Poly(A)-Specific Ribonuclease (PARN-1) Function in Stage-Specific mRNA Turnover in Trypanosoma brucei
Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5′ to 3′ exonuclease decay or 3′ to 5′ exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher plants. Trypanosomatids possess three different PARN genes, PARN-1, -2, and -3, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite Trypanosoma brucei. Here we show that T. brucei PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability in vivo, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production.
EUKARYOTIC CELL, Sept. 2011, p. 1230–1240 Vol. 10, No. 9
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Poly(A)-Speciﬁc Ribonuclease (PARN-1) Function in Stage-Speciﬁc
mRNA Turnover in Trypanosoma brucei
Christopher J. Utter,‡ Stacey A. Garcia,‡ Joseph Milone, and Vivian Bellofatto*
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry-New Jersey Medical School,
Newark, New Jersey 07103
Received 29 April 2011/Accepted 27 June 2011
Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes.
Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including dead-
enylation-dependent decapping, followed by 5ⴕto 3ⴕexonuclease decay or 3ⴕto 5ⴕexosome-mediated decay. In
trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed
genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-speciﬁc RNase
(PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher
plants. Trypanosomatids possess three different PARN genes, PARN-1,-2, and -3, each of which is expressed
at the mRNA level in two life-cycle stages of the human parasite Trypanosoma brucei. Here we show that T.
brucei PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability in vivo,we
overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life.
Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-speciﬁc coat
proteins. These data suggest that PARN-1 functions in stage-speciﬁc protein production.
Regulation of gene expression in the protozoan parasite
Trypanosoma brucei allows the organism to adapt and survive
during its life cycle in two very different environments, the
mammalian bloodstream and the tsetse ﬂy. Expression of nu-
merous protein-coding genes is regulated posttranscriptionally,
particularly at the level of mRNA stability (4, 11, 25). For
example, differential mRNA stability accounts for the stage-
speciﬁc expression of procyclins, hexose transporters, and
phosphoglycerate kinases (6, 20, 27, 28, 53).
In the well-studied Saccharomyces and mammalian systems,
mRNA decay is a tightly controlled, multistep, and multipath-
way process. Various cis-acting elements, embedded in speciﬁc
mRNAs, are recognized by RNA-binding proteins (7, 52, 55),
which stabilize mRNAs or recruit RNases to carry out mRNA
degradation and inhibit translation (8, 34, 39, 47). The removal
of the mRNA 3⬘poly(A) tail by 3⬘to 5⬘exoribonucleases
(deadenylases) is often the rate-limiting step in mRNA degra-
dation in vertebrates and thus a key point in regulation of
mRNA turnover (19, 40).
A number of different deadenylases exist in eukaryotes, in-
cluding poly(A)-speciﬁc RNase (PARN), the CCR4/CAF1/
NOT complex, and the PAN2/PAN3 complex (reviewed in
reference 23). The speciﬁc role of each of these proteins re-
mains largely unknown, although evidence suggests that each
enzyme may recognize a particular set of mRNA substrates
(46). PARN functions in the targeted degradation of speciﬁc
mRNAs in humans, Xenopus, and higher plants (1, 3, 22, 31,
32, 36, 58). To date, no PARN-encoding genes have been
characterized in any single-cell eukaryote (56). In humans,
PARN initiates decay of mRNAs containing AU-rich elements
or nonsense codons. In Xenopus, PARN regulates oocyte mat-
uration, whereas in Arabidopsis, PARN regulates embryogen-
The trypanosome genome encodes homologs to the deade-
nylation enzymes PARN, CAF1/NOT, and PAN2/PAN3, al-
though a CCR4 homolog is absent (50, 51). Investigation of T.
brucei CAF1/NOT1 showed that these two proteins were es-
sential, whereas PAN2 depletion studies were less conclusive
We set out to study PARN in T. brucei on the basis of our
discovery of deadenylation activity in cytoplasmic extracts from
this organism (42). T. brucei possesses three PARN homologs
(PARN-1,-2, and -3), each of which is transcribed in both the
procyclic (Pro) and bloodstream form (BF) stages of the par-
asite life cycle (30; this work). We chose PARN-1 to initiate
our studies of PARN-dependent mRNA decay in trypano-
somes. We veriﬁed that PARN-1 is a functional deadenylase,
and we overexpressed PARN-1 in situ to identify transcripts
that are targeted for degradation by this enzyme. A subset of
mRNAs targeted for PARN-1-dependent degradation in T.
brucei was identiﬁed using microarray studies and quantitative
real-time PCR (qRT-PCR). Analysis of the genes coding for
several of these mRNAs suggests that PARN-1 contributes to
regulating differential gene expression.
MATERIALS AND METHODS
Culturing and transfection of parasites. T. brucei Lister 427 procyclic cells
were cultured in SDM-79 medium containing 10% fetal calf serum (FCS) at 26°C
in 5% CO
. Procyclic 29-13 cells were cultured in the presence of 15 g/ml G418
and 50 g/ml hygromycin to maintain expression of T7 RNA polymerase and the
tetracycline (Tet) repressor (57). Transfected parasites containing the integrated
plasmid were selected for by the addition of 2.5 g/ml phleomycin (2). Lister 427
BF cells were grown in HMI-9 medium containing 10% FCS and 10% Serum
* Corresponding author. Mailing address: Department of Microbi-
ology and Molecular Genetics, University of Medicine and Dentistry-
New Jersey Medical School, Newark, NJ 07103. Phone: (973) 972-
4483, ext. 2-4406. Fax: (973) 972-3644. E-mail: email@example.com.
† Supplemental material for this article may be found at http://ec
‡ C.J.U. and S.A.G. contributed equally to this study.
Published ahead of print on 8 July 2011.
Plus (SAFC Biosciences) at 37°C in 5% CO
(26). Single-marker BF cells (a gift
from G. Cross) were cultured in the presence of 2.5 g/ml G418 to maintain the
T7 RNA polymerase and Tet repressor (57), and transfected parasites were
obtained using an Amaxa system and selected for using 2.5 g/ml phleomycin.
Plasmid constructs. The pSAP1 vector containing the streptavidin-binding
protein-protein A tandem afﬁnity puriﬁcation (TAP) tag was a generous gift
from Larry Simpson. The 630-bp open reading frame (ORF) was PCR ampliﬁed
to add HindIII and KpnI sites upstream and a BamHI site downstream of the tag.
The product was ligated into the pLEW111 expression vector using the HindIII
and BamHI sites to produce pLEW111-TAP. The PARN-1 ORF was PCR
ampliﬁed, and KpnI sites were added to each end. PARN-1 was ligated into
pLEW111-TAP using KpnI to produce a Tet-inducible, TAP-tagged PARN-1
protein expression vector.
RNA analysis. Total RNA was extracted from procyclic cells grown to 8 ⫻10
cells/ml and BF cells grown to 1 ⫻10
cells/ml using Qiagen’s RNeasy minikit.
RNA was isolated from the total RNA using Qiagen’s Oligotex mRNA
For Northern blot analysis, 10 g of total RNA was separated on a formaldehyde-
1.2% agarose gel in morpholinepropanesulfonic acid (MOPS) buffer (49). RNA was
hydrolyzed and transferred to a nylon membrane overnight by capillary diffusion in
3 M NaCl–0.01 N NaOH. Radiolabeled probes were generated from 25 ng of PCR
product using a Megaprime DNA labeling system (Amersham). Each probe was
speciﬁc for a particular PARN gene; gene-speciﬁc primers for ampliﬁcation were, for
TTTGC-3⬘, amplifying the region between nucleotides (nt) 960 and 1761 of the
ORF; for PARN-2,5⬘-GGTTTCAACATTTTTGGAAG-3⬘and 5⬘-CTACCCACT
AAGACGGTAAA-3⬘, amplifying the region between nt 710 and 1530 of the ORF;
and for PARN-3,5⬘-GTTACCACCGGAGGCTACGACTC-3⬘and 5⬘-CTACTTC
TCAGTCAAATGTT-3⬘, amplifying the region between nt 1032 and 1872 of the
ORF. Probes were hybridized to the membrane overnight in hybridization buffer
(5⫻SSC [1⫻SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% sodium
pyruvate, 5⫻Denhardt’s solution, 0.5% SDS, 100 g/ml heparin, 0.1% diethyl
pyrocarbonate) and washed three times (0.5⫻SSC, 15 min). Radiolabeled bands
were analyzed by a PhosphorImager apparatus (Molecular Dynamics).
S100 protein extracts. Extracts were prepared as described previously (42).
Brieﬂy, 2 liters of T. brucei procyclic cells were grown to 2 ⫻10
cultures were harvested by centrifugation (1,100 ⫻g, 20 min, 4°C). Cell pellets
were washed three times in cold 1⫻phosphate-buffered saline (PBS) and resus-
pended in three pellet volumes of hypotonic lysis buffer (10 mM HEPES-KOH,
pH 7.9, 10 mM KCl, 1.5 mM MgCl
, 1 mM dithiothreitol [DTT], 0.05% NP-40,
1M protease inhibitors), incubated on ice for 10 min, and then sheared using
a tight-ﬁtting 7-ml Dounce homogenizer. The cellular suspension was centri-
fuged (12,000 ⫻g, 10 min, 4°C), and the supernatant was transferred to a new
tube. The supernatant was adjusted to 275 mM KCl and centrifuged (100,000 ⫻
g, 1 h, 4°C). The S100 sample was collected and dialyzed in buffer D (20 mM
HEPES-KOH, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT,
1M protease inhibitors) for4hat4°C. The sample was aliquoted into 200-l
samples, quick-frozen, and stored at ⫺80°C. Protein concentrations were deter-
mined by Bradford assay. Extracts typically yield 3 ml at 10 g/l.
TAP puriﬁcation. Procyclic parasites overexpressing PARN-1 (PARN-1 OvEx
cells) were grown to 1 ⫻10
cells/ml in 1 liter culture medium. Expression of
TAP-tagged PARN protein was induced with 500 g/ml tetracycline for 18 h.
Cells were harvested by centrifugation, and S100 protein extracts were prepared
as described above. TAP-tagged protein was puriﬁed as described previously (12,
29). All steps were performed at 4°C. Brieﬂy, 2 ml of S100 extract was adjusted
to 10 ml with IgG-binding buffer (10 mM Tris HCl, pH 8.0, 150 mM NaCl, 2 mM
EDTA, 0.1% NP-40, 1 mM DTT, 10% glycerol, Roche complete protease in-
hibitor cocktail tablet). One milliliter rabbit IgG agarose beads (Sigma) was
washed with 10 ml IgG-binding buffer and incubated (1 h) in a 0.8- by 4-cm
Poly-prep chromatography column (Bio-Rad). After incubation, the column was
placed in a stand and opened to allow unbound protein to ﬂow through. The
protein-bead mixture was washed twice with 10 ml IgG-binding buffer and once
with 10 ml tobacco etch virus (TEV)-cleavage buffer (10 mM Tris HCl, pH 8.0,
150 mM NaCl, 0.5 mM EDTA, 0.1% NP-40, 1 mM DTT, 5% glycerol). Beads
were resuspended with 3 ml TEV-cleavage buffer containing 200 units of AcTEV
protease (Invitrogen) and incubated (3 h). Protein was eluted from the column
by gravity ﬂow, and 1 ml TEV-cleavage buffer was added to the column to elute
any residual protein. Four milliliters of eluted protein was adjusted to 10 ml with
IgG-binding buffer. One milliliter streptavidin Sepharose high-performance
beads (GE Healthcare) was washed with 10 ml IgG-binding buffer and incubated
with the extracts (1 h) in the Poly-prep chromatography column. The column was
then drained and washed twice with 10 ml IgG-binding buffer. Beads were
resuspended in 1.5 ml elution buffer (40 mM Tris HCl, pH 8.0, 150 mM NaCl, 0.5
mM EDTA, 0.1% NP-40, 1 mM DTT, 10% glycerol, 2 mM biotin [Sigma]).
Eluted protein was collected, and elution was repeated with another 1.5 ml of
elution buffer. Protein samples were dialyzed in 1 liter buffer D (4 h). The
samples were aliquoted, quick-frozen, and stored at ⫺80°C.
Nuclear fractionation. Log-phase procyclic cells were harvested by centrifu-
gation (900 ⫻g, 15 min, 4°C), and the pellet was washed twice with wash buffer
(20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 3 mM MgCl
, 2.5 mM DTT, 1 M
protease inhibitors). Pellet was resuspended in hypotonic buffer (10 mM HEPES,
pH 7.9, 10 mM KCl, 2.5 mM MgCl
, 1 mM EDTA, 2.5 mM DTT, 1 M protease
inhibitors) and incubated on ice for 10 min. Cells were lysed in 0.2% NP-40
followed Dounce homogenization in a tight-ﬁtting 7-ml Dounce homogenizer.
An aliquot was saved as total lysate for immunoblots. Total lysate was layered on
hypotonic buffer plus 0.8 M sucrose and centrifuged in a swing-out rotor (8,000 ⫻
g, 10 min, 4°C). The top layer was collected and saved as the cytoplasmic fraction
for immunoblots, and the pellet was collected and saved as the nuclear fraction
for immunoblots. Figure 2A contains protein fractions from equal number of
) in each of the nine lanes.
Mitochondrial fractionation. Mitochondria were isolated from T. brucei cells
as described previously, with some exceptions (45). Brieﬂy, 1 liter of procyclic
cells was harvested by centrifugation (6,000 ⫻g, 10 min, 4°C) and washed twice
in PBS. Cells were resuspended in hypotonic lysis buffer (1 mM Tris-Cl, pH 8.0,
1 mM EDTA), incubated on ice for 5 min, and then sheared using a tight-ﬁtting
7-ml Dounce homogenizer. An aliquot was saved as total lysate for immunoblots.
Sucrose was added to the lysate at a ﬁnal concentration of 0.25 M, and the lysate
was centrifuged at 15,000 ⫻gfor 10 min at 4°C. Supernatant was removed and
saved as the cytoplasmic fraction for immunoblots. The pellet was resuspended
in STM buffer (20 mM Tris-Cl, pH 8.0, 250 mM sucrose, 2 mM MgCl
), and the
suspension was incubated with 1/200 volume DNase I for 60 min. Two milliliters
of STE buffer (20 mM Tris-Cl, pH 8.0, 250 mM sucrose, 2 mM EDTA) was
added to the lysate to stop the DNase I reaction, and lysate was centrifuged at
15,000 ⫻gfor 10 min at 4°C. The supernatant was removed, and the pellet was
resuspended in 0.4 ml of 70% Percoll and homogenized in a 2-ml Dounce
homogenizer. The resuspended pellet was layered under a 15 to 40% linear
Percoll gradient and centrifuged at 103,000 ⫻gfor 60 min at 4°C. The middle
layer was collected and was washed twice with STE buffer by centrifugation
(32,530 ⫻gfor 15 min at 4°C). The washed pellet was saved as the mitochondrial
fraction for immunoblots. In Fig. 2B and C, each of the lanes containing total
lysate represents 2 ⫻10
cell equivalents, each of the lanes containing cytoplas-
mic extract represents 2 ⫻10
cell equivalents, and each of the lanes containing
mitochondrial proteins represents a 50-fold higher cell equivalent to compensate
for the decreased protein yield during mitochondrial fractionation.
Antibodies, Western analysis, and immunodepletion. The C-terminal 133
amino acids of the PARN-1 open reading frame were cloned into the pGEX-6P-1
bacterial expression vector and transformed into Escherichia coli BL21. Protein
expression was induced overnight and puriﬁed by a glutathione S-transferase
column. The puriﬁed protein was used to generate polyclonal antibody in rabbits
(Lampire). Antibody was puriﬁed from sera by afﬁnity chromatography with
puriﬁed bacterium-expressed protein. Western analysis was performed by sepa-
rating proteins on a 10% SDS-polyacrylamide gel and transferring the proteins
to a polyvinylidene diﬂuoride membrane. PARN-1 protein was identiﬁed using
puriﬁed polyclonal antibody, and the TAP tag alone was identiﬁed using perox-
idase antiperoxidase antibody (Sigma). Bound antibody was detected with anti-
rabbit IgG (from donkey) linked to horseradish peroxidase, followed by chemi-
luminescence (Amersham). For PARN-1 immunodepletion experiments, 100 l
of resuspended protein A-Sepharose beads was incubated with 150 l of poly-
clonal antibody. Beads were washed and split into three tubes. Approximately 1
g of TAP tag-puriﬁed PARN-1 protein was added to the ﬁrst set of beads and
the mixture was incubated (1 h, 4°C). The antibody-bead complex was then
removed from the sample by centrifugation, and the extract was depleted for two
additional rounds. The depleted extracts were aliquoted into 50-l samples,
quick-frozen, and stored at ⫺80°C.
Preparation of RNA substrates. RNA substrates were prepared as previously
described (18). DNA templates were prepared from the pGEM4 vector (Pro-
mega). Vector was digested with HindIII, and an A
taining a 22-nt adapter sequence was ligated downstream. A 65-bp sequence
from the pGEM vector containing the SP6 promoter was PCR ampliﬁed along
with the 3⬘A
sequence. The 22-nt adapter sequence was removed by SspI
digestion for the pGEM
substrate but was not removed for the pGEM
22-nt adapter sequence substrate. For the A
substrate lacking a poly(A) tail, an
HindIII-digested vector was used.
One microgram of DNA template was used to make internally labeled RNA
with a Riboprobe in vitro transcription system (Promega). RNA was capped by
the addition of 5 mM m
GTP cap analog and labeled with 50 Ci [alpha-
VOL. 10, 2011 mRNA TURNOVER IN T. BRUCEI 1231
P]UTP. The reaction mixtures were incubated (1 h, 37°C), and the RNA
products were puriﬁed by phenol-chloroform extraction, followed by ethanol
precipitation. Samples were resuspended in 10 l TBE (Tris-borate-acetate)-
urea loading buffer and separated on a 5% acrylamide–7 M urea gel. Radiola-
beled RNA bands were excised from the gel and eluted in 400 l HSCB buffer
(50 mM Tris-HCl, pH 7.6, 400 mM NaCl, 0.1% SDS) with 50 g proteinase K
overnight at room temperature. Samples were phenol-chloroform extracted,
ethanol precipitated, and resuspended in RNase-free water.
In vitro deadenylase assays. Assays were adapted from the previously de-
scribed protocol (17). For assays using S100 protein extracts, 14-l reaction
mixtures containing 80 g dialyzed S100 extract, 100,000 cpm labeled RNA (⬃50
fmol), 2.3% polyvinyl alcohol, 3.2 mM MgCl
, and 500 ng poly(A) were pre-
pared. For assays using TAP tag-puriﬁed PARN-1 protein, 15-l reaction mix-
tures contained ⬃50 ng puriﬁed PARN-1 protein, 100,000 cpm labeled RNA
(⬃50 fmol), 2.2% polyvinyl alcohol, 3 mM MgCl
,2g bovine serum albumin
(BSA), and 16 units RNase inhibitor (Amersham). All reaction mixtures were
incubated at 26°C, and the reactions were stopped by the addition of 300 l
HSCB2 (20 mM Tris, pH 8.0, 400 mM NaCl, 0.1% SDS, 20 mM EDTA, 10 g
glycogen). RNA products were puriﬁed by phenol-chloroform extraction, fol-
lowed by ethanol precipitation. RNA was resuspended in 10 l TBE-urea load-
ing buffer and separated on a 5% acrylamide–7 M urea gel. Gels were analyzed
by a PhosphorImager apparatus (Molecular Dynamics) and quantitated by
Microarray analysis. Procyclic cells were grown to a density of 8 ⫻10
cells/ml. PARN-1 and TAP tag overexpression was induced for 48 h with 500
ng/ml tetracycline. RNA was extracted as previously described. Four micrograms
RNA was reverse transcribed for 16 h at 42°C in a reaction mixture
containing 12 g of random hexamers (Invitrogen), 50 mM Tris-HCl, pH 8.3, 75
mM KCl, 3 mM MgCl
, 10 mM DTT, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM
dGTP, 0.17 mM dTTP, 1 mM 5-(3-aminoallyl)-dUTP, 0.33 mM KPO
RNaseOUT (Invitrogen), and 800 U SuperScript III reverse transcriptase (In-
vitrogen). The steady-state lengths of poly(A) tails on T. brucei mRNAs are 150
to 200 nt. The RNA in the cDNA-RNA mixture was hydrolyzed in 100 mM
EDTA and 200 mM NaOH. The reaction mixture was incubated (15 min, 65°C),
and then the pH was adjusted to ⬃7.0 with 333 mM Tris (pH 7.0). cDNA was
puriﬁed using a QIAquick PCR puriﬁcation kit (Qiagen) and dried to ⬃1lby
a SpeedVac apparatus. cDNA was resuspended in 100 mM sodium carbonate
buffer (pH 9.3) and labeled with Cy3 or Cy5 monoreactive dye (GE Healthcare).
Two reaction mixtures were incubated for2hatroom temperature and stopped
with 80 mM sodium acetate (pH 5.2). Labeled cDNAs were puriﬁed using the
QIAquick PCR puriﬁcation kit, combined, and dried to ⬃1l by the SpeedVac
apparatus. Samples were resuspended in hybridization buffer containing 40%
formamide, 5⫻SSC, 0.1% SDS, and 0.6 g/l salmon sperm DNA and heated
(twice, 5 min, 95°C). T. brucei DNA microarrays, version 3, obtained from the
Pathogen Functional Genomic Research Center, were prehybridized at (2 h,
42°C) in 5⫻SSC, 0.1% SDS, and 1% BSA. Slides were washed in water and then
dried using centrifugation. Samples were applied to the microarray slides, cov-
ered with a 24- by 60-cm glass coverslip (Fisher), and placed in a chamber
(HybChamber; GeneMachines). The chamber was placed in a 42°C water bath,
covered in aluminum foil, and incubated for 16 h. Slides were washed (2⫻SSC
and 0.1% SDS, 0.1⫻SSC and 0.1% SDS, 0.1⫻SSC) and dried by centrifugation.
Slides were scanned using a GenePix 4000A microarray scanner (Axon Instru-
ments), and data were analyzed using GenePixPro (version 6.0; Axon Instru-
ments) and MultiExperiment Viewer (version 4.1) software (the Institute for
Genomic Research [TIGR]). Using this analysis, we determined that PARN-1
mRNA levels were increased ⬃27-fold following induction with tetracycline.
Quantitative real-time PCR. qRT-PCR experiments were carried out using an
iScript one-step RT-PCR kit with SYBR green (Bio-Rad). The Primer sets are
shown in Fig. S3 in the supplemental material. Brieﬂy, 100 ng total RNA was
added to a 25-l reaction mixture containing 1⫻SYBR green reaction mixture,
300 nM forward and reverse primers, and 0.5 l iScript reverse transcriptase.
Reactions were run in a RotorGene 3000 real-time cycler (Corbett Research).
Cycling conditions were as follows: 50°C for 10 min for cDNA synthesis, 5 min at
95°C for reverse transcriptase inactivation, and 40 cycles of 10 s at 95°C and 30 s
at 55°C, followed by melt curve analysis from 55°C to 99°C. To assay for mRNA
stability, cells were treated with 10 g/ml actinomycin D, total RNA was ex-
tracted at 0, 30, 60, 120, and 180 min, and mRNA levels were analyzed by
qRT-PCR. All mRNA levels were normalized to that of 7SL RNA, and the
percentage of mRNA remaining from the starting amount (time zero) was
calculated. For the mRNA decay kinetics, the percentage of mRNA remaining
was plotted versus time. An exponential trendline was ﬁt to each set of data, and
half-lives were calculated using the trendline equation (y⫽e
Generation of double-knockout cell lines. The 500-bp region upstream of the
PARN-1 ORF was PCR ampliﬁed from genomic DNA with the addition of a
BglII site downstream with primers JM300 (5⬘-CACCGAATTCGTACTCTTC
TCTAAATTCGTTTC-3⬘) and JM301 (5⬘-CACCAGATCTAGTTACACTTGG
GCTAATGC-3⬘). The 500-bp region downstream of the PARN-1 ORF was PCR
ampliﬁed with the addition of a MluI site upstream with primers JM302 (5⬘-C
JM303 (5⬘-CACCGAATTCGGACGTTGGTCTTATGAAC-3⬘). The phleo-
mycin resistance ORF was PCR ampliﬁed with a BglII site upstream and MluI
site downstream of the ORF using primers JM304 (5⬘-CACCAGATCTATG
GCCAAGTTGACCAGTGCC-3⬘) and JM305 (5⬘-CACCACGCGTTCAGTC
CTGCTCCTCGGCCAC-3⬘). The neomycin resistance open reading frame
was PCR ampliﬁed with a BglII site upstream and MluI site downstream of
the ORF using primers JM204 (5⬘-CACCAGATCTCGGAAAGGGAGAGA
AAC-3⬘) and JM205 (5⬘-CACCACGCGCCCAGTGGATTGAATTG-3⬘).
Upstream and downstream sequences were ligated to either the phleomycin
or neomycin resistance open reading frames using the BglII and MluI sites.
The ligated constructs were PCR ampliﬁed using primers JM300 and JM305,
and the PCR product was inserted in TOPO vector pCR 2.1.
The phleomycin resistance plasmid was digested with EcoRI and transfected
into 427 procyclic T. brucei cells. After 24 h, 1 g/ml phleomycin was added to
cultures to select transfected cells. After these single-knockout cell lines were
veriﬁed by restriction digestion, cells were transfected with a NotI-digested
neomycin resistance plasmid. After 24 h, 15 g/ml G418 was added to the
cultures to screen for cells containing deletions of both PARN-1 alleles. Double-
knockout strains were conﬁrmed by PCR of genomic DNA and Northern blot-
Microarray data accession number. Microarray data were deposited into the
NCBI GEO database (accession no. GSE20593).
T. brucei encodes three PARN homologs. The amino acid
sequence of Homo sapiens PARN (HsPARN) was used to iden-
tify possible PARN homologs in the T. brucei genome (Fig. 1A)
(5). Three genes were designated T. brucei PARN-1 (TbPARN-
1), TbPARN-2, and TbPARN-3 on the basis of their relative
homology to HsPARN. PARN proteins are members of the
DEDD exoribonuclease superfamily and the DEDDh family,
characterized by (i) three exonuclease (Exo) motifs (Exo I, II,
and III; Fig. 1A, gray boxes; see Fig. S1A in the supplemental
material), (ii) four invariant acid amino acids necessary for
catalytic activity (Fig. 1A, asterisks; see Fig. S1A in the sup-
plemental material), (iii) a ﬁfth invariant acidic residue located
between Exo II and III (DTK, where the boldface D indicates
the ﬁfth invariant acidic residue; Fig. 1A, triangle; see Fig. S1A
in the supplemental material), and (iv) a conserved histidine
within the Exo III motif (Fig. 1A, ﬁlled circle; see Fig. S1A in
the supplemental material) (48, 59). Because the TbPARNs
and HsPARN are highly divergent within their C termini, we
restricted the comparison to the N termini. The N terminus of
TbPARN-1 is 30% identical and 17% similar in amino acid
sequence to the HsPARN sequence, TbPARN-2 is 27% iden-
tical and 15% similar, and TbPARN-3 is 18% identical and
We determined whether each of the three T. brucei PARN-
coding genes produces mRNAs in parasites using Northern
blot analysis (Fig. 1B). DNA probes were targeted to the di-
vergent, terminal 800 bp of each ORF to distinguish among the
three PARN genes. Northern blot analysis showed that
PARN-1,2-, and -3 are transcribed in procyclic parasites, which
is the life-cycle stage in the tsetse ﬂy midgut, and in the blood-
stream-form parasites, which is the replicating life-cycle stage
in the mammalian host. In addition, the steady-state levels of
each PARN mRNA do not differ in a stage-speciﬁc manner.
1232 UTTER ET AL. EUKARYOT.CELL
PARN mRNA is present at low steady-state levels compared to
beta-tubulin mRNA quantities, as determined in the Northern
blot and qRT-PCR analyses (Fig. 1B and data not shown). The
absolute levels of PARN-1, -2, and -3 proteins were not com-
pared. We conclude that T. brucei constitutively expresses mul-
tiple PARN genes.
We aligned TbPARN-1 with the HsPARN and Arabidopsis
thaliana PARN (AtPARN) to uncover similarities that might
suggest functional conservation, unrelated to the three exonu-
clease motifs, among PARNs from different organisms (see
Fig. S1 in the supplemental material). TbPARN-1, as well as
TbPARN-2 and -3, lacks the RNA recognition motif (RRM)
and the nuclear localization signal (NLS) found in HsPARN.
The RRM in HsPARN enhances deadenylase activity by in-
teracting with the 5⬘cap of mRNA (14, 43). The absence of an
RRM in TbPARN proteins is consistent with our observation
that deadenylation in the cytoplasm does not require a 5⬘cap
on the RNA substrate (42). Moreover, the absence of an NLS
in all three TbPARN proteins is consistent with our observa-
tions that TbPARN-1 resides primarily in the cytoplasm in
procyclic parasites (Fig. 2). The lack of an NLS in AtPARN
correlates with its predominantly cytoplasmic localization (9,
48). Thus, TbPARN-1 likely functions without a 5⬘-cap depen-
dency and in the parasite’s cytoplasm.
PARN-1 is nonessential for parasite viability. To identify
the role of PARN-1 in T. brucei, we deleted the two allelic
copies of this gene using homologous recombination (see Fig.
S2A in the supplemental material). Each allele was replaced
with a drug resistance cassette. Northern blot analysis demon-
strated the absence of PARN-1 mRNA in the double knockout
(see Fig. S2B in the supplemental material). Western analysis
demonstrated the absence of PARN-1 protein in the double
knockout (see Fig. S2C in the supplemental material). Cell
viability, growth rate, and microscopic analysis of two clonal
cell lines showed no growth alterations compared to wild-type
parasites (see Fig. S2D in the supplemental material). These
results prove that PARN-1 is a nonessential gene in cultured
procyclic T. brucei.
To explore the combined necessity of PARN-1, -2, and -3 in
parasites, we simultaneously depleted all three PARNs using
RNA interference in procyclic and bloodstream-form parasites
(data not shown). PARN-1 and PARN-2 were shown to be
decreased at the mRNA level by Northern analysis, and
PARN-3 was shown to be decreased at the protein level by
Western analysis using anti-PARN-3 antibody (data not
shown). Neither growth rates nor gross morphology was af-
fected in PARN-depleted procyclic forms, and growth rates
were only slightly affected (⬃10% slower than control rates) in
PARN-depleted BF parasites. Because decreased amounts of
all three PARNs did not affect cell growth, we conclude that
PARN proteins either are not essential for T. brucei viability or
are sufﬁcient to sustain cell viability at low levels.
PARN-1 is a deadenylase in vitro.Puriﬁed PARN-1 was used
in RNase assays to assess its deadenylase activity. To obtain
puriﬁed protein, the PARN-1 ORF was tagged with a TAP tag
and transfected on a Tet-inducible expression vector to pro-
duce the PARN-1 OvEx cell line. TAP-tagged PARN-1 was
expressed and puriﬁed from S100 extracts using a two-step
afﬁnity method that employed IgG and streptavidin chroma-
tography (Fig. 3A). A synthetic, radiolabeled 60-nt poly(A) tail
) was used as substrate. As expected, RNA-A
trimmed, in an apparently distributive manner, in the presence
of puriﬁed PARN-1 (fraction shown in Fig. 3A, lane 8) to
produce an RNA lacking a poly(A) tail, RNA-A
, after 10 min
(Fig. 3B, lanes 1 to 5). To ensure that we were speciﬁcally
assaying PARN-1 activity, we tested eluate (Fig. 3A, lane 8,
and C, lane 1) that was PARN-1 depleted using PARN-1-
speciﬁc polyclonal antibody or mock depleted using nonspe-
ciﬁc antibody (Fig. 3C, lanes 2 to 7). Following depletion of
PARN-1 protein using PARN-1-speciﬁc antibodies, RNA-A
degradation was abolished (Fig. 3D, lanes 6 to 10). This was
not the case in the mock depletion of PARN-1 (Fig. 3D, lanes
1 to 5). Thus, PARN-1 has deadenylase activity.
To test whether PARN-1 was an adenosine-speciﬁc exonu-
clease, PARN-1 activity on RNA-A
was evaluated in the
presence of poly(A) or poly(C) competitor (Fig. 3E). Poly(A)
competitor inhibited RNA-A
deadenylation (Fig. 3E, lanes 2
to 5), whereas poly(C) competitor had no effect on RNA-A
deadenylation (Fig. 3E, lanes 6 to 9). In addition, we tested
PARN-1 activity using a 22-heteronucleotide sequence added
3⬘to the poly(A) tail of RNA-A
⫹22) as substrate
FIG. 1. T. brucei PARN-1 is a member of a small family of related
PARN proteins. (A) Representation of the amino acid sequence of the
open reading frames shows that each of the three proteins contains the
conserved Exo I, Exo II, and Exo III domains (gray boxes). The four
residues of the DEDD motif required for catalytic activity are noted
with asterisks, and a ﬁfth conserved aspartate residue is shown by open
triangles. The ﬁlled circles indicate the histidine in Exo III indicative of
DEDDh subfamily members (59). The divergent C termini are shown
as a crosshatched box (PARN-1), a hatched box (PARN-2), and a
stippled box (PARN-3). The drawing is to scale. Chr, chromosome.
(B) Northern blot of total RNA shows transcript levels of the three
PARN genes in Pro and BF cells. Arrows indicate that PARN-1 mi-
grates as a 2.1-kb mRNA, PARN-2 as a 2.05-kb mRNA, and PARN-3
as a 3.0-kb mRNA. Ethidium bromide staining of the formaldehyde gel
shows 18S and the two main fragments of 28S rRNAs used as loading
controls. The TriTryp gene numbers are as follows: PARN-1,
Tb927.8.2850; PARN-2, Tb927.10.8360; PARN-3, Tb09.211.4350.
VOL. 10, 2011 mRNA TURNOVER IN T. BRUCEI 1233
(Fig. 3B, lanes 6 to 10). PARN-1 did not degrade this RNA.
Thus, PARN-1 appears to be exclusively a deadenylase.
The catalytic activity of most deadenylases requires divalent
cations (13, 15, 59). To determine whether PARN-1 activity
has this requirement, enzyme activity was tested in the pres-
ence and absence of Mg
(Fig. 3F). When Mg
deadenylation was inhibited, demonstrating that Tb-
PARN-1 activity is dependent upon a divalent cation, such as
, for its deadenylation activity.
Cells overexpressing PARN-1 have increased deadenylase
activity. To examine the role of PARN-1 in procyclic T. brucei,
we determined whether overexpression of PARN-1 resulted in
increased deadenylation. PARN-1 OvEx cells and a control
culture expressing the TAP tag alone were induced, and S100
protein extracts were prepared (Fig. 4B). Both cultures grew at
the same rate, indicating that there was no gross effect of
PARN-1 overexpression on cell growth (data not shown).
Deadenylation rates were measured using the in vitro deadeny-
lation assay (Fig. 4A). The PARN-1 OvEx cell extract rapidly
, and nearly all RNA-A
converted to RNA-A
after 15 min (Fig. 4A, lanes 5 to 8). In
contrast, control extracts deadenylated RNA-A
rates, and 45 min was required to convert nearly all RNA-A
substrate to RNA-A
(Fig. 4A, lanes 1 to 4). A graphic repre-
sentation of the data is shown in Fig. 4C. Other deadenylases
were unaffected by PARN-1 overexpression; thus, their activ-
ities were the same in PARN-1 OvEx cell and control extracts.
Therefore, these data indicate that induced overexpression of
PARN-1 protein in parasites enhances deadenylase activity.
A subset of procyclic mRNAs is reduced in PARN-1 OvEx
parasites. To determine the effect of PARN-1 overexpression
on global mRNA steady-state levels, the mRNA expression
proﬁle of PARN-1 OvEx cells was examined by microarray
analysis. Three clones each from the PARN-1 OvEx and con-
trol cell lines were used for analysis, and each experiment was
run in duplicate, with dye labeling reversed between dupli-
cates. Heat maps of the six arrays are shown (Fig. 5A). Twenty-
nine protein-coding genes had their mRNAs decreased (Fig.
5B). Within this gene set, 4 genes encode T. brucei alanine-rich
proteins (BARPs), 2 genes encode acidic phosphatases, 2
genes encode bona ﬁde ribosomal subunits, and 12 genes en-
code hypothetical proteins. Eight protein-coding genes had
their mRNAs increased. This set includes PARN-1, as ex-
pected. All of the misregulated mRNAs appear to be RNA
polymerase II-dependent genes, except for Tb927.4.1200,a
putative expression site-associated gene (ESAG), which is usu-
ally transcribed by RNA polymerase I. Thus, a limited number
of mRNAs expressed in procyclic parasites are regulated, at
least in part, by PARN-1.
PARN-1 affects the steady-state level and decay rate of at
least four different procyclic mRNAs. To conﬁrm the microar-
ray data indicating that the levels of speciﬁc mRNAs were
reduced in PARN-1 OvEx parasites, qRT-PCR was performed
on a subset of genes (Fig. 5B, asterisks, and 6A). Primer sets
and ampliﬁed RNA regions are shown in Fig. S4 in the sup-
plemental material. BARP mRNA levels were reduced ⬃3.2-
fold in cells overexpressing PARN-1, determined using a
primer set that recognized sequences common to all BARP
FIG. 2. PARN-1 is primarily cytoplasmic. (A) PARN-1 is absent from the nucleus. Fractionation of procyclic cells by sucrose cushion separates
the nuclear fraction from the cytoplasmic fraction. Antibody against the RNA polymerase II largest subunit (RPB1) was used as a nuclear control,
and antibody against translation elongation factor 2 (EF-2) was used as a nuclear control. (B) PARN-1 is absent from the mitochondria in wild-type
cells. Fractionation of procyclic cells by a Percoll gradient separates the mitochondrial fraction from the cytoplasmic fraction. Antibody against
elongation factor 2 was used as a cytoplasmic control, and antibody against guide RNA-binding complex proteins 1 and 2 (GRBC1 and -2) was
used as a mitochondrial control. (C) TAP-tagged PARN-1 is primarily cytoplasmic in cells in which PARN-1 is overexpressed. Antibody against
GRBC1 and -2 was used as a mitochondrial protein control.
1234 UTTER ET AL. EUKARYOT.CELL
isoforms (Fig. 6A). mRNA levels of the BARP isoform
Tb09.244.2520 were reduced ⬃5.9-fold in cells overexpressing
PARN-1. The mRNA levels of two conserved hypothetical
proteins (designated p28 and p16 to reﬂect their molecular
masses) were reduced ⬃2-fold in PARN-1 OvEx cells. The
acidic phosphatase (Acid phos) mRNA transcribed from
Tb927.5.630 was reduced ⬃2-fold in PARN-1 OvEx cells. Di-
hydroxyacetone phosphate acyltransferase (DHAPAT) mRNA
steady-state levels, which were unchanged in the microarray
data set, were the same in PARN-1 OvEx and control cell lines.
Thus, the qRT-PCR data and the microarray data are consis-
tent with each other and conﬁrm that a subset of mRNAs is
regulated by PARN-1.
To determine whether the reduced levels of BARP, p28,
p16, and Acid phos mRNAs in PARN-1 OvEx cells were
caused by increased rates of mRNA decay, we measured
mRNA amounts at 0 and 2 h following RNA synthesis inhibi-
tion by actinomycin D (Fig. 6B). DHAPAT mRNA was used to
FIG. 3. Puriﬁed PARN-1 has deadenylase activity in vitro. (A) A silver-stained 10% SDS-acrylamide gel shows the puriﬁcation of TAP-tagged
PARN-1 from procyclic PARN-1 OvEx cell S100 protein extract. Fractions applied to the IgG resin (lanes 1 to 4) ﬂowed through the column (FT)
and eluted in the wash or after TEV protease cleavage (eluate). Eluted protein was applied to streptavidin resin (lanes 5 to 8), and the ﬂowthrough,
wash, and protein eluted with biotin are shown. In each case, the material retained on the resin following elution is shown (beads). Highly enriched
PARN-1 is indicated by an arrow. A molecular size marker (lane M) is shown. (B) Time course analysis of puriﬁed PARN-1 incubated with
radiolabeled RNAs. Lanes 1 to 5, RNA-A
substrate incubated with 50 ng enzyme; lanes 6 to 10, RNA-A
⫹22 substrate incubated with 50 ng
enzyme; lane 11, deadenylated RNA (RNA-A
). About 1% of input material contained an unblocked 3⬘end, due to substrate puriﬁcation
procedures, and was deadenylated as expected. (C) A Western blot, using anti-PARN-1 antibody, identiﬁes puriﬁed PARN-1 samples that
underwent three rounds of depletion (depl) with either PARN-1-speciﬁc antibody (lanes 5 to 7) or nonspeciﬁc antibody (lanes 2 to 4). Lane 1,
starting sample; asterisk, clipped PARN-1 protein that has lost its streptavidin-binding protein tag. (D) Time course analysis of depleted PARN-1
samples incubated with radiolabeled RNA. Lanes 1 to 5, RNA-A
substrate incubated with mock-depleted sample; lanes 6 to 10, RNA-A
substrate incubated with PARN-1-depleted sample. (E) PARN-1 activity in the presence of poly(A) and poly(C) competitor. Lanes 2 to 5,
deadenylation of RNA-A
substrate in the presence of increased concentrations of poly(A) after 30 min; lanes 6 to 9, deadenylation of RNA-A
substrate in the presence of increased concentrations of poly(C) after 30 min. Amounts of competitor added are 0 ng, 50 ng, 100 ng, and 500 ng
in lanes 2 to 5, respectively, and lanes 6 to 9, respectively. (F) PARN-1 activity in the presence (⫹) and absence (⫺)ofMg
and EDTA after
30 min. Reaction products from deadenylase assays (B, D, E, and F) were separated usinga7Murea–5% polyacrylamide denaturing gel. There
are no radioactive signals running faster than the RNA-A
species in the experiments. In panels B, D, E, and F the RNA-A
migration was slower
near the outside edges of the gels. In each case, the RNA-A
is clearly marked.
VOL. 10, 2011 mRNA TURNOVER IN T. BRUCEI 1235
represent the mRNAs that were unaffected by PARN-1 over-
expression in the microarray study. As expected, the amount of
DHAPAT mRNA remaining was similar in the PARN-1 OvEx
and control cell lines (30% versus 31%). The amount of
mRNA remaining from all BARP isoforms was lower in
PARN-1 OvEx cells (8%) than control cells (18%) after 2 h.
Similarly, the amount of mRNA remaining from the BARP
isoform Tb09.244.2520 was also decreased in PARN-1 OvEx
cells (2%) relative to control cells (6%). In addition, the
amount of p28 mRNA remaining was lower in PARN-1 OvEx
cells (8%) than control cells (21%). Unexpectedly, the p16
mRNA amounts remaining in PARN-1 OvEx cells (23%) were
close to those in control cells (19%), and the Acid phos mRNA
amounts remaining in PARN-1 OvEx cells (37%) was greater
than those remaining in control cells (27%). A direct measure
of mRNA half-life was done for BARP (using the primers
speciﬁc for the isoform Tb09.244.2520), p28, and control mes-
sage DHAPAT (Fig. 6C). The degradation kinetics were ex-
ponential, as expected, and are shown for BARP mRNA (see
Fig. S4 in the supplemental material). The half-life of BARP
mRNA was decreased from 22 min to 15 min and the half-life
of p28 mRNA was decreased from 60 min to 33 min after
PARN-1 overexpression. DHAPAT, the control mRNA, was
unaffected by PARN-1 overexpression, maintaining a half-life
of 100 min under the two different conditions. In summary, the
overexpression of PARN-1, as analyzed by qRT-PCR and
mRNA decay proﬁles, resulted in an increase in the mRNA
decay rates of BARP and p28 mRNAs.
Herein we show that trypanosomatids possess three different
PARN genes, PARN-1,-2, and -3. Each PARN gene is ex-
pressed at the mRNA level in two life-cycle stages of the
human parasite Trypanosoma brucei. PARN-1 is an active
deadenylase and appears to regulate a subset of mRNAs, in-
cluding a family of stage-speciﬁc coat proteins, the BARPs.
PARN proteins are members of the DEDD RNase super-
family, characterized by three exonuclease motifs that contain
four invariant acidic amino acids. Most eukaryotes possess a
single PARN gene. The three T. brucei PARNs, PARN-1, -2,
and -3, each contain all three exonuclease motifs. Moreover,
each possesses the conserved acidic amino acids, suggesting
that all three PARNs are active in deadenylation. Other mem-
bers of the trypanosomatid family also possess three PARN
FIG. 4. Overexpression of PARN-1 enhances deadenylation in cytoplasmic extracts. (A) Time course of PARN-1 OvEx cell and control S100
extracts incubated with radiolabeled RNA. Enzyme activity was assayed from TAP tag alone and TAP-tagged PARN-1 protein in S100 extracts
from Tet-induced (OvEx) and uninduced (basal) cultures. Lanes 1 to 4, RNA-A
substrate incubated with S100 extracts from control cells induced
with Tet (control cells produce a Tet-regulated streptavidin-binding protein and protein A TAP tag alone); lanes 5 to 8, RNA-A
incubated with S100 extracts from PARN-1 OvEx cells after Tet induction. Reaction products were separated ona7Murea–5% polyacrylamide
denaturing gel. (B) Western blot showing protein amounts in uninduced and induced (basal and OvEx) S100 extracts. Control and PARN-1 OvEx
cell extracts are included. Control cells contain a conditionally expressed TAP tag, which is detected with secondary antibody. Loading controls
show that equal amounts of protein were applied to each gel lane. (C) The distance (in millimeters) that the majority of the RNA migrated, relative
to the input RNA-A
marker migration, is graphed as a function of deadenylation assay reaction time. The experiments were done three or more
times, and the results of a representative experiment are shown.
1236 UTTER ET AL. EUKARYOT.CELL
FIG. 5. Overexpression of PARN-1 decreases the steady-state mRNA levels of a subset of genes in T. brucei. (A) Heat map showing the change in
steady-state mRNA levels after induction of PARN-1 deadenylase. Data were determined by microarray analysis. The data are arranged by the systematic
gene name, which is based on chromosomal gene location. Columns 1 to 6 present the results for the 6 independent experiments. RNAs are represented
as lines colored relative to their expression levels, as indicated in the heat map key on the left of the map. Green indicates an increase in mRNA levels
in PARN-1 OvEx cells, red indicates a decrease in mRNA levels in PARN-1 OvEx cells, and black indicates no change. Each gene on the microarray
was represented in duplicate; thus, for each experiment, mRNA levels were obtained from both points and averaged. In the case of dye ﬂips, values were
multiplied by ⫺1 to allow numerical comparisons of all six arrays. Heat maps were generated in the MultiExperiment Viewer (MeV) program (version
4.1; TIGR), with genes arranged by identiﬁer. The locus on chromosome (Chr) 9 containing the BARP genes is indicated with an arrow. (B) Summary
of the gene loci encoding the mRNAs that were decreased or increased in PARN-1 OvEx cells. Column 1, systematic gene name, as designated in
GeneDB; column 2, description of the gene product; column 3, number of independent experiments (n/6) in which the gene-encoding mRNA was
decreased at least 1.5-fold; column 4, average fold decrease in PARN-1 OvEx cells among the six experiments; rows 1 to 24, genes located on the locus
on chromosome 9 between Tb09.244.2400 and Tb09.244.2860; asterisks, genes further analyzed by qRT-PCR experiments; shaded gray, genes with
reduced mRNA levels in procyclic parasites relative to other life-cycle stages (30, 44, 54). Microarray data were deposited into the NCBI GEO database
(accession no. GSE20593). Eleven of the 14 BARP ORFs were present in triplicate on the microarray slide.
homologs, indicating multiple roles for PARNs in mRNA reg-
ulation during the complex life cycle of these organisms.
The sequences at the C terminus of all three T. brucei PARNs
and that of the active AtPARN are highly divergent from the
sequence at the C terminus of human PARN. In human PARN,
the C terminus binds cap 0 (m
GpppG) of mRNA during deade-
nylation (14, 21, 38). Trypanosome mRNAs contain a cap 4 (a
hypermethylated form of m
GpppAACU). Thus, the divergence
from the human PARN in the C termini of the three T. brucei
PARNs may reﬂect an interaction between at least one of them
and the unique trypanosome cap structure.
Deletion of PARN-1 was not lethal to cultured procyclic
FIG. 6. PARN-1 overexpression affects the steady-state levels and mRNA decay of speciﬁc genes. (A) Steady-state mRNA levels of genes in
PARN-1 OvEx cells (light bars) and control cells (dark bars) determined by qRT-PCR. mRNA amounts were normalized to that of 7SL RNA,
and levels from control cells were set equal to 100%. BARP (all), primers used to recognize all BARP isoforms; BARP (2520), BARP isoform
Tb09.244.2520. (B) Percent mRNA remaining before actinomycin D treatment (dark bars) and 2 h after actinomycin D treatment (light bars), as
determined by qRT-PCR. (C) A log scale shows the degradation kinetics of BARP, p28, and DHAPAT (control) mRNAs in PARN-1 OvEx cells.
Actinomycin D (10 g/ml) was added at time zero, and mRNA was isolated at the indicated time points and quantitated using qRT-PCR. Control
cells in each graph show cells induced to overexpress the TAP tag alone. The quantitated results are expressed as the mean and 1 standard deviation
for at least three independent experiments performed using two different RNA preparations. Although the degradation kinetics appear to be
biphasic, overall half-lives (t
s) are calculated on the basis of changes in total mRNA levels and are therefore plotted on a semilog scale.
1238 UTTER ET AL. EUKARYOT.CELL
parasites. This result was not surprising, as functional PARN is
also not essential for viability in cultured HeLa cells, Xenopus
oocytes, or Saccharomyces pombe (10, 31). However, PARN
may be required for cellular processes involved in develop-
ment. For example, embryonic development is stymied in Ara-
bidopsis lacking PARN (48).
T. brucei PARN-1 participates in regulating speciﬁc mRNAs,
as determined by our microarray and qRT-PCR data. Over-
expression of PARN-1 affected BARP and p28 mRNA abun-
dance and decay. In humans, PARN plays a role in regulating
speciﬁc mRNAs via targeting to AU-rich element-containing
mRNAs (35). In Xenopus and Arabidopsis, PARN plays a role
in embryogenesis, targeting different subsets of mRNAs at
speciﬁc stages of development (31, 48). Similarly, PARN-1 may
regulate BARPs in a life-stage-speciﬁc manner in T. brucei.
BARP mRNA is present only at low levels in the procyclic life
stage, where we have conﬁrmed that the PARN-1 protein is
present (data not shown). These low BARP levels are possibly
due to PARN-1-mediated decay. In contrast, BARP protein is
highly expressed in epimastigotes (54), and we predict that
PARN-1-mediated decay of BARP mRNA is decreased in this
Other mRNA decay studies in trypanosomes have begun to
characterize CAF1, PAN2, DHH1, XRNA, DCP1/2, and
DCPS-like activities and the exosome in T. brucei (16, 24, 33,
37, 41, 50, 51). CAF1 and PAN2 appear to affect the decay of
stable mRNAs since depletion of either protein resulted in an
increased poly(A) tail length in bloodstream-form parasites
(50, 51). In addition, depletion of the CAF1, PAN2, or XRNA
protein decreased the decay rate of the unstable EP1 procyclin
mRNA in bloodstream-form cells. Also, recent studies of
DHH1 show that this RNA helicase has a selective role in
modulating levels of developmentally regulated mRNAs (33).
On the basis of our ﬁndings that PARN-1 is a nonessential
protein for cultured procyclic parasites and appears to regulate
the steady-state levels of a subpopulation of cellular mRNAs,
we propose that PARN-1 operates as a regulatory enzyme to
control the levels of a subset of mRNAs during the parasite life
cycle. The paradigm for differential deadenylase targeting of
speciﬁc mRNAs includes RNA-binding proteins that recruit
deadenylases via cis-acting elements in speciﬁc mRNAs. Inter-
estingly, Roditi and colleagues have shown that the BARP 3⬘
untranslated region (UTR) contains cis-acting elements that
contribute to mRNA instability in procyclic parasites (54).
PARN-1 may participate in the regulation of BARP mRNA
turnover by interacting with 3⬘UTR-protein complexes. Thus,
it will be interesting to see if the set of three PARN deadeny-
lases, recruited by different RNA-binding proteins, serves to
modulate stage-speciﬁc mRNA decay during the parasite life
In summary, we present herein the ﬁrst characterization of
PARN in a single-cell eukaryote. PARN-mediated degrada-
tion of stage-speciﬁc BARP messages suggests a role for
PARN in T. brucei development much like the developmental
role of PARN in multicell eukaryotes. Interestingly, T. cruzi
and Leishmania spp. also encode PARN homologues. We
speculate that these human-infective trypanosomes, and pos-
sibly other pathogenic single-cell eukaryotes, utilize PARN to
regulate gene expression during development.
We thank David Wah and Mahi Banday for critical reading of the
manuscript. We thank Ruslan Aphasizhev for anti-guide RNA-binding
complex protein antibodies. We gratefully appreciate the ﬁgure illus-
trations by Timothy Linteau and Han Wu. We appreciate the work of
Harleen Jammu for anti-PARN-1 production.
This work was supported by National Institutes of Health, NIAID
(grant AI535835 to V.B.), and a Foundation of UMDNJ grant to V.B.
1. Balatsos, N. A., P. Nilsson, C. Mazza, S. Cusack, and A. Virtanen. 2006.
Inhibition of mRNA deadenylation by the nuclear cap binding complex
(CBC). J. Biol. Chem. 281:4517–4522.
2. Banerjee, H., et al. 2009. Identiﬁcation of the HIT-45 protein from Trypano-
soma brucei as an FHIT protein/dinucleoside triphosphatase: substrate spec-
iﬁcity studies on the recombinant and endogenous proteins. RNA 15:1554–
3. Belostotsky, D. A., and L. E. Sieburth. 2009. Kill the messenger: mRNA
decay and plant development. Curr. Opin. Plant Biol. 12:96–102.
4. Berberof, M., et al. 1995. The 3⬘-terminal region of the mRNAs for VSG and
procyclin can confer stage speciﬁcity to gene expression in Trypanosoma
brucei. EMBO J. 14:2925–2934.
5. Berriman, M., et al. 2005. The genome of the African trypanosome Trypano-
soma brucei. Science 309:416–422.
6. Blattner, J., and C. E. Clayton. 1995. The 3⬘-untranslated regions from the
Trypanosoma brucei phosphoglycerate kinase-encoding genes mediate de-
velopmental regulation. Gene 162:153–156.
7. Brewer, G. 1991. An A ⫹U-rich element RNA-binding factor regulates
c-myc mRNA stability in vitro. Mol. Cell. Biol. 11:2460–2466.
8. Chen, C. Y., and A. B. Shyu. 1995. AU-rich elements: characterization and
importance in mRNA degradation. Trends Biochem. Sci. 20:465–470.
9. Chiba, Y., et al. 2004. AtPARN is an essential poly(A) RNase in Arabidopsis.
10. Chou, C. F., et al. 2006. Tethering KSRP, a decay-promoting AU-rich ele-
ment-binding protein, to mRNAs elicits mRNA decay. Mol. Cell. Biol.
11. Clayton, C. E. 2002. Life without transcriptional control? From ﬂy to man
and back again. EMBO J. 21:1881–1888.
12. Das, A., and V. Bellofatto. 2009. The non-canonical CTD of RNAP-II is
essential for productive RNA synthesis in Trypanosoma brucei. PLoS One
13. Daugeron, M. C., F. Mauxion, and B. Seraphin. 2001. The yeast POP2 gene
encodes a nuclease involved in mRNA deadenylation. Nucleic Acids Res.
14. Dehlin, E., M. Wormington, C. G. Korner, and E. Wahle. 2000. Cap-depen-
dent deadenylation of mRNA. EMBO J. 19:1079–1086.
15. Dlakic, M. 2000. Functionally unrelated signalling proteins contain a fold
similar to Mg
-dependent endonucleases. Trends Biochem. Sci. 25:272–
16. Estevez, A. M., T. Kempf, and C. Clayton. 2001. The exosome of Trypano-
soma brucei. EMBO J. 20:3831–3839.
17. Ford, L. P., J. Watson, J. D. Keene, and J. Wilusz. 1999. ELAV proteins
stabilize deadenylated intermediates in a novel in vitro mRNA deadenyla-
tion/degradation system. Genes Dev. 13:188–201.
18. Ford, L. P., and J. Wilusz. 1999. An in vitro system using HeLa cytoplasmic
extracts that reproduces regulated mRNA stability. Methods 17:21–27.
19. Fritz, D. T., N. Bergman, W. J. Kilpatrick, C. J. Wilusz, and J. Wilusz. 2004.
mRNA decay in mammalian cells: the exonuclease perspective. Cell
Biochem. Biophys. 41:265–278.
20. Furger, A., N. Schurch, U. Kurath, and I. Roditi. 1997. Elements in the 3⬘
untranslated region of procyclin mRNA regulate expression in insect forms
of Trypanosoma brucei by modulating RNA stability and translation. Mol.
Cell. Biol. 17:4372–4380.
21. Gao, M., D. T. Fritz, L. P. Ford, and J. Wilusz. 2000. Interaction between a
poly(A)-speciﬁc RNase and the 5⬘cap inﬂuences mRNA deadenylation rates
in vitro. Mol. Cell 5:479–488.
22. Garneau, N. L., J. Wilusz, and C. J. Wilusz. 2007. The highways and byways
of mRNA decay. Nat. Rev. Mol. Cell Biol. 8:113–126.
23. Goldstrohm, A. C., B. A. Hook, and M. Wickens. 2008. Regulated deadeny-
lation in vitro. Methods Enzymol. 448:77–106.
24. Haile, S., A. M. Estevez, and C. Clayton. 2003. A role for the exosome in the
in vivo degradation of unstable mRNAs. RNA 9:1491–1501.
25. Hendricks, E. F., and K. Matthews. 2007. Post-transcriptional control of
gene expression in African trypanosomes, p. 209–237. In J. D. Barry, R.
McCulloch, J. C. Mottram, and A. Acosta-Serrano (ed.), Trypanosomes;
after the genome. Horizon Bioscience, Norwich, United Kingdom.
26. Hirumi, H., and K. Hirumi. 1989. Continuous cultivation of Trypanosoma
brucei blood stream forms in a medium containing a low concentration of
serum protein without feeder cell layers. J. Parasitol. 75:985–989.
27. Hotz, H. R., C. Hartmann, K. Huober, M. Hug, and C. Clayton. 1997.
VOL. 10, 2011 mRNA TURNOVER IN T. BRUCEI 1239
Mechanisms of developmental regulation in Trypanosoma brucei: a polypy-
rimidine tract in the 3⬘-untranslated region of a surface protein mRNA
affects RNA abundance and translation. Nucleic Acids Res. 25:3017–3026.
28. Hotz, H. R., P. Lorenz, R. Fischer, S. Krieger, and C. Clayton. 1995. Role of
3⬘-untranslated regions in the regulation of hexose transporter mRNAs in
Trypanosoma brucei. Mol. Biochem. Parasitol. 75:1–14.
29. Ibrahim, B. S., et al. 2009. Structure of the C-terminal domain of transcrip-
tion factor IIB from Trypanosoma brucei. Proc. Natl. Acad. Sci. U. S. A.
30. Jensen, B. C., D. Sivam, C. T. Kifer, P. J. Myler, and M. Parsons. 2009.
Widespread variation in transcript abundance within and across develop-
mental stages of Trypanosoma brucei. BMC Genomics 10:482.
31. Kim, J. H., and J. D. Richter. 2006. Opposing polymerase-deadenylase
activities regulate cytoplasmic polyadenylation. Mol. Cell 24:173–183.
32. Korner, C. G., et al. 1998. The deadenylating nuclease (DAN) is involved in
poly(A) tail removal during the meiotic maturation of Xenopus oocytes.
EMBO J. 17:5427–5437.
33. Kramer, S., et al. 2010. The RNA helicase DHH1 is central to the correct
expression of many developmentally regulated mRNAs in trypanosomes.
J. Cell Sci. 123:699–711.
34. Lai, W. S., et al. 1999. Evidence that tristetraprolin binds to AU-rich ele-
ments and promotes the deadenylation and destabilization of tumor necrosis
factor alpha mRNA. Mol. Cell. Biol. 19:4311–4323.
35. Lai, W. S., E. A. Kennington, and P. J. Blackshear. 2003. Tristetraprolin and
its family members can promote the cell-free deadenylation of AU-rich
element-containing mRNAs by poly(A) RNase. Mol. Cell. Biol. 23:3798–
36. Lejeune, F., X. Li, and L. E. Maquat. 2003. Nonsense-mediated mRNA
decay in mammalian cells involves decapping, deadenylating, and exonucleo-
lytic activities. Mol. Cell 12:675–687.
37. Li, C. H., et al. 2006. Roles of a Trypanosoma brucei 5⬘33⬘exoribonuclease
homolog in mRNA degradation. RNA 12:2171–2186.
38. Martinez, J., Y. G. Ren, P. Nilsson, M. Ehrenberg, and A. Virtanen. 2001.
The mRNA cap structure stimulates rate of poly(A) removal and ampliﬁes
processivity of degradation. J. Biol. Chem. 276:27923–27929.
39. Mauxion, F., C. Faux, and B. Seraphin. 2008. The BTG2 protein is a general
activator of mRNA deadenylation. EMBO J. 27:1039–1048.
40. Meyer, S., C. Temme, and E. Wahle. 2004. mRNA turnover in eukaryotes:
pathways and enzymes. Crit. Rev. Biochem. Mol. Biol. 39:197–216.
41. Milone, J., J. Wilusz, and V. Bellofatto. 2002. Identiﬁcation of mRNA de-
capping activities and an ARE-regulated 3⬘to 5⬘exonuclease activity in
trypanosome extracts. Nucleic Acids Res. 30:4040–4050.
42. Milone, J., J. Wilusz, and V. Bellofatto. 2004. Characterization of deadeny-
lation in trypanosome extracts and its inhibition by poly(A)-binding protein
Pab1p. RNA 10:448–457.
43. Nilsson, P., et al. 2007. A multifunctional RNA recognition motif in poly(A)-
speciﬁc RNase with cap and poly(A) binding properties. J. Biol. Chem.
44. Nolan, D. P., et al. 2000. Characterization of a novel alanine-rich protein
located in surface microdomains in Trypanosoma brucei. J. Biol. Chem.
45. Panigrahi, A. K., A. Schnaufer, and K. D. Stuart. 2007. Isolation and com-
positional analysis of trypanosomatid editosomes. Methods Enzymol. 424:1–
46. Parker, R., and H. Song. 2004. The enzymes and control of eukaryotic
mRNA turnover. Nat. Struct. Mol. Biol. 11:121–127.
47. Peng, S. S., C. Y. Chen, N. Xu, and A. B. Shyu. 1998. RNA stabilization by
the AU-rich element binding protein, HuR, an ELAV protein. EMBO J.
48. Reverdatto, S. V., J. A. Dutko, J. A. Chekanova, D. A. Hamilton, and D. A.
Belostotsky. 2004. mRNA deadenylation by PARN is essential for embryo-
genesis in higher plants. RNA 10:1200–1214.
49. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory
manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
50. Schwede, A., et al. 2008. A role for Caf1 in mRNA deadenylation and decay
in trypanosomes and human cells. Nucleic Acids Res. 36:3374–3388.
51. Schwede, A., et al. 2009. The role of deadenylation in the degradation of
unstable mRNAs in trypanosomes. Nucleic Acids Res. 37:5511–5528.
52. Tadauchi, T., K. Matsumoto, I. Herskowitz, and K. Irie. 2001. Post-tran-
scriptional regulation through the HO 3⬘-UTR by Mpt5, a yeast homolog of
Pumilio and FBF. EMBO J. 20:552–561.
53. Tetaud, E., M. P. Barrett, F. Bringaud, and T. Baltz. 1997. Kinetoplastid
glucose transporters. Biochem. J. 325(Pt 3):569–580.
54. Urwyler, S., E. Studer, C. K. Renggli, and I. Roditi. 2007. A family of
stage-speciﬁc alanine-rich proteins on the surface of epimastigote forms of
Trypanosoma brucei. Mol. Microbiol. 63:218–228.
55. Vlasova, I. A., and P. R. Bohjanen. 2008. Posttranscriptional regulation of
gene networks by GU-rich elements and CELF proteins. RNA Biol. 5:201–
56. Wiederhold, K., and L. A. Passmore. 2010. Cytoplasmic deadenylation: reg-
ulation of mRNA fate. Biochem. Soc. Trans. 38:1531–1536.
57. Wirtz, E., S. Leal, C. Ochatt, and G. A. Cross. 1999. A tightly regulated
inducible expression system for conditional gene knock-outs and dominant-
negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 99:89–
58. Yamashita, A., et al. 2005. Concerted action of poly(A) nucleases and de-
capping enzyme in mammalian mRNA turnover. Nat. Struct. Mol. Biol.
59. Zuo, Y., and M. P. Deutscher. 2001. Exoribonuclease superfamilies: struc-
tural analysis and phylogenetic distribution. Nucleic Acids Res. 29:1017–
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