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identified on Northern blots, and treatment with debranching enzyme eliminated
these branched RNAs (
Bektesh and Hirsh, 1988
). The 3
0
hydroxyl of the freed leader
then attacks the phosphodiester bond at the 3
0
splice site, covalently attaching itself
to the downstreamRNA as a spliced leader and becoming the first exon of the mature
mRNA. This results in a trans-spliced RNA molecule, complete with the TMG-
capped spliced leader transferred from the SL snRNP (
Liou and Blumenthal, 1990;
Thomas et al., 1988; Van Doren and Hirsh, 1990
). This trans-spliced mRNA mol-
ecule is exported from the nucleus for translation. Because eukaryotic mRNA does
not typically contain a TMG cap, specialized isoforms of the eukaryotic initiation
factor eIF4E have evolved to recognize the TMG cap and initiate translation from
these mRNA molecules (
Keiper et al., 2000; Wallace et al., 2010
).
4. Properties of the Outron
The RNA between the transcriptional start site and the 3
0
splice site used in trans-
splicing is the outron (
Conrad et al., 1991
). Several studies have characterized the
features of an outron that mark pre-mRNAs for trans-splicing. An intron lacking a 5
0
splice site can act as an outron when placed in the 5
0
UTR of a synthetic construct
(
Conrad et al., 1991
). Furthermore, if a 5
0
splice site was constructed upstream of the
3
0
splice site used in trans-splicing, RNA produced from the construct was cis-
spliced at the trans-splice site. The outron, in effect, became an exon and an intron
(
Conrad et al., 1993a
). Several constructs in which increasingly long artificial out-
rons, each composed only of AU-rich sequence and a 3
0
splice site, were placed
upstream of a gene were found to be trans-spliced efficiently if the outron was at least
51 nt in length (
Conrad et al., 1995
). These studies were interpreted to mean that an
outron contains no sequences or features necessary for trans-splicing, although a
loose consensus could have been inadvertently contained in the synthetic sequence.
In addition, there appears to be a minimal length requirement, as has been observed
for introns. Apparently, a 3
0
splice site preceded by an adequately sized AU-rich
sequence with no upstream 5
0
splice site is sufficient to promote trans-splicing.
However, a recent bioinformatic analysis of information content of C. elegans out-
rons has identified a very weak consensus sequence, UUUUCUUU, termed the Ou
element, centered about 50 nt upstream from the trans-splice site (
Graber et al.,
2007
). The functional significance of this motif remains to be investigated.
The removal and subsequent destruction of the 5
0
section of many pre-mRNA
molecules by trans-splicing has complicated the task of determining the transcrip-
tional start site of trans-spliced genes. Like cis-splicing, trans-splicing is a very
efficient process (
Blumenthal and Steward, 1997; Cramer et al., 2001
). Also, most
C. elegans genes do not have obvious TATA boxes to indicate the approximate
location of transcription start sites. For these reasons, the transcriptional start site is
known for only a handful of trans-spliced genes (e.g.,
Kramer et al., 1990; Park and
Kramer, 1990
). Traditional RNA-end identificationmethods, such as 5
0
RACE, SAGE,
TEC-RED, and even primer extension can identify the presence and location of the
spliced leader on mRNA molecules, but, since trans-splicing most likely happens
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