Abstract
THE U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA1–5. Mammalian Ul snRNP contains a 165-nucleo-tide long RNA molecule and at least 11 proteins: the Ul-specific 70K proteins A and C, and the common U snRNP proteins (B′, B, Dl, D2, D3, E, F and G). One of the functions of Ul snRNP is recognition of the 5′ splice site, an event that requires both Ul RNA and Ul proteins6–10. The 70K protein is the only heavily phosphorylated Ul protein in the cell11,12. Isolated Ul snRNPs are associated with a kinase activity that selectively phosphorylates the 70K protein in vitro in a reaction requiring ATP. Here we investigate the role of phosphorylation of the 70K protein in the splicing of pre-mRNA. The 70K protein on Ul snRNPs was phosphorylated in vitro with either ATP, or with ATP-γS, which gave a thiophosphorylated product that was resistant to dephos-phorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous Ul snRNPs were complemented with Ul snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated Ul snRNPs were used instead, splicing was completely inhibited, although formation of the mature spliceosome was unaffected. Our data show that the state of phosphorylation of the Ul-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.
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Tazi, J., Kornstädt, U., Rossi, F. et al. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature 363, 283–286 (1993). https://doi.org/10.1038/363283a0
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DOI: https://doi.org/10.1038/363283a0
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