Stem-loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly.
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.
Department of Basic Medical Sciences, University of Arizona, College of Medicine-Phoenix, Phoenix, Arizona 85004, USA; Department of Microbiology, Immunology, and Molecular Genetics.
Shalini
S
Sharma
Molecular Biology Interdepartmental Graduate Program.
Somsakul Pop
SP
Wongpalee
Department of Biological Chemistry.
Ajay
A
Vashisht
Department of Biological Chemistry.
James A
JA
Wohlschlegel
Department of Microbiology, Immunology, and Molecular Genetics, Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, California 90095, USA.
Douglas L
DL
Black
D006367:HeLa Cells; D006801:Humans; D055029:Inverted Repeat Sequences / Q000235:genetics; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D022821:RNA Splice Sites; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology*; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; C088050:SF3A1 protein, human; C405483:U1 small nuclear RNA
RNA–protein interaction; alternative splicing; gene expression; pre-mRNA splicing; ribonucleoprotein; snRNA; spliceosome