Abstract
Spliceosome assembly follows a well conserved pathway of subunit addition that includes both small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splicing factors. Clf1p is an unusual splicing factor composed almost entirely of direct repeats of the tetratricopeptide repeat (TPR) protein-binding motif. Here we show that the Clf1p protein resides in at least two multisubunit protein complexes, a small nuclear RNA-free structure similar to what was reported as the Prp19p complex (nineteen complex; NTC) and an RNP structure that contains the U2, U5, and U6 small nuclear RNAs. Thirty Ccf (Clf1p complex factor) proteins have been identified by mass spectroscopy or immune detection as known or suspected components of the yeast spliceosome. Deletion of TPR1 or TPR2 from an epitope-tagged Clf1p protein (i.e. Clf1Delta2-TAP) destabilizes Clf1p complexes assembled in vivo, causing the release of the Cef1p and Prp19p NTC factors and decreased association of the Rse1p, Snu114p, and Hsh155p snRNP proteins. In vitro, temperature inactivation of Clf1Delta2p impairs the prespliceosome to spliceosome transition and prevents Prp19p recruitment to the splicing complex. These and related data support the view that the poly-TPR Clf1p splicing factor promotes the functional integration of the U4/U6.U5 tri-snRNP particle into the U1-, U2-dependent prespliceosome.
Highlights
Tri-small nuclear ribonucleoprotein (snRNP) particle account for all of the small nuclear RNAs (snRNAs) and the majority of proteins known to act in splicing
We show that the Clf1p protein resides in at least two multisubunit protein complexes, a small nuclear RNA-free structure similar to what was reported as the Prp19p complex and an RNP structure that contains the U2, U5, and U6 small nuclear RNAs
We show that the poly-tetratricopeptide repeat (TPR) protein, Clf1p, plays a critical role in the union of the two large RNP “halves” of the spliceosome, namely the U1/U2-dependent prespliceosome and the U4/U6.U5 trisnRNP particle
Summary
5Ј-GGGGAGAGGATCGACTAATATTGATATA-3Ј 5Ј-GGCAGTCGGTTTCTCTTCAAATA-3Ј 5Ј-ATGGAAAAGAGAAGATGGAAAAAG-3Ј 5Ј-TCAGGTTGACTTCCCCGCGGAATT-3Ј 5Ј-TACGTATAGTTCCAAGAACTTTGATGGTTTTG-3Ј 5Ј-GCCAAAAAATGGAAACAGGAACAGGCA-3Ј 5Ј-AAGCTTCGGAATGATATGAGGGAA-3Ј 5Ј-GGATCCCGGGTCTTTTGGCGCTTAAGTTGG-3Ј spliceosome transition even in the presence of stable U4/U6.U5 tri-snRNP complexes [10], suggesting that Clf1p acts in the recruitment or retention of the U4/U6.U5 tri-snRNP particle. Whereas Clf1p functions prior to stable spliceosome formation, the NTC is reported to bind the splicing complex later, during or after the U4 snRNA release from the mature spliceosome [20]. This inconsistency and results from the Schizosaccharomyces pombe system that cast doubt on the RNA-free status of the NTC [21] stimulated a detailed analysis of Clf1p function in the spliceosome assembly pathway. Removal of TPR elements from the Clf1p amino terminus results in the conditional release of several essential splicing factors and blocks the recruitment of Prp19p to the splicing apparatus. These observations provide evidence that the N-terminal TPR elements of Clf1p recruit splicing factors required for the successful integration of the two large RNP halves of the spliceosome
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