eprintid: 17057
rev_number: 14
eprint_status: archive
userid: 1221
dir: disk0/00/01/70/57
datestamp: 2014-06-20 06:18:22
lastmod: 2014-06-20 07:51:57
status_changed: 2014-06-20 06:18:22
type: doctoralThesis
metadata_visibility: show
creators_name: Klein, Cornelia Andrea
title: The role of ZC3H32 in Trypanosoma brucei
subjects: 570
divisions: 140001
adv_faculty: af-14
abstract: In
the
protozoan
parasite
Trypanosoma
brucei
gene
expression
is
mostly
regulated
on
the
post-­‐transcriptional
level.
C3H
zinc
fingers
have
shown
to
play
an
important
role
in
this
process.
So
far,
all
described
C3H
zinc
finger
in
Trypanosoma
brucei
have
shown
to
stabilize
their
targets.
This
work
focuses
on
the
zinc
finger
protein
ZC3H32.
A
large-­‐scale
isolation
of
free
and
membrane-­‐bound
polyribosomes
from
proyclic
and
bloodstream
trypanosomes,
which
is
also
described
in
this
thesis,
suggested
that
this
protein
associates
with
polyribosomes.
However,
small-­‐scale
polyribosome
isolations
followed
by
Western
blotting
revealed
that
only
a
minor
fraction
of
it
is
actually
associated
with
polyribosomes.
Immunofluorescence
showed
that
ZC3H32
localizes
to
the
cytoplasm.
A
yeast-­‐two-­‐
hybrid
screen
identified
it
as
a
putative
binding
partner
of
MKT1.
This
interaction
was
confirmed
by
co-­‐immunoprecipitation.
RNAi,
as
well
as
knock-­‐out
studies,
showed
that
ZC3H32
is
essential
in
the
bloodstream
form.
Northern
blotting,
as
well
as
a
SILAC
screen
by
Urbaniak
et
al.
indicate
that
it
is
also
enriched
in
this
life-­‐cycle
stage
as
compared
to
procyclics.
Artificial
tethering
of
ZC3H32
to
a
reporter
RNA
lead
to
the
RNA’s
degradation,
suggesting
that
this
protein
has
a
destabilizing
effect.
Tethering
of
ZC3H32
fragments
revealed
that
both
its
N-­‐terminal,
as
well
as
its
C-­‐terminal
region
are
able
to
generate
this
destabilization
effect,
while
the
middle
region,
containing
the
zinc
finger
domains,
can’t.
RNA
isolation
from
polyribosomal
fractions
showed
that
tethering
of
ZC3H32
also
decreases
the
translation
of
the
reporter
RNA.
High-­‐throughput
sequencing
of
poly-­‐A+
RNA
from
a
ZC3H32-­‐RNAi
cell
line
revealed
20
RNAs
that
were
up-­‐regulated
upon
ZC3H32
knock-­‐down
and
thus
might
be
putative
targets.
The
upregulation
was
confirmed
for
three
of
these
candidate
RNAs.
The
majority
of
the
putative
ZC3H32
targets
play
a
role
in
the
trypanosome’s
energy
metabolism
and
15
of
them
are
up-­‐regulated
in
procyclics.
These
results
suggest
that
ZC3H32
might
be
involved
in
the
stage-­‐specific
regulation
of
these
RNAs
in
the
bloodstream
form.
date: 2014
id_scheme: DOI
id_number: 10.11588/heidok.00017057
ppn_swb: 165829324X
own_urn: urn:nbn:de:bsz:16-heidok-170572
date_accepted: 2014-06-11
advisor: HASH(0x564e1c294638)
language: eng
bibsort: KLEINCORNETHEROLEOFZ2014
full_text_status: public
citation:   Klein, Cornelia Andrea  (2014) The role of ZC3H32 in Trypanosoma brucei.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/17057/1/Doktorarbeit%20Cornelia%20Klein.pdf