trans

POST-TRANSCRIPTIONAL REGULATION IN KINETOPLASTIDS 75
the glycosome, whereas in procyclics, PGK is
predominantly cytosolic. There are three PGK
genes, in a head-to-tail arrangement on chromosome
I (Figure 4.3A). Early experiments
showed that all three of these genes are constitutively
transcribed, and the presence of transcripts
that bridged the gap from one gene to
the next was demonstrated by RT-PCR (Figure
4.3A). The central gene of the three, PGKB,
encodes a protein which is very like PGKs of
other eukaryotes. PGKB is found in the trypanosome
cytosol and both it and its RNA are
expressed almost exclusively in procyclic trypanosomes.
The first gene, PGKA, has an internal
insertion in the protein sequence. PGKA
protein is in the glycosome and is found in low
abundance in both bloodstream and procyclic
forms. Finally, the PGKC mRNA and protein
are found almost exclusively in bloodstream
forms; PGKC is targeted to the glycosome via
a peptide extension at the C-terminus. The
expression of cytosolic PGK in bloodstream
forms inhibits parasite growth, presumably
because the flow of intermediates in the glycosome
is disrupted. Thus the mechanisms that
prevent PGKB production in bloodstream trypanosomes
are essential for parasite virulence.
Splicing and polyadenylation
The first task after polycistronic transcription
is to separate the individual open reading
frames (Figure 4.3B). In theory this could be
achieved by a single cleavage in the RNA precursor.
In practice, two cleavages occur: one at
the 5-end, with associated trans-splicing, and
a further upstream cleavage with polyadenylation
of the 3-end (Figure 4.3B). The signal for
these reactions is the 5-splice site, which normally
contains a polypyrimidine tract. It signals
the trans-splicing of a short capped leader
sequence (around 40 nt) to the 5-end of the
downstream RNA. The polyadenylation of the
preceding mRNA is spatially and temporally
(and therefore in all probability mechanistically)
linked to the splicing, occuring 100–300 nt
upstream depending on species.
As trans-splicing is essential for mRNA
production, it seems an obvious candidate for
developmental regulation. There are often several
possible AG acceptor dinucleotides downstream
of the splicing signal, which results in
alternative 5-untranslated regions of different
lengths. Some trypanosome intergenic regions
contain multiple polypyrimidine tracts, which
may be recognized with varying efficiency by
the splicing machinery. Such variable splicing
can produce non-functional RNAs lacking
a complete open reading frame. It can also
result in mRNAs with long 5-untranslated
regions containing very short open reading
frames upstream of the main reading frame.
Examples of both types have been seen, but
there is no firm evidence so far that differential
splicing is important in developmental
regulation. The paucity of examples may simply
reflect the very small number of genes that
has been examined, or it may be that the coupling
of splicing and polyadenylation limits
this type of regulation. A failure of splicing
in one transcript could result in a failure to
polyadenylate the preceding one, and thus
preclude independent regulation of adjacent
genes.
Although no examples of developmental
regulation of splicing have been found, it is clear
that some splice sites are more efficient than
others. For example, the main reason for the
low abundance of PGKA (Figure 4.3) is that the
splice site of the PGKA mRNA is very inefficient.
The regulation of the other two genes will be
discussed later.
So far, the only gene that has been found to
contain an intron and be subjected to cissplicing
is that encoding poly(A) polymerase.
The mRNA is also trans-spliced, and the
MOLECULAR BIOLOGY