trans

80 POST-TRANSCRIPTIONAL REGULATION
Studies of constitutively expressed genes of
T. brucei have revealed that their mRNA halflives
are somewhat longer in procyclic forms
than in bloodstream forms. How can this be
reconciled with the fact that mRNA levels are
the same? The discrepancy is explained by the
fact that mRNA abundance is affected not only
by degradation, but also by the dilution factor
in the cells (the volume of procyclics is 2–3
times greater than that of bloodstream forms)
and the rate at which the cells are growing and
dividing.
The effects of protein synthesis
inhibition
The inhibition of protein synthesis usually
increases the abundance of several unstable
mRNAs in trypanosomatids. Experimentally,
cycloheximide is used, which arrests translating
ribosomes without release of the mRNA.
Under these circumstances, stabilization of
RNA can be interpreted in several ways: either
the stable association with polysomes inhibits
degradation by preventing nuclease access,
or degradation requires active ongoing translation,
or degradation requires an unstable
protein. In T. brucei the results have been
confirmed using a variety of inhibitors, including
puromycin, which results in chain termination
and release of transcripts from the
ribosome. In this case, the first explanation
can be ruled out. All authors seeing such
results have preferred the third explanation:
that degradation is effected by an unstable
protein. This could of course be a specific protein
interacting with particular mRNAs. However,
the disappearance of a component of
the general degradation machinery would also
have much more dramatic effects on unstable
mRNAs than on stable ones: in yeast, cycloheximide
inhibits decapping. We have found that
the abundance of many mRNAs is affected
to some extent by cycloheximide treatment,
which leads us to prefer this less specific explanation.
Protein synthesis inhibition does not always
result in increases in mRNA abundance.
Indeed, opposite effects may be seen with
stable and unstable mRNAs. In bloodstream
T. brucei, EP mRNAs are stabilized, as are
ESAG5, ESAG6, ESAG7, and TUB mRNAs,
but VSG mRNA decreases. Conversely, in
T. cruzi amastigotes, cycloheximide treatment
resulted in a 3-fold decrease in amastin RNA
and a 7-fold increase in tuzin mRNA, whereas
in epimastigotes (where both mRNAs are less
abundant than in amastigotes) amastin
mRNA was not significantly affected and tuzin
mRNA increased 4.3-fold. The MSPL mRNA of
L. chagasi is very stable in virulent (recently
isolated) log-phase promastigotes and less
stable and abundant in stationary phase.
Cycloheximide increased the abundance 4–6-
fold in all growth phases but, in contrast, prevented
stabilization of L. infantum HSP70
(genes 1–5) mRNA upon a temperature shift
from 26°C to 37°C. No mechanistic conclusions
can be drawn from these (and other)
rather disparate results at this time.
Heat shock gene control and
stress effects
Heat shock proteins (HSP) are important in
protecting eukaryotic and prokaryotic cells
from the effects of moderate rises in temperature.
In particular, many HSPs have a chaperone
function: that is, they are able to assist in
the folding of newly synthesized proteins, and
the refolding of proteins that have become
denatured. In nearly all organisms, transcription
of HSP genes is stimulated by a moderate
(non-lethal) heat shock, and the mechanisms
of the control have been extensively studied in
both prokaryotes and eukaryotes. The heat
MOLECULAR BIOLOGY