Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

Shalini Sharma, Somsakul Pop Wongpalee, Ajay Vashisht, James A. Wohlschlegel, Douglas L. Black

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2518-2531
Number of pages14
JournalGenes and Development
Volume28
Issue number22
DOIs
StatePublished - Nov 15 2014

Fingerprint

U2 Small Nuclear Ribonucleoproteins
Small Nuclear Ribonucleoproteins
Spliceosomes
RNA Splice Sites
Proteins
Introns
U1 Small Nuclear Ribonucleoproteins
Isotope Labeling
RNA Precursors
Alternative Splicing
Biotin
U1 small nuclear RNA
Mass Spectrometry
Cell Culture Techniques
RNA
Amino Acids
Messenger RNA

Keywords

  • Alternative splicing
  • Gene expression
  • Pre-mRNA splicing
  • Ribonucleoprotein
  • RNA–protein interaction
  • snRNA
  • Spliceosome

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Medicine(all)

Cite this

Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly. / Sharma, Shalini; Wongpalee, Somsakul Pop; Vashisht, Ajay; Wohlschlegel, James A.; Black, Douglas L.

In: Genes and Development, Vol. 28, No. 22, 15.11.2014, p. 2518-2531.

Research output: Contribution to journalArticle

Sharma, Shalini ; Wongpalee, Somsakul Pop ; Vashisht, Ajay ; Wohlschlegel, James A. ; Black, Douglas L. / Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly. In: Genes and Development. 2014 ; Vol. 28, No. 22. pp. 2518-2531.
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