trans

42 RNA PROCESSING
utilize artificial gRNAs and editing substrates,
it proved difficult to distinguish between these
potential roles.
Development of in vitro editing
systems and the current model
for kRNA editing
An important breakthrough in the field was
provided by the development by Stuart and
colleagues of in vitro systems capable of supporting
editing of defined substrates using
cognate gRNAs. This allowed confirmation of
the hypothesis that gRNAs do indeed direct the
insertion and deletion of uridines and set the
stage for specific tests of each model. Initially,
the efficiency of these systems was so low that
intermediates and products could only be characterized
by RT-PCR analysis, but eventually
the efficiency reached levels allowing the use
of isotopically labeled substrates and gRNAs.
This was a critical advance, since it allowed
the direct analysis of reaction intermediates
and products.
The power of this approach can be illustrated
by comparing the predicted outcomes of experiments
utilizing end-labeled molecules for
each of the models depicted in Figure 2.7. For
example, if a 5 end-labeled gRNA (* in Figure
2.7) is used, the transesterification model predicts
that this short RNA would become incorporated
into a larger gRNA-mRNA chimera
during the course of the reaction, whereas
in the cleavage–ligation model the size of
the labeled gRNA would remain unchanged.
Addition of a 3 end-labeled pre-edited mRNA
( in Figure 2.7) would also distinguish
between these models, with the cleavage–
ligation model predicting the production of a
smaller 3 cleavage fragment rather than the
larger chimera anticipated by the transesterification
model. Similarly, because the size of
the 5 cleavage intermediate is only expected
to change in the cleavage–ligation model (upon
addition or deletion of uridines), a 5 endlabeled
pre-edited mRNA (■ in Figure 2.7)
could also be used to differentiate between
these two potential mechanisms.
Data derived from such labeling experiments
strongly support the original cleavage–ligation
model for kRNA editing shown in Figure 2.7A.
Short, 3 cleavage intermediates are produced
in these in vitro assays, and uridines are added
to or removed from the 5 cleavage intermediates.
Although chimeric gRNA-mRNA molecules
have been observed in these experiments,
the kinetics of the appearance of these chimeras
has led to the conclusion that these hybrid molecules
are rare, non-productive side-products
rather than authentic intermediates in the
editing pathway.
Proteins involved in kRNA editing
The cleavage–ligation model of uridine insertion/deletion
predicts the existence of a
number of kinetoplastid proteins, including
endonuclease(s), TUTase, an exonuclease (for
U deletion from the 5 cleavage product),
and RNA ligase(s). These activities have been
detected in, and in some cases purified from,
mitochondrial lysates. Roles for additional proteins
in regulation, assembly of the editing
machinery, processivity, and gRNA unwinding
have been postulated, and candidates for many
of these activities have also been identified.
A number of these enzymatic activities cosediment
on glycerol gradients (fractionating
between 10–40 S, depending on conditions and
species), suggesting that they act as part of one
or more large complexes. A combination of biochemical
(fractionation, UV crosslinking, RNA
binding, adenylation/deadenylation, and helicase
assays), genetic (gene disruption, replacement,
knockouts), and antibody (inhibition,
immunoprecipitation, immunofluorescence
MOLECULAR BIOLOGY