trans

TRANS-SPLICING 33
snRNPs (small nuclear ribonucleoproteins).
Thus, the spliceosome rivals the ribosome in
complexity.
Unlike the ribosome however, the spliceosome
does not exist as a preformed entity, but
rather forms anew on each intron. Spliceosome
assembly involves the ordered recognition of
three conserved sequence elements within
the intron, the 5 splice site (splice donor), the
branch point region, and the 3 splice site
(splice acceptor): First, U1 snRNP recognizes
the 5 splice site via a base-pairing interaction;
U1 snRNP then promotes U2 snRNP binding
to the branch point region, an interaction that
also involves base pairing. Subsequent to
engagement of the substrate by U1 and U2
snRNPs, U4, U5 and U6 snRNPs join to form
a complete spliceosome. After assembly, but
prior to catalysis, a complex series of RNA/
RNA rearrangements occurs, resulting in
destabilization of U1 and U4 snRNPs. Following
spliceosome maturation, catalysis commences.
The actual removal of intervening sequences
occurs through two consecutive transesterification
reactions (the replacement of one
phosphodiester with another). In the first transesterification,
the 2 hydroxyl of the branch
point adenosine attacks the 5 splice site.
This reaction liberates the 5 exon and creates
the so-called lariat intermediate. The
second transesterification then occurs; here
the free 3 hydroxyl of the 5 exon attacks
the 3 splice site resulting in ligated exons
and release of intron in the form of a lariat
(Figure 2.1). Several lines of evidence suggest
(but have not definitively proven) that the
catalytic moieties of the spliceosome are its
RNA constituents. It therefore seems likely that
the spliceosome (like the ribosome) is a
ribozyme.
As noted above, the sequence elements
defining the 5 and 3 splice sites used in transsplicing
conform to consensus cis splice sites.
Furthermore, the intermediates and products
of a trans-splicing reaction are analogous to
those produced by cis-splicing (Figure 2.1).
Accordingly, it seemed likely that the fundamental
mechanism of trans-splicing (i.e. consecutive
trans-esterification reactions) would
be identical to that of cis-splicing. Moreover, it
also seemed likely that the catalytic machinery
(the spliceosome) would be similar if not
identical. These predictions were borne out in
studies both in trypanosomes and nematodes
(see below). Here, it should be noted that
experimental systems useful for studying
trans-splicing are quite limited. Most of our
understanding of the cis-splicing mechanism
has derived from biochemical studies in cellfree
systems derived from either mammalian
cells or yeast. In addition, yeast is genetically
tractable and genetic approaches have been
extremely valuable in dissecting the splicing
mechanism in this organism.
The vast majority of organisms that carry
out trans-splicing are not amenable to either
genetic analysis or biochemical manipulation.
However, certain in vivo approaches (e.g.
transfection, gene inactivation and permeabilization)
are available in trypanosomes, and biochemical
approaches are possible in extracts
derived from homogenized embryos of the
parasitic nematode, Ascaris; these extracts
faithfully and efficiently catalyze both cis and
trans-splicing. Essentially all of our understanding
of the mechanism of trans-splicing
has been obtained in these two systems, and
while it seems likely that results obtained in
nematodes and trypanosomes will apply to
other systems, this has not been proven. The
following discussion makes the assumption
that the mechanism of trans-splicing is similar
in all organisms that carry out the reaction. It
is now clear that cis and trans-splicing are
identical in terms of the chemistry of catalysis.
Furthermore, both reactions occur in large
MOLECULAR BIOLOGY