trans

RNA EDITING 41
5
Editing
block C
Editing
block B
Editing
block A
3
gRNA A
5
3
gRNA B
gRNA C
Fully edited RNA
FIGURE 2.8
Sequential use of different guide RNAs accounts for the 3 to 5 polarity of editing.
(Figure 2.7B) was proposed shortly thereafter,
based largely on the exciting developments in
the fields of catalytic RNAs and RNA splicing.
This model predicted the existence of hybrid
RNAs consisting of 5 gRNA sequences covalently
attached to mRNA sequences at their 3
ends. The presence of such ‘chimeras’ in kinetoplastid
RNAs, albeit at very low abundance
(1 per cell), further intensified the debate
regarding the relative merits of individual
models.
Each of the proposed mechanisms served to
explain many of the observations present in
the literature. For instance, in all models of
gRNA-directed editing the apparent 3 to 5
polarity could be explained by sequential utilization
of gRNAs (Figure 2.8). In a pre-edited
mRNA requiring the utilization of many overlapping
gRNAs, only the most 3 gRNA binding
site would be present, with the anchor regions
for the subsequent gRNAs being created by
insertion and deletions of Us within the preceding
editing block. Likewise, examples of misediting
could be explained by ‘mis-guiding’,
i.e. the use of the wrong gRNA for U insertiondeletion
within a given region.
These models also proved extremely valuable
in directing experimental approaches,
since each made specific predictions regarding
reaction intermediates (see below). The most
obvious difference between the models was the
role of the oligoU tail of gRNAs, which in the
cleavage–ligation model serves as a means of
stabilizing the editing complex, perhaps retaining
the 5 cleavage product after endonuclease
cleavage, while in the transesterification model
it provides the attacking group in the first transesterification
reaction and serves as a reservoir
for added or deleted uridine residues. However,
in the absence of an assay system that could
MOLECULAR BIOLOGY