U1 snRNA Directly Interacts with Polypyrimidine Tract-Binding Protein during Splicing Repression

Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Molecular cell (Impact Factor: 14.02). 03/2011; 41(5):579-88. DOI: 10.1016/j.molcel.2011.02.012
Source: PubMed


Splicing of the c-src N1 exon is repressed by the polypyrimidine tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5' splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5' splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.

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Available from: Christophe Maris, Jan 05, 2016
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    • "The pairing of splice sites across the intron and the formation of the A complex are appealing steps for the regulation of alternative pre-mRNA splicing. We previously found that the regulator PTBP1 contacts SL4 within an exon complex repressed for splicing (Sharma et al. 2011). Interestingly, the PTBP1-interacting sites on SL4 and the regions required for splicing are nonoverlapping. "
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    ABSTRACT: The pairing of 5' and 3' splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem-loop 4 (SL4) of the U1 snRNA, which recognizes the 5' splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3' splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5' and 3' splice site complexes within the assembling spliceosome. © 2014 Sharma et al.; Published by Cold Spring Harbor Laboratory Press.
    Preview · Article · Nov 2014 · Genes & Development
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    • "PTB can repress splicing via interaction with U1 (45) or U2 snRNA (46). Repression of the C-SRC N1 exon involves interactions of the N-terminal RRMs 1 and 2 of PTB with stem-loop 4 of U1 snRNA bound at the 5′ splice site (45). If PTB acted in a similar fashion at FAS exon 6, we would have expected the RNA binding mutations of RRMs 1 and 2 to have led to a larger loss of function. "
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    ABSTRACT: Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.
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    • "Three of the four proteins from the RNAcompete set showing significant improvements over the sequence models (PTB, RBM4 and U1A) are known to recognize stem-loop structures [51-53]. For PTB, it was determined by ITC, gel shift assays and NMR studies that the two RRM domains bind a stem-loop structure of U1 snRNA [51]. For RBM4, information about possible targets is scarce; however, in one case it was reported that the target of RBM4 is a cis-regulatory element that was predicted to be a stem-loop structure [52]. "
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    ABSTRACT: We present GraphProt, a computational framework for learning sequence- and structure-binding preferences of RNA-binding proteins (RBPs) from high-throughput experimental data. We benchmark GraphProt, demonstrating that the modeled binding preferences conform to the literature, and showcase the biological relevance and two applications of GraphProt models. First, estimated binding affinities correlate with experimental measurements. Second, predicted Ago2 targets display higher levels of expression upon Ago2 knockdown, whereas control targets do not. Computational binding models, such as those provided by GraphProt, are essential to predict RBP-binding sites and affinities in all tissues. GraphProt is freely available at
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