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 between 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 formation in yeast, little is known about the interactions that connect the two snRNP particles in human tri-snRNPs. Here, we describe the molecular characterisation of a 102kD protein form HeLa tri-snRNPs. The 102kD protein exhibits 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 is considered to be a U4/U6-specific protein, the human 102kD protein was found 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 protein specifically and exclusively immunoprecipitate free U5 snRNPs, but not [U4/U6.U5] tri-snRNPs, from HeLa nuclear extract, suggesting that the C-terminal 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 interacts within the tri-snRNP with both the U5 and U4/U6 snRNPs, thus bridging the two particles. Consistent with this idea, we show that in vitro translated U5-102kD protein binds to purified 13S U4/U6 snRNPs, which contain, in addition to the Sm proteins, all known U4/U6-specific proteins.
Makarov
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EM
Institut für Molekularbiologie und Tumorforschung, Emil Mannkopff-Str. 2, Philipps-Universität, 35037, Germany.
Makarova
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OV
Achsel
T
T
Lührmann
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R
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D019281:Dimerization; D020224:Expressed Sequence Tags; D005656:Fungal Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011233:Precipitin Tests; D011485:Protein Binding; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors
D002352:Carrier Proteins; D005656:Fungal Proteins; C065573:PRP6 protein, S cerevisiae; C408605:PRPF6 protein, human; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors