Through a combination of in vitro snRNP reconstitution, photocross-linking
and immunoprecipitation techniques, we have investigated the interaction of
proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and
U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate spe
cifically with this spliceosomal snRNA, three were shown to contact the RNA
directly, and to maintain contact as the U6 RNA is incorporated into tri-s
nRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6,
which is in the conserved region that contributes to the formation of the
catalytic core of the spliceosome. Other tri-snRNP-specific contacts were a
lso detected, indicating the dynamic nature of protein interactions with th
is important snRNA, The uridine-rich extreme 3' end of U6 RNA was shown to
be essential but not sufficient for the association of the Lsm proteins. In
terestingly, the Lsm proteins associate efficiently with the 3' half of U6,
which contains the 3' stem-loop and uridine-rich 3' end, suggesting that t
he Lsm and Sm proteins may recognize similar features in RNAs.