Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome.
Several protein kinases, including SRPK1 and SRPK2, have been implicated in spliceosome assembly and catalytic activation. However, little is known about their targets. Here we show that SRPK1 is predominantly associated with U1 small nuclear ribonucleoprotein (snRNP), whereas SRPK2 associates with the U4/U6-U5 tri-snRNP. RNAi-mediated depletion in HeLa cells showed that SRPK2 is essential for cell viability, and it is required for spliceosomal B complex formation. SRPK2 knock down results in hypophosphorylation of the arginine-serine (RS) domain-containing human PRP28 protein (PRP28, also known as DDX23), and destabilizes PRP28 association with the tri-snRNP. Immunodepletion of PRP28 from HeLa cell nuclear extract and complementation studies revealed that PRP28 phosphorylation is required for its stable association with the tri-snRNP and for tri-snRNP integration into the B complex. Our results demonstrate a role for SRPK2 in splicing and reveal a previously unknown function for PRP28 in spliceosome assembly.
Mathew
Rebecca
R
Department of Cellular Biochemistry MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
Hartmuth
Klaus
K
Möhlmann
Sina
S
Urlaub
Henning
H
Ficner
Ralf
R
Lührmann
Reinhard
R
D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism
D034741:RNA, Small Interfering; D017412:Ribonucleoprotein, U1 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; C087728:SRPK1 protein, human; D017346:Protein Serine-Threonine Kinases; C110522:SRPK2 protein, human; C498290:DDX23 protein, human; D053487:DEAD-box RNA Helicases