trans

UNUSUAL MODES OF TRANSCRIPTION IN TRYPANOSOMATIDS AND NEMATODES 49
Trans-splicing of an already capped Spliced
Leader (SL) sequence, which comprises the 5
terminal region of the SL RNA, to the 5 end of
a mRNA is an alternative, post-transcriptional
mode of capping and, in combination with
polyadenylation, allows the excision of individual
mRNA molecules from polycistronic
precursors. Accordingly, polycistronic transcription
of protein coding genes has been
found in those organisms harboring transsplicing
of SLs. In trypanosomatids, protein
coding genes are tandemly linked, separated
by short intergenic regions and, as has been
demonstrated by various experimental procedures,
are transcribed polycistronically. The
organization of the Leishmania major Friedlin
chromosome 1 sheds some light on the extent
of trypanosomatid transcription units. The
300-kbp long chromosome encodes 79 genes.
Of those, 50 genes are tandemly located on
one strand and the remaining 29 genes sit in
tandem on the other strand. Both gene arrays
are arranged head-to-head and, most likely,
each represents a single transcription unit.
However, it is not clear where transcription
starts or how RNA pol II is recruited to the
DNA. In nematodes, as has been estimated in
the free-living worm Caenorhabditis elegans,
about 25% of all genes are organized in polycistronic
transcription units. In contrast to trypanosomatid
units, these units are defined
and consist of 2–8 genes.
-Amanitin-resistant transcription of
genes encoding the major cell surface
antigens in T. brucei
Cotranscriptional capping locks mRNA synthesis
to RNA pol II-mediated transcription,
because the capping enzyme directly and
specifically interacts with the phosphorylated,
carboxy-terminal domain of the RNA
pol II largest subunit. Post-transcriptional
trans-splicing of a capped Spliced Leader,
however, uncouples this linkage and raises the
possibility that other RNA pols are recruited
for mRNA production. The ability of RNA pol I
to efficiently synthesize functional mRNA in
combination with trans-splicing was demonstrated
in T. brucei by transient, rDNA promoterdirected
reporter gene expression. Evidence
that T. brucei utilizes RNA pol I for transcription
of some endogenous protein coding genes
was first obtained by nuclear run-on experiments
in which transcription elongation on a
variant surface glycoprotein (VSG) gene was
resistant to -amanitin. This mushroom toxin
is a strong inhibitor of RNA pol II and a moderate
inhibitor of RNA pol III, but does not
affect RNA pol I transcription. VSG is the
constituent of the cell surface coat in bloodstream
form trypanosomes, and a VSG gene is
expressed from a telomeric VSG gene expression
site (VSG ES) which harbors a single
VSG gene and several associated genes. VSG
ESs are not the only units transcribed by an
-amanitin-resistant RNA pol in T. brucei.
Procyclin gene expression sites (procyclin ES),
encoding the major cell surface antigens of
insect form trypanosomes (procyclics), are
located at chromosome-internal positions and
are transcribed by an analogous enzyme activity.
Furthermore, metacyclic trypanosomes,
in the salivary gland of the tsetse, express
VSG genes in an -amanitin-resistant manner
from monocistronic transcription units called
metacyclic (m)VSG ES. Transcription of other
protein coding genes, however, is highly sensitive
to -amanitin and, therefore, mediated by
RNA pol II.
-Amanitin-resistant transcription of protein
coding genes is not restricted to T. brucei,
but has also been observed in the parasites
Trichomonas vaginalis and Entamoeba
histolytica. However, in these organisms resistance
appears to be a general phenomenon
MOLECULAR BIOLOGY