trans

30 RNA PROCESSING
as Variant Surface Glycoproteins (or VSGs),
are the main target of the host immune
response. In order to understand how VSG
expression was controlled and how one VSG
was ‘switched’ for another during the course
of infection (see Chapter 5), an early priority
was to obtain cDNA and genomic clones
encoding VSGs.
Comparison of such clones revealed that the
cDNA and genomic sequences were collinear
except that the cDNA contained a 39 nucleotide
(nt) sequence at its 5 end not found in the
genomic clone. Interestingly, the genomic
sequence at the point of divergence from the
cDNA corresponded to a potential splice
acceptor site (3 splice site). It was well established
that most genes in higher eukaryotes
contain intervening sequences (introns) that
must be removed by splicing to create functional
mRNAs. Accordingly, it seemed likely
that the 39 nucleotide sequence found at the 5
end of VSG cDNA clones was derived by splicing.
However, extensive analysis of chromosomal
DNA sequences upstream of the VSG gene
did not reveal the 39 nucleotide sequence.
Where then did this sequence come from?
Surprisingly, it was shown that the sequence
was present in the genome in multiple
copies, organized within a tandem repeat.
Furthermore, RNA analysis revealed that these
repeated units were transcribed to yield a small
non-polyadenylated RNA (140 nt) with the
39 nt sequence at its 5 end. Strikingly, the 39 nt
sequence was immediately upstream of a
consensus splice donor site (5 splice site).
Collectively, all of these observations were consistent
with the possibility that the mature VSG
mRNA was generated by splicing of the small
RNA to the pre-VSG mRNA. The joining of two
separate molecules by splicing (trans-splicing)
was unprecedented. However, definitive proof
that trans-splicing indeed occurs in trypanosomatids
came with the demonstration that
intermediates predicted from such a reaction
actually existed (Figure 2.1).
It is now clear that trans-splicing is not confined
to VSG mRNAs. Indeed, available evidence
indicates that all mRNAs in trypanosomes
receive the 39 nt sequence (now known as the
spliced leader, SL) from the small RNA (SL RNA)
via trans-splicing. The biological roles and
mechanism of this unusual RNA processing
reaction are discussed below.
Phylogenetic distribution and
evolutionary origin of trans-splicing
For some time after its discovery, transsplicing
was thought to be idiosyncratic to trypanosomes.
This notion was dispelled when
a careful analysis of actin genes and their
mRNAs was performed in the free-living soil
nematode, C. elegans. Here, it was observed
that mRNAs derived from three of four actin
genes contained a common 22 nt sequence at
their 5 ends. Similar studies to those described
above in trypanosomes showed that the 22 nt
sequence was acquired via trans-splicing from
a small SL RNA. Subsequently, trans-splicing
was shown to be a common feature of gene
expression in all nematodes, both parasitic
and free-living. In these organisms, it is now
evident that most (but not all) pre-mRNAs
receive the SL sequence. The fact that some
mRNAs in nematodes are not subject to transsplicing
contrasts with the situation in trypanosomatids
(see above). A second difference
between nematode and trypanosomatids is
the prevalence of conventional (cis-) introns;
such introns are extremely rare in trypanosome
genes but quite abundant in nematodes. The
significance (if any) of this difference is not yet
known.
Trypanosomatids branched extremely early
in eukaryotic evolution. Nematodes, although
MOLECULAR BIOLOGY