptional activators to a promoter controls the splicing efficiency of a constitutively spliced Drosophila doublesex intron; strong activators induce higher levels of splicing than weak activators (3). This result is not merely an indirect effect of the relative concentration of splicing precursors since the efficiency of splicing does not correlate with the level of transcripts (3).…”

mentioning

confidence: 76%

“…The MAZ4 Effect Is Promoter-dependent-Alternative splicing decisions have previously been shown to be dependent upon transcription by altering promoter structure (3)(4)(5)(6)(7)(8). To confirm that the MAZ4 effect on splicing was dependent on transcription, the FGFR2 wild-type minigene and insertion constructs were cloned downstream of the adenovirus major late (ADML) promoter.…”

Section: Construction Of Co-transcriptional Splicing Reportermentioning

confidence: 99%

See 1 more Smart Citation

MAZ Elements Alter Transcription Elongation and Silencing of the Fibroblast Growth Factor Receptor 2 Exon IIIb

Robson-Dixon¹,

García-Blanco

2004

Journal of Biological Chemistry

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The fibroblast growth factor receptor 2 (FGFR2) gene exons IIIb and IIIc are alternatively spliced in a mutually exclusive and cell type-specific manner. FGFR2 exon choice depends on both activation and silencing. Exon IIIb silencing requires cis-acting elements upstream and downstream of the exon. To examine the influence of transcription on exon IIIb silencing, the putative RNA polymerase II (RNAPII)-pausing MAZ4 element was inserted at different positions within the FGFR2 minigene construct. MAZ4 insertions 5 to the upstream silencing elements or between exon IIIb and downstream silencing elements result in decreased silencing. An insertion 3 of the downstream silencing elements, however, has no effect on splicing. An RT-PCR elongation assay shows that the MAZ4 site in these constructs is likely to be a RNAPII pause site. Insertion of another RNAPII pause site into the minigene has a similar effect on exon IIIb silencing. Transfection of in vitro transcribed RNA demonstrates that the cell type specificity of FGFR2 alternative splicing requires co-transcriptional splicing. Additionally, changing the promoter alters both FGFR2 minigene splicing and the MAZ4 effect. We propose that RNAPII pauses at the MAZ4 elements resulting in a change in the transcription elongation complex that influences alternative splicing decisions downstream.The processes of transcription and splicing are spatially and temporally linked within the cell (1, 2), and these links may be critical for both constitutive splicing and for the regulation of alternative splicing. Recruitment of several different transcriptional activators to a promoter controls the splicing efficiency of a constitutively spliced Drosophila doublesex intron; strong activators induce higher levels of splicing than weak activators (3). This result is not merely an indirect effect of the relative concentration of splicing precursors since the efficiency of splicing does not correlate with the level of transcripts (3). Similar promoter-dependent effects on splicing have been demonstrated for the alternatively spliced ED1 exon of the fibronectin gene (4 -8). Transcription factors that alter the rate of transcript initiation have no effect on splicing, but transcription factors that alter elongation rate affect ED1 inclusion (6, 7). Conversely, splicing can also influence the efficiency of transcription. Promoter proximal splice sites can enhance gene transcription in vivo, an effect dependent upon the splicing factor U1snRNP (9). Additionally, transcript initiation and elongation can both be enhanced by splicing factors in vitro (10 -12). Thus there is a two-way functional influence between transcription and splicing, each process is capable of influencing the other.One possible mechanism for the effect of different transcriptional activators on splicing is that transcription factors may recruit splicing factors to the transcription complex thus impacting splicing decisions. Indeed, several splicing factors are associated with general transcription factors (e.g. hnRNPF ...

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confidence: 76%

Section: Construction Of Co-transcriptional Splicing Reportermentioning

confidence: 99%

MAZ Elements Alter Transcription Elongation and Silencing of the Fibroblast Growth Factor Receptor 2 Exon IIIb

Robson-Dixon¹,

García-Blanco

2004

Journal of Biological Chemistry

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“…Recognition of the 3Ј-terminal exons has been postulated to involve an interaction of splicing and polyadenylation factors, and a growing number of reports support this view (42)(43)(44)(45)(46)(47)(48)(49)(50). Because the RS domain of SR proteins mediates protein-protein interactions that are important for spliceosome assembly The cells were fixed and immunostained with antibodies against nuclear markers (red).…”

Section: Cf I M 68 Interacts With the 25-kda Protein Via Its Rrm-mentioning

confidence: 99%

Distinct Sequence Motifs within the 68-kDa Subunit of Cleavage Factor Im Mediate RNA Binding, Protein-Protein Interactions, and Subcellular Localization

Dettwiler

Aringhieri

Cardinale

et al. 2004

Journal of Biological Chemistry

140

219

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Eukaryotic mRNA precursors (pre-mRNAs) 1 are synthesized and processed in the nucleus prior to their export to the cytoplasm where they serve as templates for protein synthesis. Transcription is coupled spatially and temporally to the capping of the pre-mRNA at the 5Ј-end, splicing, and 3Ј-end formation. The mature 3Ј-ends of most eukaryotic mRNAs are generated by endonucleolytic cleavage of the primary transcript followed by the addition of a poly(A) tail to the upstream cleavage product (for reviews see Refs. 1 and 2). In mammals, these reactions are catalyzed by a large multicomponent complex that is assembled in a cooperative manner on specific cis-acting sequence elements in the pre-mRNA. The cleavage and polyadenylation specificity factor (CPSF) (3) recognizes the highly conserved hexanucleotide AAUAAA, whereas the cleavage stimulation factor (CstF) (4) binds a more degenerate GUor U-rich element downstream of the poly(A) site. It has been suggested that in vivo CPSF and CstF may become associated with each other prior to pre-mRNA binding, recognizing the two elements in a concerted manner (5). In addition, the cleavage reaction requires mammalian cleavage factor I (CF I m ), cleavage factor II m (CF II m ), and poly(A) polymerase (PAP). After the first step of 3Ј-end processing, CPSF remains bound to the upstream cleavage fragment and tethers PAP to the 3Ј-end of the pre-mRNA (6). In the presence of the nuclear poly(A)-binding protein 1 (PABPN1), PAP elongates the poly(A) tail in a processive manner (6). These factors are both necessary and sufficient to reconstitute cleavage and polyadenylation in vitro. However, the other proteins involved in either transcription, such as the C-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (7-9).Three major polypeptides of 25, 59, and 68 kDa and one minor polypeptide of 72 kDa copurify with CF I m activity from HeLa cell nuclear extract (10). Reconstitution of CF I m activity with recombinant proteins suggests that CF I m is a heterodimer consisting of the 25-kDa subunit and one of the larger polypeptides (11). All of the three larger proteins appear to be highly related in their amino acid sequence. Moreover, all of the CF I m subunits are only present in metazoan organisms. The primary sequence of the 25-kDa polypeptide contains a NUDIX motif (12). The amino acid composition of the 68-kDa protein has a domain organization that is reminiscent of spliceosomal SR proteins. Members of the SR family of splicing factors contain one or more N-terminal RNA recognition motifs (RRMs) that function in sequence-specific RNA binding and a C-terminal domain rich in alternating arginine and serine residues, referred to as RS domain, which is required for proteinprotein interactions with other RS domains (13). In the 68-kDa protein, the RRM and the RS-like domain are separated by a

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“…In a subsequent study by the same investigators, it was demonstrated that factors influencing pol II processivity, including transcriptional activators, influence EDI inclusion levels, apparently by enhancing or delaying the transcription of cis-acting splicing-regulatory sequences that modulate the selection of adjacent splice sites . We recently showed that strong transcriptional activators can result in the increased pre-mRNA splicing of constitutive introns, as well as increased cleavage levels (Rosonina et al 2003). These effects of strong activators were shown to be dependent on the pol II CTD.…”

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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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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Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

Cited by 62 publications

References 45 publications

MAZ Elements Alter Transcription Elongation and Silencing of the Fibroblast Growth Factor Receptor 2 Exon IIIb

MAZ Elements Alter Transcription Elongation and Silencing of the Fibroblast Growth Factor Receptor 2 Exon IIIb

Distinct Sequence Motifs within the 68-kDa Subunit of Cleavage Factor Im Mediate RNA Binding, Protein-Protein Interactions, and Subcellular Localization

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

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