trans

RNA EDITING 39
entire transcript results in the creation of long,
translatable mRNAs. Perhaps the most spectacular
example of this type of ‘pan-editing’ is
provided by the 969 nt T. brucei coxIII mRNA,
which contains 547 U insertions and 41 U deletions
relative to the maxicircle genome. The
end result of RNA editing is the creation of
open reading frames predicted to encode polypeptides
homologous to mitochondrial proteins
found in other organisms. These changes,
which involve the addition of 1–8 Us at insertion
sites and the removal of 1–5 Us at deletion
sites, often result in the creation (or removal)
of initiation and termination codons.
The frequency of uridine insertion and deletion
varies considerably among different groups
of kinetoplastid protozoa, with more extensive
editing generally found in early-diverging
branches of the phylogenetic tree. For instance,
12 of 17 maxicircle genes in both T. brucei and
Leishmania tarentolae are edited, but T. brucei
editing involves the insertion of 3030 U residues
and the deletion of 322 Us, whereas L. tarentolae
kRNAs are subject to ‘only’ 348 U insertions
and 56 U deletions. At first it was thought that
editing might have arisen as an adaptation to
a parasitic lifestyle, but this seems not to be
the case, since bodonids, a free-living group of
kinetoplastid protists, also edit their RNAs in
a similar manner. Interestingly, many editing
events are developmentally regulated (see
Figure 2.6), but the extent to which these alterations
are used to regulate gene expression is
still unclear.
Potential clues as to mechanism
Despite the lack of obvious patterns or
sequence contexts surrounding editing sites,
mechanistic hints began to emerge upon characterization
of the steady-state RNAs present
in kinetoplasts. The identification of a substantial
pool of partially edited RNAs that were
processed only near their 3 ends suggested
that editing occurs with a 3 to 5 polarity, a feature
that has since been confirmed (see below).
The key to the mechanism was the discovery
of guide RNAs (gRNAs) by Blum, Bakalara,
and Simpson. These small (55–70 nt) transcripts
are perfectly complementary to short
stretches of fully edited mRNAs if non-Watson–
Crick G-U pairs are allowed, suggesting that
gRNAs are the source of the ‘missing’ information.
(Despite the fact that only A-U and G-C
pairs are normally used during RNA synthesis,
such G-U ‘wobble’ pairs are common in structured
RNAs and are sometimes used in codon
recognition by tRNAs during translation.)
Further characterization of these small RNAs led
to the recognition that they contain three functionally
distinct regions: (i) an anchor region
(5–12 nt at the 5 end of the gRNA) that pairs
with the mRNA just 3 of an editing site, (ii) a
25–35 nt long guiding region that specifies 1–20
sites of U insertion/deletion using both standard
A-U pairs and G-U wobble pairs, and (iii) a
non-encoded 3 oligoU tail of 5–24 nt (Figure
2.7). Many, but not all, gRNAs are encoded
within minicircle sequences, with the number
of gRNAs derived from each minicircle varying
between species.
Editing models
The discovery of gRNAs, partially-edited potential
intermediates, and ‘mis-edited’ RNAs
containing odd junction sequences led to the
elaboration of several editing models. Each
required pairing of a specific gRNA with a region
of the pre-edited mRNA just downstream of an
editing site, cleavage of the RNA chain, addition
(or deletion) of uridines based on the
sequence of the guiding region of the gRNA,
and reformation of the altered mRNA. These
models differed, however, in the means by
which these steps were accomplished. Most
MOLECULAR BIOLOGY