The human homologue of the yeast splicing factor Prp6p contains multiple TPR elements and is stably associated with the U5 snRNP via protein-protein interactions.

An essential step of pre-mRNA spliceosome assembly is the interaction betwe en the snRNPs U4/U6 and U5, to form the [U4/U6.U5] tri-snRNP. While the tri -snRNP protein Prp6p appears to play an important role for tri-snRNP format ion in yeast, little is known about the interactions that connect the two s nRNP particles in human tri-snRNPs. Here, we describe the molecular charact erisation of a 102kD protein form HeLa tri-snRNPs. The 102kD protein exhibi ts a significant degree of overall homology with the yeast Prp6p, including the conservation of multiple tetratrico peptide repeats (TPR), making this the likely functional homologue of Prp6p. However, while the yeast Prp6p i s considered to be a U4/U6-specific protein, the human 102kD protein was fo und to be tightly associated with purified 20 S U5 snRNPs. This association appears to be primarily due to protein-protein interactions. Interestingly , antibodies directed against the C-terminal TPR elements of the 102kD prot ein specifically and exclusively immunoprecipitate free U5 snRNPs, but not [U4/U6.U5] tri-snRNPs, from HeLa nuclear extract, suggesting that the C-ter minal region of the 102kD protein is covered by U4/U6 or tri-snRNP-specific proteins. Since proteins containing TPR elements are typically involved in multiple protein-protein interactions, we suggest that the 102kD protein i nteracts within the tri-snRNP with both the U5 and U4/U6 snRNPs, thus bridg ing the two particles. Consistent with this idea, we show that in vitro tra nslated U5-102kD protein binds to purified 13S U4/U6 snRNPs, which contain, in addition to the Sm proteins, all known U4/U6-specific proteins. (C) 200 0 Academic Press.