IN-VITRO RECONSTITUTION OF MAMMALIAN U1 SNRNPS ACTIVE IN SPLICING - THE U1-C PROTEIN ENHANCES THE FORMATION OF EARLY (E) SPLICEOSOMAL COMPLEXES

We have established an in vitro reconstitution/splicing complementatio n system which has allowed the investigation of the role of mammalian U1 snRNP components both in splicing and at the early stages of splice osome formation, U1 snRNPs reconstituted from purified, native snRNP p roteins and either authentic or in vitro transcribed U1 snRNA restored both early (E) splicing complex formation and splicing activity to U1 -depleted extracts, In vitro reconstituted U1 snRNPs possessing an m(3 )G or ApppG cap were equally active in splicing, demonstrating that a physiological cap structure is not absolutely required for U1 function , However, the presence of an m(7)GpppG or GpppG cap was deleterious t o splicing, most likely due to competition for the m(7)G cap binding p roteins, No significant reduction in splicing or E complex formation w as detected with U1 snRNPs reconstituted from U1 snRNA lacking the RNA binding sites of the U1-70K or U1-A protein (i.e., stem-loop I and II , respectively), Complementation studies with purified HeLa U1 snRNPs lacking subsets of the U1-specific proteins demonstrated a role for th e U1-C, but not U1-A, protein in the formation and/or stabilization of early splicing complexes, Studies with recombinant U1-C protein mutan ts indicated that the N-terminal domain of U1-C is necessary and suffi cient for the stimulation of E complex formation.