The 65 and 110 kDa SR-related proteins of the U4/U6 center dot U5 tri-snRNP are essential for the assembly of mature spliceosomes

The association of the U4/U6(.)U5 tri-snRNP with pre-spliceosomes is a poor ly understood step in the spliceosome assembly pathway. We have identified two human tri-snRNP proteins (of 65 and 110 kDa) that play an essential rol e in this process. Characterization by cDNA cloning of the 65 and 110 kDa p roteins revealed that they are likely orthologues of the yeast spliceosomal proteins Sad1p and Snu66p, respectively. Immunodepletion of either protein from the HeLa cell nuclear extracts inhibited pre-mRNA splicing due to a b lock in the formation of mature spliceosomes, but had no effect on the inte grity of the U4/U6(.)U5 tri-snRNP, Spliceosome assembly and splicing cataly sis could be restored to the respective depleted extract by the addition of recombinant 65 or 110 kDa protein. Our data demonstrate that both proteins are essential for the recruitment of the tri-snRNP to the pre-spliceosome but not for the maintenance of the tri-snRNP stability. Moreover, since bot h proteins contain an N-terminal RS domain, they could mediate the associat ion of the tri-snRNP with pre-spliceosomes by interaction with members of t he SR protein family.