Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly
- Shalini Sharma1,2,
- Somsakul Pop Wongpalee3,
- Ajay Vashisht4,
- James A. Wohlschlegel4 and
- Douglas L. Black2,5
- 1Department of Basic Medical Sciences, University of Arizona, College of Medicine-Phoenix, Phoenix, Arizona 85004, USA;
- 2Department of Microbiology, Immunology, and Molecular Genetics,
- 3Molecular Biology Interdepartmental Graduate Program,
- 4Department of Biological Chemistry,
- 5Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, California 90095, USA
- Corresponding authors: dougb{at}microbio.ucla.edu, shalinijs{at}email.arizona.edu
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.
Keywords
- RNA–protein interaction
- alternative splicing
- gene expression
- pre-mRNA splicing
- ribonucleoprotein
- snRNA
- spliceosome
Footnotes
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Supplemental material is available for this article.
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.248625.114.
- Received July 4, 2014.
- Accepted October 10, 2014.
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