Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing.
The U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA. Mammalian U1 snRNP contains a 165-nucleotide long RNA molecule and at least 11 proteins: the U1-specific 70K proteins A and C, and the common U snRNP proteins (B', B, D1, D2, D3, E, F and G). One of the functions of U1 snRNP is recognition of the 5' splice site, an event that requires both U1 RNA and U1 proteins. The 70K protein is the only heavily phosphorylated U1 protein in the cell. Isolated U1 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 U1 snRNPs was phosphorylated in vitro with either ATP, or with ATP-gamma S, which gave a thiophosphorylated product that was resistant to dephosphorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous U1 snRNPs were complemented with U1 snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated U1 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 U1-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.
UA CNRS 1191, Génétique Moléculaire Université Montpellier II, France.
J
J
Tazi
U
U
Kornstädt
F
F
Rossi
P
P
Jeanteur
G
G
Cathala
C
C
Brunel
R
R
Lührmann
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism
D012322:RNA Precursors; D017412:Ribonucleoprotein, U1 Small Nuclear; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D010770:Phosphotransferases