trans

76 POST-TRANSCRIPTIONAL REGULATION
cis-splicing acceptor site can function as a
trans-splicing acceptor. It is not known whether
this alternative splicing has a regulatory role.
The SIRE transposable element of T. cruzi
contains splice sites of moderate efficiency.
The element is distributed throughout the
genome, preferentially in intergenic regions,
and SIRE sequence is present in many mature
mRNAs, either 5- or 3- to the open reading
frame. The presence of the SIRE element in an
intergenic region can therefore influence the
location and efficiency of both splicing and
polyadenylation, influencing expression of
both upstream and downstream genes.
Before leaving the issue of mRNA processing,
it should be mentioned that the editing of
mitochondrial transcripts exhibits considerable
developmental regulation. This is described
elsewhere in this volume (Chapter 2).
Cell-cycle control and the
5-untranslated region
The insect kinetoplastid Crithidia fasciculata
can be synchronized in culture, unlike
the trypanosomes and mammalian-infective
Leishmanias. This makes it possible to study
the regulation of gene expression through the
cell cycle. As would be expected from other
systems, mRNAs linked to nuclear and mitochondrial
DNA replication, such as those
encoding kinetoplast topoisomerase II (TOP2),
dihydrofolate reductase-thymidylate synthase
(DHFRTS) and the large (RPA1) and middle
(RPA2) subunits of a nuclear single-strand DNA
binding protein all begin to accumulate prior
to S phase then decline rapidly. At the protein
level (for TOP2), this results in a stepwise accumulation
of protein within the population, as
maximal protein synthesis corresponds to the
maximal mRNA level, but there is no subsequent
protein degradation. Examination of
the 5-untranslated regions of these mRNAs
revealed an octamer consensus sequence,
CATAGAAA, which was present several times.
Experiments with transfected reporter genes
bearing 5-untranslated regions from TOP2 and
RPA1 showed that the sequence was necessary
and sufficient for appropriate cell-cycle
regulation. Intriguingly, the octamers also
functioned if they were transposed to the 3untranslated
region.
To find proteins involved in the regulation
of cycling, radioactively labeled RNAs containing
the octamer recognition sequence were
incubated with Crithidia extracts. When the
mixture was loaded on a polyacrylamide gel,
the resulting RNA–protein complexes could
be seen because they migrated more slowly
than the labeled RNA alone (a ‘gel-shift’ experiment).
The bound proteins could be covalently
linked to the RNA by UV irradiation. These
experiments revealed that there were indeed
some binding activities present that showed
the expected pattern of cell-cycle regulation.
The strongest candidate so far is a 38 kDa protein.
Activity was found in nuclear, not cytoplasmic
extracts, suggesting that regulation
operates at the level of mRNA splicing, export
or degradation in the nucleus. It is also possible
that the proteins also operate in the cytosol,
but are not detected there because they were
already bound to target RNAs in the extracts.
These issues can be addressed once the genes
encoding the binding proteins have been
cloned.
Control by 3-untranslated regions:
RNA degradation
At the time of writing more than 30 trypanosomatid
genes had been examined to find out
how regulation operates. To find the sequences
responsible for regulation, 5-untranslated
regions, 3-untranslated regions, or both have
been attached to reporter genes such as
MOLECULAR BIOLOGY