Characterization of the essential pre-mRNA splicing factor PSF: investigation of RNA binding specificity and splicing-related complex formation
PSF (PTB-associated splicing factor) has been implicated in both early and late steps of pre-mRNA splicing as well as multiple nuclear events including transcription regulation, nuclear RNA retention/export, topoisomerase activity, and DNA recombination. We found that PSF and its closely related protein p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with the U4/U6.U5 tri-snPNP. Further analysis of the association of PSF with U5 snRNP resulted in the interesting discovery that a portion of PSF is contained within a large multi-snRNP complex (PCC complex). The formation of the PCC complex occurs when HeLa cell nuclear extracts are adjusted to splicing conditions but surprisingly does not require the addition of pre-mRNA nor ATP hydrolysis. Sedimentation analyses demonstrated that the complex contains all of the five splicing snRNPs and has a size close to the spliceosome. Approximately 70 proteins were identified in the PCC complex by mass spectrometry analysis, and 70% of them are either known spliceosomal proteins or proteins related to splicing. This suggests that penta-snRNPs and/or pre-formed spliceosomes may exist in mammalian cells.