Nucleic Acids Research, Vol 24, Issue 23 4614-4623, Copyright © 1996 by Oxford University Press
CL Will, S Rumpler, J Klein Gunnewiek, WJ van Venrooij and R Luhrmann
We have established an in vitro reconstitution/splicing complementation
system which has allowed the investigation of the role of mammalian U1
snRNP components both in splicing and at the early stages of spliceosome
formation. U1 snRNPs reconstituted from purified, native snRNP proteins 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 m3G 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 m7GpppG or GpppG cap was deleterious to splicing, most likely due to
competition for the m7G cap binding proteins. No significant reduction in
splicing or E complex formation was 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 the U1-C, but not U1-A, protein in the formation
and/or stabilization of early splicing complexes. Studies with recombinant
U1-C protein mutants indicated that the N-terminal domain of U1-C is
necessary and sufficient for the stimulation of E complex formation.
ARTICLES
In vitro reconstitution of mammalian U1 snRNPs active in splicing: the U1-C protein enhances the formation of early (E) spliceosomal complexes
Institut fur Molekularbiologie und Tumorforschung, Philipps Universitat Marburg, Germany.
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