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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    • "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.
    Genes & Development 11/2014; 28(22):2518-31. DOI:10.1101/gad.248625.114 · 10.80 Impact Factor
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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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    • "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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