Biology Reference
In-Depth Information
mechanisms (
Luo et al., 2006
). Whatever the mechanism, within operons, this
termination signal either never occurs or is inhibited. Polymerase continues to
transcribe downstream genes and only terminates after the last gene in the operon
(
Haenni et al., 2009
).
Several features of the operon help carry out the processing. The nucleotides
between the polyadenylation site of an upstream gene and the trans-splice site of a
downstream gene constitute the ICR. Typically, an ICR is around 100 nt long,
although examples of significantly longer ICRs have been observed
(
Blumenthal et al., 2002
). This region and the nearby upstream 3
0
end processing
signals have been extensively examined for sequence elements and motifs that
enable RNA processing machinery to suspend transcription termination and cor-
rectly SL2 trans-splice downstream genes (
Huang et al., 2001
). An upstream poly-
adenylation site is not necessary to specify downstream SL2 trans-splicing, although
it does appear to play some role in favoring SL2 over SL1 (
Kuersten et al., 1997;
Spieth et al., 1993
). Removal of the AAUAAA still allows SL2 trans-splicing, but
SL1 trans-splicing increases.
The fact that the AAUAAA is somehow involved and the strong tendency for the
ICR to be just about 100 bp long suggested the 3
0
end formation machinery might be
involved in the specification of SL2 trans-splicing. It is possible that the AAUAAA
specifies downstream transcription termination, and the short ICR ensures that
downstream trans-splicing occurs before termination occurs (
Spieth et al., 1993
).
Also, analysis of the SL2 snRNP showed that it coimmunoprecipitates with a protein
component of the 3
0
end processing machinery, named for its mammalian ortholog-
cleavage stimulation factor, 64kd subunit (CstF-64) (
Evans et al., 2001
).
Importantly, residues in the third stem-loop, necessary for SL2 trans-splicing spec-
ificity (
Evans and Blumenthal, 2000
), were also required for CstF-64 interaction.
Analysis of the sequence within the ICR identified the uridylate-rich (Ur) element
(
Huang et al., 2001
). It was initially thought to have no consensus sequence and be
identifiable only based on its nucleotide content. The mutation of this element
resulted in complete loss of downstream RNA. If 3
0
end formation was prevented,
downstream RNA did accumulate, but if the Ur element was absent, only SL1 trans-
splicing was observed. Further in vivo analysis showed that, in an operon construct
driven by a heat shock promoter, uncapped RNA extending from the 5
0
end of the Ur
element through the downstream gene could be detected (
Liu et al., 2003
), indicating
that this element likely transiently stabilized downstream pre-mRNA to allow SL2
trans-splicing to occur. This stability could be recapitulated by the replacement of
the Ur element with MS2 coat protein binding sites combined with expression of the
MS2 coat protein.
Given the established relationship between the SL2 snRNP and CstF64, it was
initially hypothesized that the Ur element served as the binding site for this proces-
sing factor. However, recent in vitro mutational analysis of both the 3
0
end formation
signals and Ur element, coupled with a bioinformatic analysis of trans-splicing
control elements have shown that CstF64 actually binds to a U-rich sequence just
downstream from the 3
0
cleavage site. The Ur element
is a separate feature
Search WWH ::
Custom Search