trans

POST-TRANSCRIPTIONAL REGULATION IN KINETOPLASTIDS 83
control alone is clearly insufficient. Next, the
3-untranslated regions from the EP1 or EP2
mRNAs were linked to reporter genes encoding
CAT or luciferase. Transient transfections
were done, and showed that the 3-untranslated
regions gave no expression in bloodstream trypanosomes.
The constructs were then transfected
into bloodstream and procyclic trypanosomes
in such a fashion that they would
integrate into the tubulin locus, where they
would be equally transcribed in both stages
(Figure 4.5B). Now, there was about 100 times
more expression of the reporter protein in procyclic
trypanosomes than in bloodstream trypanosomes,
but the reporter gene mRNA was
detectable and regulated only 11-fold. That
means there must also be about 9-fold regulation
of translation. Thus EP gene expression is
regulated roughly 10-fold at each of three levels:
transcription, RNA degradation, and translation
(Figure 4.5A, B).
The 3-untranslated regions of the different
EP and GPEET genes are not conserved, apart
from a U-rich 26-mer and a 16-mer stem-loop
(Figure 4.4A). The 16-mer enhances translation
in procyclic forms by an unknown mechanism,
but plays no role in developmental regulation.
The mechanism of the translational enhancement
is unknown but the stem-loop structure
is important. We have attempted to find proteins
that bind to this sequence using a yeast
three-hybrid screen, with no success at all.
Deletions of, and point mutations in, the
U-rich 26-mer abolished or reduced regulation.
In particular, an EP1 3-untranslated
region lacking the 26-mer, ep126, gave mRNA
and protein expression that was 30–40% of
the level obtained using a control ACT 3untranslated
region. The results of enzymatic
and chemical probing experiments indicated
that the 26-mer is normally in a singlestranded
conformation. Examination of the
PGKB 3-untranslated region, which can also
mediate procyclic-specific expression, revealed
a similar U-rich sequence, with interspersed
As, which is predicted to be single-stranded.
Specific deletion of this U-rich domain from
the PGKB 3-untranslated region abolished
stage-specific regulation at both the RNA and
protein levels.
Both the EP1 26-mer and the PGKB regulatory
sequence bear a strong resemblance to mammalian
AU-rich elements (AREs). Interestingly,
another control element of similar sequence,
which gives preferential expression in epimastigotes
and amastigotes, has been identified
in the 3-untranslated region of small
mucin gene transcripts in T. cruzi. It will be
interesting to see to what extent the degradation
mechanisms are also similar.
In order to compare the degradation mechanisms
of the constitutive ACT mRNA with the
regulated EP1 mRNA, CAT transgenes bearing
EP1, ACT or ep126 3-untranslated regions
were placed downstream of a bacteriophage T7
promoter and integrated into the genome of
trypanosomes that expressed the bacteriophage
polymerase. This resulted in the production of
sufficiently large amounts of RNA to enable
detailed study of the structure of the CAT mRNA
during degradation. To distinguish events at
the 5 end from those at the 3 end, the resulting
mRNAs were cut into specific pieces
(Figure 4.5C). Oligonucleotides that hybridized
to the CAT gene were added, together with
RNAaseH, which digests RNA/DNA hybrids.
Thus the RNA was cut only where the oligonucleotides
were hybridized, into three pieces
representing the 5 end, 3 end, and a middle
portion. The length of the poly(A) tail was
estimated by adding oligo d(T) with RNAaseH,
which removed the poly(A) tail, then measuring
the difference in size between poly(A)
and poly(A)-fragments (Figure 4.5C). All three
RNAs had poly(A) tails approximately 200
nucleotides long, in both bloodstream and
MOLECULAR BIOLOGY