trans

RNA EDITING 43
microscopy) approaches are currently being
used to define the proteins involved in the
editing pathway. However, given the complexity
of the system, the large number of components,
and the potential overlap between activities
responsible for U insertion and deletion, thus
far it has been quite difficult to definitively
assign a function (or functions) to most of the
proteins under study.
Editing is an essential process
Because no ‘correct’ copies of most mitochondrial
genes have been found, it has long been
assumed that editing is required for expression
of a number of kinetoplastid gene products.
However, due to the highly hydrophobic
nature of the proteins encoded by these edited
mRNAs, direct evidence for translation of
the products of editing has only lately been
achieved. Very recently, gene knockout experiments
have been used to demonstrate that the
gene encoding an RNA ligase associated with
editing complexes is essential for both RNA
editing and survival of the bloodstream form
of T. brucei, indicating that RNA editing is both
a critical aspect of gene expression and an
inviting target for intervention.
Outstanding questions
Although the general outlines of the editing
mechanism are now clear, many important
challenges still remain. Work is continuing on
the identification and characterization of the
components of the editing apparatus and
the determination of their functional roles in
the editing process. The current in vitro systems
are somewhat limited in that they support
only a single round of nucleotide insertion or
deletion and that only a subset of substrates
are efficiently edited. Examination of the roles of
proteins, such as helicases, that may contribute
to editing processivity will require the use of
assay systems that are capable of carrying out
editing at consecutive sites, and eventually,
sequential use of multiple gRNAs.
The assembly and disassembly of the editing
machinery is another issue that has yet to
be seriously addressed. Although it is clear that
targeting of editing sites is accomplished by
annealing of the gRNA anchor to its cognate
mRNA binding site, the steps involved in this
initial recognition are unknown. It seems likely
that gRNAs are utilized as ribonucleoprotein
particles and it is possible that they are actively
recruited to specific sites by assembly factors.
Many kinetoplastid proteins have been shown
to crosslink to gRNAs, with postulated roles
including gRNA maturation, folding, stability,
and turnover as well as more direct involvement
in the editing process. Other related
questions include (i) the point at which the rest
of the editing machinery is added, (ii) whether
the components of the editing apparatus are
added as a pre-formed complex, as small subassemblies,
or individually, (iii) whether or not
enzymatic activities required for uridine insertion
and deletion are present in the same complexes,
and (iv) the extent of overlap between
insertion and deletion activities (for example,
are separate endonucleases and/or RNA ligases
required for insertion and deletion?). Ultimately,
in vitro reconstitution of a processive
editing activity from purified components will
be needed to definitively answer these difficult
questions.
Finally, the role of editing in the regulation of
kinetoplastid gene expression has not been fully
explored. Mitochondrial function is drastically
reduced in bloodstream parasites and many
aspects of RNA metabolism are developmentally
regulated. There is a differential abundance
of individual mRNAs and gRNAs at various life
cycle stages, and the extent of both editing
and polyadenylation vary with developmental
MOLECULAR BIOLOGY