trans

34 RNA PROCESSING
spliceosomal complexes that share most essential
components in common. In this regard,
U2, U4, U5 and U6 snRNPs are required for
both types of splicing as are most, if not all,
non-snRNP associated factors. The most striking
difference between the two types of splicing
is that U1 snRNP, an essential cofactor for
cis-splicing, is not involved in trans-splicing.
As noted above, the function of U1 snRNP is to
recognize cis 5 splice sites; it also helps U2
snRNP associate with the branch point region
and it is of central importance in the joining of
5 and 3 splice sites prior to catalysis.
How these functions are performed in
trans-splicing and how the SL RNA (which
contains the 5 splice site) efficiently associates
with the pre-mRNA (which contains the
3 splice site) have been the most intriguing
questions with regard to the mechanism of
trans-splicing. Recent biochemical analysis
has begun to provide answers to these longstanding
questions.
Early on, it was recognized that the SL RNA
did not function as a naked RNA, but rather as
a ribonucleoprotein particle that shares striking
similarities with the small RNAs that are
cofactors for cis-splicing. Specifically, a subset
of those RNPs (U1, U2, U4 and U5) are known
as Sm-snRNPs because they bind a common
set of seven proteins known as Sm proteins
(Sm simply stands for an antigenic determinant).
All known SL RNPs are Sm snRNPs but
unlike the cis-spliceosomal Sm snRNPs, which
are stable cofactors, SL RNPs are consumed
during the trans-splicing reaction.
We now know that assembly into an SmsnRNP
is a necessary prerequisite for SL RNP
function in trans-splicing. Indeed, the fact that
all SL RNAs known are Sm-snRNPs is one of
the strongest arguments for a common evolutionary
ancestor for trans-splicing (see above).
We also know that assembly into an Sm-snRNP
promotes the association of two additional SL
RNP-specific proteins. These proteins in turn
are essential for SL RNP function. First, they
facilitate association of the SL RNP with U4,
U5 and U6 snRNPs such that the SL RNA’s 5
splice site is recognized independent of U1
snRNP. Second, they provide the mechanism
whereby the SL RNP and pre-mRNA associate.
In this regard, one SL RNP-specific protein
interacts specifically with a protein associated
with the branch point region of the pre-mRNA,
and this interaction is required for transsplicing.
The mechanisms of cross-intron bridging
in cis-splicing and ‘intron-intron’ bridging
in trans-splicing are illustrated schematically
in Figure 2.3. The two functions of the SL RNP
specific proteins adequately explain how
trans-splicing can proceed without U1 snRNP.
Perhaps more importantly, the mechanistic
details of trans-splicing provide an object lesson
in how the same fundamental problem
(i.e. splice site recognition and juxtaposition)
can be solved in different ways. Finally, because
the SL RNP-specific proteins are unambiguously
absent in organisms that do not carry
out trans-splicing (e.g. human hosts) they provide
attractive targets for chemotherapeutic
intervention.
Prior to leaving a discussion of mechanism,
it is necessary to consider how the 3 splice
site of the pre-mRNA is recognized. In cissplicing
(as noted above) 3 splice site recognition
is coupled to and facilitated by 5 splice
site recognition. Obviously, this cannot apply
in trans-splicing, where splice sites are recognized
independently. We now know that 3
splice site identification in trans-splicing is
determined by sequence elements present in
the downstream exon. These elements, known
as exonic splicing enhancers, serve as bindings
sites for proteins known as SR splicing factors.
These SR proteins (so called because they possess
domains rich in serine arginine repeats)
recruit specific proteins to the 3 splice site
MOLECULAR BIOLOGY