p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

Abstract

Modulation of the interaction between U1 snRNP and the 5 splice site (5ss) is a key event that governs 5ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5ss during early stage of splicing. However, dissociation of the U1 from the 5ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.To remove introns, a large RNA-protein complex, known as the spliceosome, must be assembled on mRNA precursors (pre-mRNA) (11,29,39). Assembly of a functional spliceosome proceeds through an ordered addition of four small nuclear ribonucleoprotein particles (snRNPs) (U1, U2, U4/U6, and U5) as well as many non-snRNP proteins. This pathway leads to the formation of several intermediate spliceosome complexes. Recognition of the 5Ј splice site (5Јss) by the U1 snRNP, along with binding of the polypyrimidine tract and branch point by U2AF65 and SF1, results in the formation of the commitment complex. Recruitment of the U2 snRNP to the commitment complex leads to the formation of complex A, or the prespliceosome. At this point, the preformed U4/U6.U5 tri-snRNPs will join into the prespliceosome, leading to the formation of the spliceosome (1, 13, 28).The spliceosome is a dynamic molecular machine. Dynamic RNA-RNA base-pairing interactions between snRNAs and pre-mRNA and among snRNAs play critical roles in the spliceosome assembly and recognition of splice sites (32,33,35). The RNA duplexes in the spliceosome complexes are generally transient and short, ranging from 3 to 7 bp. Many of these RNA-RNA interactions are mutually exclusive. One example is the RNA-RNA interactions that occur at the 5Јss. The 5Јss is first recognized by interactions of 5 to 7 bp between the 5Јss and 5Ј-end of the U1 snRNA. This early U1-5Јss duplex is unwound to expose the same 5Јss sequence for pairing with the U6 snRNA prior to the first chemical reaction of splicing (28). The U6 snRNA is associated with U4 by two RNA helices before joining the prespliceosome. The two RNA helices between U6 and U4 must be unwound before U6 pairing with the 5Јss (2, 30, 40). Three events, unwinding of the U1-5Јss duplex, unwinding of the U4/U6 helices, and formation of the U6-5Јss duplex, are tightly coupled. How does the spliceosome coordinate these RNA-RNA interactions precisely in time and space? Presumably, protein factors are ne...

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