A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2

Shen, Haihong; Kan, Julie L.C.; Ghigna, Claudia; Biamonti, Giuseppe; Green, Michael R.

doi:10.1261/rna.5229704

Cited by 50 publications

(60 citation statements)

References 35 publications

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“…This complex is not splicing-specific, as it assembles not only on T7 pre-mRNAs but also on T7 RNAs lacking functional splice sites (Konarska and Sharp 1986;Michaud and Reed 1991;Bennett et al 1992). It is well known that at least some hnRNP proteins inhibit splicing and that these proteins compete with essential splicing factors for binding to pre-mRNA in vitro (Caputi et al 1999;Wagner and Garcia-Blanco 2001;Zhu et al 2001;Black 2003;Rooke et al 2003;Fu 2004;Shen et al 2004;Wang et al 2004;Han et al 2005;Rothrock et al 2005). Consistent with this conclusion, previous studies indicate that the H complex is not a functional precursor to the spliceosome (Michaud andReed 1991, 1993).…”

Section: Discussionmentioning

confidence: 62%

Functional coupling of RNAP II transcription to spliceosome assembly

Das¹,

Dufu²,

Romney³

et al. 2006

Genes Dev.

104

117

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The pathway of gene expression in higher eukaryotes involves a highly complex network of physical and functional interactions among the different machines involved in each step of the pathway. Here we established an efficient in vitro system to determine how RNA polymerase II (RNAP II) transcription is functionally coupled to pre-mRNA splicing. Strikingly, our data show that nascent premessenger RNA (pre-mRNA) synthesized by RNAP II is immediately and quantitatively directed into the spliceosome assembly pathway. In contrast, nascent pre-mRNA synthesized by T7 RNA polymerase is quantitatively assembled into the nonspecific H complex, which consists of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and is inhibitory for spliceosome assembly. Consequently, RNAP II transcription results in a dramatic increase in both the kinetics of splicing and overall yield of spliced mRNA relative to that observed for T7 transcription. We conclude that RNAP II mediates the functional coupling of transcription to splicing by directing the nascent pre-mRNA into spliceosome assembly, thereby bypassing interaction of the pre-mRNA with the inhibitory hnRNP proteins.

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Section: Discussionmentioning

confidence: 62%

Functional coupling of RNAP II transcription to spliceosome assembly

Das¹,

Dufu²,

Romney³

et al. 2006

Genes Dev.

104

117

View full text Add to dashboard Cite

show abstract

“…Structurally, PTB consists of four RNA recognition motif (RRM) domains, and its optimal binding site determined by SELEX comprises motifs such as UCUU in a generally pyrimidine-rich context (Perez et al 1997a). Motifs identical or similar to this have been found to act as splicing silencer elements in a wide range of pre-mRNA substrates and are often located adjacent to exons that are included with neuronal or muscle specificity (Mulligan et al 1992;Ashiya and Grabowski 1997;Chan and Black 1997;Perez et al 1997a;Gooding et al 1998;Southby et al 1999;Zhang et al 1999;Wagner and Garcia-Blanco 2002;Shen et al 2004;Izquierdo et al 2005;Sauliere et al 2006). Structurefunction analysis initially indicated that RRMs 3 and 4 are principally responsible for RNA binding, while the N-terminal half of the protein might be responsible for PTB dimerization (Perez et al 1997b;Oh et al 1998).…”

Section: Introductionmentioning

confidence: 99%

Repression of α-actinin SM exon splicing by assisted binding of PTB to the polypyrimidine tract

Matlin

Southby²,

Gooding³

et al. 2007

RNA

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Polypyrimidine tract binding protein (PTB) acts as a regulatory repressor of a large number of alternatively spliced exons, often requiring multiple binding sites in order to repress splicing. In one case, cooperative binding of PTB has been shown to accompany repression. The SM exon of the a-actinin pre-mRNA is also repressed by PTB, leading to inclusion of the alternative upstream NM exon. The SM exon has a distant branch point located 386 nt upstream of the exon with an adjacent 26 nucleotide pyrimidine tract. Here we have analyzed PTB binding to the NM and SM exon region of the a-actinin pre-mRNA. We find that three regions of the intron bind PTB, including the 39 end of the polypyrimidine tract (PPT) and two additional regions between the PPT and the SM exon. The downstream PTB binding sites are essential for full repression and promote binding of PTB to the PPT with a consequent reduction in U2AF 65 binding. Our results are consistent with a repressive mechanism in which cooperative binding of PTB to the PPT competes with binding of U2AF 65 , thereby specifically blocking splicing of the SM exon.

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“…However, other studies failed to find increased ESE-dependent U2AF cross-linking (Guth et al 1999;Kan and Green 1999;Li and Blencowe 1999), possibly due to different experimental conditions (Guth et al 2001). In one instance, an ESE promoted splicing by counteracting the negative effect of a downstream splicing silencer (Kan and Green 1999;Shen et al 2004a). Another alternative to the SR/U2AF protein-protein interaction model for ESE-mediated splicing activation is based on a recent report that a tethered RS domain specifically contacts the branchpoint (Shen et al 2004b).…”

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confidence: 99%

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin

Webb¹,

Romfo²,

Heeckeren³

et al. 2004

Genes Dev.

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Discrete sequence elements known as exonic splicing enhancers (ESEs) have been shown to influence both the efficiency of splicing and the profile of mature mRNAs in multicellular eukaryotes. While the existence of ESEs has not been demonstrated previously in unicellular eukaryotes, the factors known to recognize these elements and mediate their communication with the core splicing machinery are conserved and essential in the fission yeast Schizosaccharomyces pombe. Here, we provide evidence that ESE function is conserved through evolution by demonstrating that three exonic splicing enhancers derived from vertebrates (chicken ASLV, mouse IgM, and human cTNT) promote splicing of two distinct S. pombe pre-messenger RNAs (pre-mRNAs). Second, as in extracts from mammalian cells, ESE function in S. pombe is compromised by mutations and increased distance from the 3 -splice site. Third, three-hybrid analyses indicate that the essential SR (serine/arginine-rich) protein Srp2p, but not the dispensable Srp1p, binds specifically to both native and heterologous purine-rich elements; thus, Srp2p is the likely mediator of ESE function in fission yeast. Finally, we have identified five natural purine-rich elements from S. pombe that promote splicing of our reporter pre-mRNAs. Taken together, these results provide strong evidence that the genesis of ESE-mediated splicing occurred early in eukaryotic evolution.[Keywords: ESE; U2AF; SR protein; Srp1p; Srp2p; ASF/SF2] Supplemental material is available at http://www.genesdev.org. Pre-messenger RNA (pre-mRNA) splicing in all eukaryotes requires conserved intronic sequence elements located at the 5Ј-and 3Ј-splice sites and branchpoint for both accurate recognition of exon/intron boundaries and catalysis of the two transesterification reactions. Nevertheless, it was appreciated early on that intronic signals alone do not suffice for efficient splicing of some vertebrate pre-mRNAs (Reed and Maniatis 1986;Nelson and Green 1988). The discovery that positively acting elements within exons designated exonic splicing enhancers (ESEs) promote splicing of upstream introns exposed additional layers of complexity in metazoan 3Ј-splice site selection (Black 2003). ESEs are found both in premRNAs that are constitutively spliced (Mayeda et al. 1999;Schaal and Maniatis 1999) and those that are subject to regulated splicing (e.g., Ryner and Baker 1991;Tian and Maniatis 1992). A large and growing body of evidence indicates that exonic splicing enhancers are widely distributed among metazoans from flies to hu-

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A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2

Cited by 50 publications

References 35 publications

Functional coupling of RNAP II transcription to spliceosome assembly

Functional coupling of RNAP II transcription to spliceosome assembly

Repression of α-actinin SM exon splicing by assisted binding of PTB to the polypyrimidine tract

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin

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