From the Department of Microbiology and Center for Microbial Pathogenesis, State University of New York at Buffalo School of Medicine, Buffalo, New York 14214
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ABSTRACT |
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Trypanosoma brucei mitochondria
possess a unique mechanism of mRNA maturation called RNA editing.
In this process, uridylate residues are inserted and deleted
posttranscriptionally into pre-mRNA to create translatable
messages. The genetic information for RNA editing resides in small RNA
molecules called guide RNAs (gRNAs). Thus, proteins in direct contact
with gRNA are likely to catalyze or influence RNA editing. Herein we
characterize an abundant gRNA-binding protein from T. brucei mitochondria. This protein, which we term RBP16 (for
RNA-binding protein of 16 kDa), binds to different gRNA molecules. The
major determinant of this interaction is the oligo(U) tail, present on
the 3'-ends of gRNAs. RBP16 forms multiple, stable complexes with gRNA
in vitro, and immunoprecipitation experiments provide
evidence for an association between RBP16 and gRNA within T. brucei mitochondria. Mature RBP16 contains a cold shock domain at
the N terminus and a C-terminal region rich in arginine and glycine.
The presence of the cold shock domain places RBP16 as the first
organellar member of the highly conserved Y-box protein family. The
arginine and glycine rich C terminus in combination with the cold shock
domain predicts that RBP16 will be involved in the regulation of gene
expression at the posttranscriptional level.
Kinetoplastid organisms possess a unique RNA processing mechanism
called kinetoplastid RNA
(kRNA)1 editing. In this
system, certain mitochondrial pre-mRNAs are posttranscriptionally
modified by the insertion and deletion of exclusively uridylate
residues to otherwise cryptic transcripts (reviewed in Refs. 1-3).
Uridylate insertion and deletion is presumably a requirement for
translation of these kinetoplast mRNAs, as it often creates start
codons, stop codons, and in many cases entire open reading frames. The
genetic information for kRNA editing is contained in short,
mitochondrially encoded transcripts called guide RNAs (gRNAs) (4, 5).
Each gRNA transfers the genetic information for specific uridylate
insertion and deletion at multiple, adjacent sites in the pre-mRNA
through base pairing interactions. In addition, multiple gRNAs are
required for the complete editing of most pre-mRNAs.
A complex of seven proteins that contains editing activity as well as
many of the enzyme activities previously postulated to be involved in
kRNA editing has been purified from Trypanosoma brucei
mitochondria (6). However, given the extensive interactions between
biological macromolecules that must take place during kRNA editing, it
is likely that proteins exist other than those found within the
purified editing complex that affect the efficiency and/or accuracy of
this process. A likely target for such protein factors is gRNA, given
the central role of gRNA in this process. Evidence for the existence of
gRNA-binding proteins in kinetoplastid mitochondria has been presented
by several laboratories (7-10). Initial experiments using gel
retardation methods revealed four ribonucleoprotein complexes that
associate specifically with gRNAs (7, 8). Subsequent UV cross-linking
experiments identified a set of mitochondrial proteins ranging from 9 to 124 kDa in T. brucei (8, 9) and from 30 to 88 kDa in
Crithidia fasciculata (10) that interact with different
gRNAs. A cDNA encoding a 21-kDa, high affinity gRNA-binding
protein, gBP21, was recently cloned from T. brucei (11).
Immunoprecipitation experiments have since revealed an association
between gBP21 and the editing machinery (12). A 90-kDa protein with
oligo(U) binding activity, TBRGG1, was found to co-sediment with
editing activity (13). Finally, from L. tarentolae,
gRNA-binding proteins of 18 and 110 kDa were identified as the ATPase
subunit b and glutamate dehydrogenase, respectively (14, 15). These
enzymes could represent a regulatory link between metabolism and RNA
editing in kinetoplastids. Because gRNAs exhibit little primary
sequence similarity, the basis of gRNA recognition by these proteins is
unclear. Common determinants on gRNA molecules that may have a role in
recognition by these proteins include a posttranscriptionally added 3'
oligo(U) tail (16) and a common secondary structure (17, 18).
To further understand the process of kRNA editing, we focused our
efforts on identifying and characterizing other protein factors that
interact specifically with gRNAs. We report here the identification of
a gene encoding a protein with an apparent molecular mass of 16 kDa
that can be UV cross-linked specifically to gRNA. We designated this
protein RBP16 (RNA-binding protein of 16 kDa). RBP16 was purified based
on its affinity for poly(U). Subsequent experiments confirmed that
RBP16 is capable of interacting with different gRNAs through the
oligo(U) tail. Furthermore, immunoprecipitation experiments provide
strong evidence that RBP16 interacts with gRNA in vivo. The
cDNA sequence of RBP16 predicts a protein with three domains: a
cleaved mitochondrial import sequence, an N-terminal cold shock domain
(CSD), and a C-terminal arginine- and glycine-rich region. The CSD of
RBP16 shows extensive homology to bacterial cold shock proteins and to
eukaryotic Y-box proteins. Bacterial cold shock proteins are proposed
to function as RNA chaperones by preventing disadvantageous RNA
secondary structures (19, 20). The eukaryotic Y-box proteins function
in both transcriptional and posttranscriptional regulation of gene
expression (21). Possible roles of RBP16 in kRNA editing and other
aspects of trypanosome mitochondrial gene expression are discussed.
Cell Culture, Mitochondrial Vesicle Isolation, and Nucleic Acid
Preparation--
Procyclic form T. brucei brucei clone
IsTaR1 stock EATRO 164 was grown as described (22). Mitochondrial
vesicles were isolated and stored using a previously described method
(23). Constructs encoding the gRNAs gA6[14] and gCYb[558], with 17 and 15 nucleotides (nt) oligo(U) tails respectively, were previously
described (8). A gA6[14] construct lacking the 3' oligo(U) tail was
generated by PCR (8). Synthetic gRNA molecules possess 10 nt of vector sequence on their 5'-ends. For competition assays, a control
transcript, approximately the size of a synthetic gRNA, was produced by
run-off transcription from pBluescript (Stratagene) linearized with
BamHI. RNAs were synthesized in vitro with T7
polymerase and gel-purified on 6% acrylamide/7 M urea.
Isolation of RBP16 Protein--
Purified mitochondrial vesicles
from approximately 4 × 1011 cells were resuspended in
Buffer A (25 mM Tris-Cl (pH 8.0), 15 mM MgOAc,
50 mM KCl) at a concentration of 1 × 1011
cells/ml. All buffers contained 1 mM phenylmethylsulfonyl
fluoride, 1.0 µg/ml leupeptin, and 5 mM iodoacetamide to
minimize proteolysis. Vesicles were lysed by addition of 0.2% Nonidet
P-40, and insoluble material was cleared from mitochondrial extracts by
centrifugation. After addition of 1 mM CaCl2,
the cleared extract was incubated with micrococcal nuclease (100 units/1 × 1011 cells) for 15 min at 27 °C.
Micrococcal nuclease was thereafter inhibited by the addition of EGTA
to 5 mM. A poly(U)-Sepharose (Amersham Pharmacia Biotech)
column with a 1-ml bed volume was equilibrated in Buffer A containing
50 mM KCl and 10% glycerol. The cleared, micrococcal
nuclease treated extract was loaded onto the column, and the column was
washed with 10 column volumes of Buffer A containing 300 mM
KCl. Bound proteins were eluted with 10 column volumes of Buffer A
containing a linear salt gradient ranging from 300 mM-800
mM KCl. Fraction volumes of 0.5 ml were collected.
Fractions were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by UV cross-linking to synthetic gA6(14) internally labeled with [ Cloning of RBP16 cDNA--
Internal peptide sequences
were used for the construction of degenerate oligonucleotides based on
previously reported T. brucei codon usage (25). Restriction
sites at the 5'-ends of oligonucleotides are indicated by underlining.
Total procyclic form cDNA was generated by reverse transcription
primed with (dT)-RXS
(5'-GAGAATTCTCGAGTCGACTTTTTTTTTTTTTTT-3'). Internal
cDNA sequence was obtained by PCR amplification of procyclic cDNA with the degenerate primers TB16-D
(5'-GCGGATCCNACYTCRAAYTCVACYTCYTGRCCNAC-3') and TB16-E
(5'-GCGGATCCGGNTTYATYGARGAYGAYGCNGAY-3'). Nested PCR was
then employed to selectively amplify 5'- and 3'-ends of RBP16 as
follows. The 5'-end was first amplified using a primer corresponding to
a portion of the splice leader sequence, E-SL22
(5'-GCGAATTCGCTATTATTAGAACAGTTTCTG-3'), and the primer
TB16K-I (5'-GCGGATCCGTTTGAAGAGCTGAGAAATGC-3'). The 5'-end
was further amplified with the primers E-SL22 (above) and TB16F-OE
(5'-GCGGATCCRTCNGCRTCRTCYTCRATRAANCC-3') using 1 µl of
the previous reaction as template. The 3'-end of RBP16 cDNA was
amplified using primer (dT)-RXS (above) and the primer TB16K-G (5'-GCGGATCCGAAGCAACACTTTGTGCATTTCTC-3'). Further
amplification of the 3'-end of RBP16 cDNA was accomplished with the
primers (dT)-RXS (above) and TB16K-J
(5'-GCGGATCCGCTCTTCAAACGGAAACGGGGGG-3') using 1 µl of
the previous reaction as template. All reaction products were digested
and ligated into the EcoRI/BamHI site of pBluescriptII SK Bacterial Expression of RBP16--
The full-length RBP16 open
reading frame (beginning with the nucleotide sequence corresponding to
the N-terminal peptide sequence) was amplified by PCR from total
procyclic cDNA using primers Tb16K-O (5'-GCGGATCCATGGGTAACAAGGGTAAGGTGATATCG-3') and Tb16K-P
(5'-GCGTCGACCTAAAAGTCATCGCTGAAGCTC-3'), which were
constructed based on the cDNA sequence. The reaction products were
inserted into the BamHI/SalI site of pMal-C2 (New England BioLabs) and transformed into E. coli strain BL21
pLysS (Novagen). Expression and purification of the resulting
maltose-binding protein (MBP) fusion was done by standard protocol
(28). The expressed fusion protein (MBP-RBP16) was further purified by
poly(U)-Sepharose chromatography.
Characterization of Nucleic Acid Binding
Properties--
Partially purified RBP16 (100 ng/reaction) and
purified MBP-RBP16 (680 ng/reaction) were analyzed by UV cross-linking
to a synthetic gRNA in the presence of various nucleic acid
competitors. Ribohomopolymers, riboheteropolymers, and the
double-stranded RNA polymer (poly(A)·U)) were purchased from Sigma.
Oligo-(dT)20 was purchased from Integrated DNA
Technologies, Inc. A Bio-Rad model GS-700 imaging densitometer was used
for the quantification of UV cross-linking signals in combination with
Molecular Analyst software (version 1.5). For gel retardation assays,
reaction conditions were identical to UV cross-linking experiments.
After incubation at room temperature for 20 min, RBP16-gRNA complexes
were separated by electrophoresis on a native 4% acrylamide gel
(acrylamide/bisacrylamide ratio 19:1) in 50 mM Tris-glycine
(pH 8.8). Shifted bands were detected by autoradiography.
Immunoprecipitation Experiments--
The MBP-RBP16 fusion
protein was used for the production of a polyclonal rabbit serum in
coordination with the State University of New York at Buffalo
Monoclonal Antibody center. Protein A-Sepharose (Amersham Pharmacia
Biotech) was used for the purification of IgG molecules from both
immune and preimmune sera. For immunoprecipitation experiments,
mitochondrial vesicles from 1 × 1010 cells were lysed
in 1 ml of buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 2.5 mM EDTA, 0.5 mM
dithiothreitol, 0.2% Nonidet P-40), and insoluble material was cleared
by centrifugation. One hundred micrograms of purified IgG from immune
or preimmune sera was added to 300 µl of extract along with 100 units
of RNAguard RNase inhibitor (Amersham Pharmacia Biotech) and incubated
for 1 h at 4 °C with gentle rocking. Reactions were added to 50 µl of protein A-Sepharose equilibrated in wash buffer (as above with
0.1% Nonidet P-40), and incubated at 4 °C for 1 h with gentle
rocking. Immune complexes were separated from supernatants by
centrifugation and washed five times with 500 µl of wash buffer. RNA
was isolated from protein A-Sepharose pellets and supernatants by
incubation for 15 min at 37 °C in wash buffer containing 0.5% SDS
and 50 µg/ml proteinase K. The reactions were extracted twice with
phenol-chloroform, and RNA was precipitated with ethanol using glycogen
as a carrier. RNA was detected by labeling the 5'-ends with
[ Identification of RBP16--
All gRNA molecules possess a 3'
oligo(U) extension that could potentially act as a protein recognition
site (16). In addition, experiments from other laboratories
demonstrated that poly(U) is an efficient competitor for the UV
cross-linking of mitochondrial proteins to gRNA (9-10). Based on these
observations, we fractionated mitochondrial proteins from procyclic
form trypanosomes according to poly(U) affinity to obtain a
chromatographic fraction enriched for gRNA-binding proteins. The
protein profile of the poly(U)-Sepharose fractionation procedure is
shown in Fig. 1A. An abundant
protein with an apparent molecular mass of 16 kDa eluted in fractions 9-16. Fractions were assayed for gRNA-binding proteins by UV
cross-linking to synthetic gA6[14] internally labeled with
[
To examine the gRNA binding capabilities of RBP16, partially purified
RBP16 was UV cross-linked to radiolabeled gA6[14] in the presence of
increasing amounts of unlabeled competitors (Fig. 2A). The quantification of UV
cross-linking signals from Fig. 2A is shown in Fig.
2B. The gA6(14) UV cross-linking signal was competed >50%
with 1000-fold molar excess unlabeled gA6[14] or gCYb[558],
indicating that RBP16 has the capacity to bind different gRNAs. To
examine the role of the oligo(U) tail in gRNA binding, the UV
cross-linking signal was competed with increasing levels of unlabeled
gA6[14]NT, a gRNA lacking the oligo(U) tail. A 4000-fold molar excess
of this RNA was required to achieve the level of competition observed
with unlabeled gA6[14]. To a slightly lesser extent, competition was
observed at a 4000-fold molar excess of unlabeled transcript from a
Bluescript plasmid. The difference in the ability of gA6[14]NT and
the pBluescript transcript to compete the gA6[14] UV cross-linking
signal was small but reproducible. This experiment demonstrated that
RBP16 preferentially binds to gRNA over a random RNA molecule of the
same size and that the major determinant of the interaction is the
oligo(U) tail.
Cloning of RBP16 cDNA--
Internal as well as N-terminal
peptide sequences were obtained from the purified protein. Using
degenerate oligonucleotide primers constructed from internal peptide
sequences, a portion of the RBP16 cDNA was amplified by PCR from
total procyclic cDNA followed by 5' and 3' amplification of
cDNA ends. The RBP16 cDNA sequence predicts an RNA molecule
with an open reading frame of 423 nt, as well as 5' and 3' untranslated
regions of 123 and 578 nt, respectively (Fig.
3A). Northern analysis of
total procyclic RNA detected a single transcript of ~1200 nt (data
not shown), which is in good agreement with our cDNA sequence.
Southern blot analysis of T. brucei genomic DNA demonstrated
that RBP16 was encoded as a single copy (data not shown). Fig.
3A shows the RBP16 cDNA sequence aligned with the
deduced amino acid sequence. The comparison of the N-terminal peptide
sequence, which begins with asparagine 18, to the expected amino acid
sequence suggests that the first 17 amino acids encode a cleaved
mitochondrial import sequence. In light of this observation, the
cDNA sequence predicts a mature protein of 13.2 kDa with a pI of
9.29. An overall positive charge of RBP16 at pH 8.8 may explain the
slightly slower electrophoretic mobility than is predicted by its
size.
Sequence comparison of RBP16 to proteins in several data bases revealed
significant homology of the N-terminal half of mature RBP16 to a motif
known as the cold shock domain (CSD). The CSD is a well conserved
nucleic acid binding domain that is homologous to the bacterial cold
shock proteins and is a component of the eukaryotic Y-box proteins
(reviewed in Ref. 30). Bacterial cold shock proteins consist entirely
of one CSD, whereas the eukaryotic Y-box proteins consist of one or
more CSDs usually with auxiliary domains. The CSD of RBP16 presumably
participates in gRNA interactions because it contains the RNP1 RNA
binding motif, conserved in all CSDs (31) (Fig. 3B, box).
Fig. 3B shows the CSD of RBP16 aligned with CspA of E. coli and the CSDs of three eukaryotic Y-box proteins. The cold
shock domain of RBP16 exhibits somewhat higher homology to prokaryotic
cold shock proteins (43-46% identity) than to eukaryotic CSDs
(33-38% identity). At the C terminus of RBP16 is a region that is
rich in arginine (14%) and glycine (32%) and may also be capable of
interacting with RNA based on an RGG type RNA binding motif (32). It is
worth noting that this domain contains an amino acid sequence (amino
acids 107-113 of the preprotein; amino acids 90-96 of the mature
protein) that is consistent with the preferred recognition motif
reported for arginine methylation of hnRNP A1 (33).
Bacterial Expression of MBP-RBP16 Fusion Protein--
For
bacterial expression of RBP16, the mature open reading frame beginning
with asparagine 18 was cloned into the pMal-C2 expression vector. Upon
induction with isopropyl-1-thio- Characterization of RBP16 Nucleic Acid Binding Properties--
UV
cross-linking competitions were used to characterize the binding of
RBP16 to various nucleic acid substrates and to compare the binding
properties of the MBP fusion protein with partially purified RBP16. The
partially purified native RBP16 and the MBP fusion protein showed
nearly identical preferences for various nucleic acid substrates (Fig.
4, B-D). These data demonstrate that the amplified cDNA
sequence encodes the RBP16 protein that was purified from trypanosome
mitochondrial vesicles. RBP16 shows affinity for poly(U) and, to a
lesser extent, poly(G) (Fig. 4B). The RBP16 UV cross-linking
signal is almost completely abolished by unlabeled poly(U) at 1000-fold
mass excess over labeled gRNA substrate and is competed with unlabeled
poly(G) to the same extent by approximately 5000-fold mass excess cold
competitor. No binding to poly(A) or poly(C) was observed. Unlabeled
poly(GU) and poly(CU) heteropolymers competed the UV cross-linking
signal as effectively as poly(U) (Fig. 4C). AU and AC
ribopolymers were ineffective in competing UV cross-linking to gA6(14).
The inability of RBP16 to bind poly(AU) maybe due to extensive
formation of double-stranded regions in this RNA. Fig. 4D
demonstrates that RBP16 can bind RNA (poly(U)) and single-stranded DNA
(oligo-(dT)20), but does not bind double-stranded RNA
(poly(A)·(U)). Although binding to oligo-(dT)20 appears
somewhat stronger than to poly(U), the significance of this result is
difficult to interpret. Given equal masses, oligo-(dT)20
contains much higher concentration of 5'- and 3'-ends than poly(U),
which is hundreds of nucleotides in length. The requirement for 5'-
and/or 3'-ends for RBP16 binding is unknown.
Gel retardation assays were used to estimate the affinity of RBP16 for
gA6[14]. When internally labeled gA6[14] was incubated with a 1 µM concentration of MBP-RBP16, no shift in the gRNA probe was observed. A single shifted band was observed when MBP-RBP16 was
added to a concentration of 2.5 µM (Fig.
5). At MBP-RBP16 concentrations of 5 µM and above, two band shifts were evident (Fig. 5). The
appearance of two shifted forms may represent two RBP16 molecules bound
to one gRNA molecule, and this possibility is currently being
investigated. No shift was seen when MBP was used in the assay (Fig.
5). This experiment demonstrated that MBP-RBP16 forms stable complexes
with gA6[14] at a minimum concentration of 2.5 µM.
Co-immunoprecipitation of gRNA with RBP16--
To demonstrate an
interaction between RBP16 and gRNA within mitochondrial vesicles, we
investigated the ability of antibodies directed against the MBP-RBP16
fusion protein to co-immunoprecipitate gRNA. Anti-RBP16 antibodies were
purified from rabbit serum using protein A-Sepharose and did not
recognize any proteins other than RBP16 in Western blots of
mitochondrial extracts (data not shown). Anti-RBP16 and preimmune IgG
were incubated with mitochondrial extract from 3 × 109 trypanosomes. Immune complexes were captured with
protein A-Sepharose and purified by centrifugation, and RNA was
isolated from reaction supernatants as well as protein A-Sepharose
pellets. RNAs were subsequently detected by labeling the 5'-ends with
either T4 kinase or guanylyl transferase. RNA was dephosphorylated
prior to labeling with T4 kinase; therefore, all RNA species should be
labeled by this method. Capping with guanylyl transferase specifically
labels 5' tri- or diphosphates and has been previously shown to label primarily the gRNA component of T. brucei mitochondrial RNA
(34). Quantification of the resulting autoradiographs indicated that anti-RBP16 antibodies co-immunoprecipitated approximately 30% of gRNA
within mitochondrial extracts (Fig.
6A). Because
immunoprecipitation experiments were performed under conditions where
approximately 90% of RBP16 was precipitated (data not shown), we
conclude that this number accurately reflects the percentage of gRNA
associated with RBP16 within mitochondrial vesicles. Anti-RBP16
antibodies also precipitated approximately 30% of the 9 S and 12 S
rRNAs (Fig. 6A, lanes 1-3). This association is likely a
result of RBP16 binding to the oligo(U) tails also present on the
3'-end of the rRNAs (35) and suggests that RBP16 may play multiple
roles in T. brucei mitochondrial gene expression. RBP16 was
not associated with tRNAs or with unidentified RNAs between the sizes
of 120 and 210 nt (Fig. 6A). The unidentified RNAs could be
rRNA degradation products or previously described tRNA precursors (36).
Preimmune IgG did not precipitate any RNA species (Fig. 6B).
Preliminary results using Northern analysis and reverse
transcription-PCR indicate that anti-RBP16 antibodies also precipitate
several mRNAs. However, we cannot distinguish a direct interaction
between mRNA and RBP16 from an indirect association mediated by
gRNA and/or ribosomes. Interestingly, anti-RBP16 antibodies did not
precipitate small (<50 nt) RNAs that were capped with guanylyl
transferase (Fig. 6A, lanes 4-6). The identities of these
RNAs are unknown; however, their size and ability to be capped with
guanylyl transferase are consistent with the truncated gRNAs lacking
correct 3'-ends and oligo(U) tails previously observed (16). Taken
together, these data support the in vitro observation that
RBP16 interacts with gRNA oligo(U) tails.
We describe here the identification and initial characterization
of RBP16, an abundant protein from T. brucei mitochondria with gRNA binding activity. Although RBP16 was initially purified from
mitochondrial vesicles, the presence of a spliced leader sequence on
RBP16 cDNAs indicates that it is encoded in the nucleus. In
addition, the N-terminal 17 amino acids of the predicted protein are
absent from the N terminus of the purified protein, indicating that
RBP16 is most likely transported into the mitochondrion via a cleaved
signal peptide. Mature RBP16 is composed of an N-terminal CSD and a
C-terminal arginine- and glycine-rich region. This structure defines
RBP16 as a member of the eukaryotic Y-box family. RBP16 is the first
member of this family to be identified in protozoans, as well as the
first organellar member of this protein family described.
The CSD was first identified as the sole component of the bacterial
cold shock proteins (37) and was later recognized as a constituent of
several eukaryotic proteins now collectively called the Y-box proteins.
The Y-box proteins are composed of a highly conserved N-terminal CSD
flanked by a more variable C-terminal domain (reviewed in Ref. 30).
Y-box proteins can possess distinct types of C-terminal domains,
including the basic and acidic islands present in many vertebrate Y-box
proteins, as well as the zinc finger motif, a characteristic of the
Caenorhabditis elegans developmental regulating protein
Lin-28 (38). The C terminus of RBP16 is a basic, 57-amino acid region
rich in arginine (14%) and glycine (32%), reminiscent of the RGG-type
RNA binding motif (32). Y-box proteins from several invertebrates
having arginine- and glycine-rich C-terminal domains have been reported
(39-42), although these proteins have significantly larger arginine-
and glycine-rich regions than RBP16. Although the functions of these
invertebrate Y-box proteins are unknown, the planarian protein DjY1 is
localized at sites of regeneration (39). The CSDs of virtually all
Y-box proteins share greater than 90% sequence homology and are
approximately 40-45% identical to the bacterial cold shock proteins.
The invertebrate Y-box proteins DjY1 (from Dugesia japonica)
and SMYB1 (from Schistosoma mansoni) are exceptions, as
their CSDs are 60-64% identical to other eukaryotic CSDs (39, 40). In
contrast, the CSD of RBP16 possesses a higher sequence identity to the
prokaryotic cold shock proteins (43-46%) than to any eukaryotic CSDs
(33-38%). This may reflect the ancient evolutionary position of the
kinetoplastids or a mitochondrial ancestry of the RBP16 gene.
The Y-box proteins are a highly conserved family of nucleic
acid-binding proteins that interact with both DNA and RNA (reviewed in
Ref. 21). The Y-box proteins function to regulate gene expression at
the transcriptional as well as posttranscriptional level, although the
mechanism by which this is achieved is largely unknown. The regulation
of gene expression is most likely accomplished through the ability of
the CSD to relieve nucleic acids of secondary structure, whereas the
C-terminal domain mediates further molecular interactions (43). The
most extensively studied Y-box protein is the FRGY2 protein, which
inhibits the translation of maternal mRNA in Xenopus oocytes until critical cellular signals are present (reviewed in Ref.
44). The translation of these messages is mediated by a
dephosphorylation event that inhibits the nucleic acid binding of FRGY2
(45). Interestingly, translational silencing is dependent on the
processing history of the message (46). This raises the possibility
that Y-box proteins are important for the coupling of mRNA
processing events to translation.
RBP16 binds to both single-stranded RNA and DNA consistent with the
nucleic acid binding properties of the Y-box proteins (reviewed in Ref.
21). We provide evidence here that at least one function of RBP16
involves RNA binding, although we cannot omit DNA as an in
vivo nucleic acid target. Competition studies show that RBP16
binds U- and G-containing RNA polymers, with a 5-fold higher affinity
for the former. RBP16 was identified based on its ability to UV
cross-link to gRNA, and subsequent experiments with gRNA deletions
suggest that the oligo(U) tail is the major determinant for this
interaction. The minimum concentration of MBP-RBP16 that is required
for stable binding to gRNA in our gel retardation experiments is 2.5 µM. The abundant nature of RBP16 suggests that its
cellular concentration may be sufficient for stable gRNA interactions
in vivo. Further evidence that RBP16 interacts with gRNA is
provided by the observation that anti-RBP16 antibodies
co-immunoprecipitate approximately 30% of native gRNAs in
mitochondrial extracts. An RNA population, characterized by its small
size (<50 nt) and ability to be labeled with guanylyl transferase, was
not associated with RBP16 in immunoprecipitation experiments. These
RNAs most likely correspond to the truncated gRNAs lacking correct
3'-ends and oligo(U) tails observed by Blum and Simpson (16). This is
consistent with the in vitro data identifying the gRNA
oligo(U) tail as the RBP16 binding determinant. The association of
RBP16 with gRNA implies that RBP16 may be involved in the process or
regulation of RNA editing. This prospect is supported by the recent
report that gBP21, a high affinity gRNA-binding protein, is associated
with RNA editing activity and a 16-kDa gRNA-binding protein (12).
Approximately 30% of the 9 S and 12 S rRNAs were also
immunoprecipitated from mitochondrial extracts with anti-RBP16
antibodies, raising the possibility that RBP16 binds the
posttranscriptionally added oligo(U) tails on these RNAs as well (35).
Preliminary evidence suggests that several mRNAs are also present
in the immunoprecipitate. RBP16 may interact with mRNA directly via
extensive U-rich regions known to be present in many of the
mitochondrial messages (47) or indirectly through interactions with
gRNA or ribosomes. The ability of RBP16 to bind to various classes of
RNA suggests that it may play multiple roles in T. brucei
mitochondrial gene expression, as has been hypothesized for Y-box
proteins in other systems (21).
It is commonly held that the CSD element of the Y-box proteins
destabilizes RNA secondary structure, exemplified by the "RNA chaperone" activity demonstrated in vitro for E. coli CspA (19). Given the extensive interactions that must occur
among nucleic acids and proteins during the process of RNA editing, it
is very likely that RNA structure is intricately involved in this
process. RBP16 may act as a gRNA chaperone by destabilizing gRNA
secondary structure or maintaining a single-stranded conformation.
Portions of both the gRNA anchor and guiding regions have been
demonstrated to be contained partly in an intramolecular duplex (17,
18). Destabilization of these structures might be necessary for initial gRNA-mRNA interactions, or interactions during uridine insertion and/or deletion. Similarly, it is postulated that partial hydrogen bonding between the oligo(U) tail and the mRNA maintains the
proximity of the pre-mRNA 5' and 3' cleavage fragments during the
course of the editing reaction (16). By discouraging intramolecular hydrogen bonds, contact between RBP16 and the gRNA oligo(U) tail could
potentially leave these bases accessible for interactions with the
pre-mRNA. Extensively U-rich regions present in many of the
mitochondrial mRNAs have the potential to form regions of largely
double-stranded character through an interaction with the poly(A)
tail.2 The resolution of
these structures could be a prerequisite for efficient editing and/or
translation. Furthermore, because the procyclic form of T. brucei exists at 27 °C, RBP16 may play an important role in the
maintenance of RNA structure in this life-cycle stage particularly. In
this manner, the function of RBP16 would be similar to that of CspA
during cold shock in bacteria.
RBP16 may also play a more regulatory role in T. brucei
mitochondrial gene expression. For example, it has been hypothesized that life cycle stage-specific editing is regulated through gRNA usage,
because gRNA abundance does not correlate to the life cycle stage-specific abundance of edited transcripts (48). gRNA usage could
be controlled in a negative manner through the sequestering or
"masking" of specific gRNAs from the editing machinery by RBP16. Alternatively, RBP16 could be a required factor for the usage of a
subset of gRNAs. Similarly, RBP16 may affect the access of certain
mRNAs to the translational or editing machinery, analogous to the
role of FRGY2 described above. Because our results indicate that
poly(U) is the main binding determinant for RBP16, an association between RPB16 and another, more sequence-specific RNA-binding protein
would most likely be required for the recognition of a subset of gRNA
or mRNA. Finally, interaction of RBP16 with both rRNA and gRNA
suggests a possible manner in which the processes of editing and
translation could be coupled through a mutually required factor. We are
currently constructing a trypanosome strain harboring deletions of both
RBP16 genes, in order to investigate these and other possible roles of
RBP16 in T. brucei mitochondrial gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP. Protein was incubated for 20 min at room temperature with 10 fmol of radiolabeled gA6[14], and UV
cross-linking was performed as described previously (8). For
determination of internal peptide sequence, poly(U)-Sepharose fractions
containing RBP16 were separated by SDS-PAGE, stained with Coomassie
Brilliant Blue, and excised from the gel. Internal peptide sequence was
obtained from trypsin digestion products of RBP16 by the Harvard
Microchemistry facility. RBP16 N-terminal sequence was obtained with a
ProSeq Protein Microsequencing after transfer to a nylon membrane.
RBP16 was further purified on poly(A)-Sepharose (Amersham Pharmacia Biotech) as follows: poly(U)-Sepharose fractions containing RBP16 were
combined and dialyzed twice against 1 liter of Buffer A containing 150 mM KCl. RBP16 was collected in the flow through fraction of a 0.5-ml poly(A)-Sepharose column equilibrated in Buffer A containing 150 mM KCl. The purification procedure was analyzed by
SDS-PAGE on a 15% polyacrylamide gel and stained with silver (24).
Protein quantification was performed with the Bio-Rad protein assay
using bovine immunoglobulin as a standard.
(Stratagene) with the exception of the internal cDNA segment, which was cloned into the EcoRI site.
Ligated products were transformed into Escherichia coli
strain DH5
(Life Technologies, Inc.), and transformants were
selected on MacConkey agar (Difco Laboratories) containing 100 µg/ml
ampicillin. Plasmid DNA was isolated from ampr colonies,
and two clones for each cDNA segment were sequenced in both
directions by automated DNA sequencing at the State University of New
York at Buffalo Center for Advanced Molecular Biology and Immunology
Nucleic Acid Sequencing facility. Sequences were analyzed using the GCG
software package (26), and sequence comparisons were done with CLUSTAL
W (27).
-32P]ATP (NEN Life Science Products; 3000 Ci/mmol)
and T4 polynucleotide kinase (Life Technologies, Inc.) following
dephosphorylation with calf intestinal alkaline phosphatase (Life
Technologies, Inc.). gRNA was identified by labeling 5'-ends with
guanylyl transferase (a generous gift from Dr. Ed Niles). Guanylyl
transferase reactions were carried out in a 15-µl volume in the
presence of 50 µCi of [
-32P]GTP (NEN Life Science
Products; 800 Ci/mmol), 50 mM Tris-Cl (pH 8.0), 1.5 mM MgCl2, 6.0 mM KCl, 2.5 mM dithiothreitol, and 25 units of RNAguard RNase
inhibitor. Labeled RNA was separated on a 6% acrylamide/7
M urea gel and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP. gA6[14] is the gRNA molecule that
specifies the editing of the 3'-most editing block of the ATPase
subunit 6 pre-mRNA (29). UV cross-linked proteins were resolved by
SDS-PAGE and detected by autoradiography (Fig. 1B). An
intense UV cross-linking signal was present in lanes 9-16,
corresponding in size and elution pattern to the 16 kDa band observed
in Fig. 1A. We named this protein RBP16. To further purify
RBP16 for UV cross-linking studies, relevant poly(U) fractions were
combined and loaded onto a poly(A)-Sepharose column, and RBP16 was
collected in the flow-through (Fig. 1C, lane A). By silver
stain, we estimated the flow-through fraction to consist of 75% RBP16.
Using this procedure, mitochondrial vesicles from 4 × 1011 cell equivalents yield approximately 40 µg of
RBP16.
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Fig. 1.
Purification of a gRNA-binding protein by
affinity chromatography on poly(U)- and poly(A)-Sepharose.
T. brucei mitochondrial extract was loaded onto a
poly(U)-Sepharose column, and the column was washed with loading buffer
containing 300 mM KCl. The column was then eluted with a
linear 300-800 mM KCl gradient in loading buffer.
A, 0.5% of the starting material (S), 0.5% of
the flow-through (F), 10% of the 300 mM wash
(W), and 10% of eluted fractions were separated by SDS-PAGE
on a 12.5% gel and stained with Coomassie Brilliant Blue. Molecular
mass standards (M) are shown on the left. An
arrow marks the position of an abundant 16-kDa protein
(RBP16). B, fractions were assayed for gRNA-binding proteins
by UV cross-linking to radiolabeled gA6[14]. Ten fmol of gA6[14]
was incubated with 4 µl each of total mitochondrial extract
(S), poly(U)-Sepharose flow-through (F), 300 mM KCl wash (W), and eluted poly(U)-Sepharose
fractions. UV cross-linking proteins were resolved by SDS-PAGE on a
15% gel and detected by autoradiography. The positions of molecular
mass markers are shown on the left. The position of gRNA UV
cross-linking activity that corresponds in size and elution pattern to
RBP16 is indicated with an arrow. C, 1 µg each
of mitochondrial lysate (L), poly(U)-purified RBP16
(U), and the subsequent poly(A)-Sepharose flow-through
(A) were separated by SDS-PAGE on a 15% gel. Proteins were
detected by staining with silver.
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Fig. 2.
gRNA specificity of RBP16 binding.
A, 100 ng of protein from the poly(A)-Sepharose flow through
was UV cross-linked to radiolabeled gA6[14] in the presence of no
competitor (N), increasing amounts of unlabeled gA6[14],
gCYb[558], gA6[14]NT, or a similarly sized transcript from a
Bluescript plasmid. Competition reactions contained either 100-, 1000-, 2000-, or 4000-fold molar excess competitor RNA as compared with the
labeled RNA substrate. B, densitometer analysis of the UV
cross-linking signal in A. Competitor levels are plotted on
the x axis against signal intensity. The UV cross-linking
signal in the presence of no competitor is defined as 100%. ,
gA6[14];
, gCYb[558];
, gA6[14]NT;
, pBluescript
transcript.
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Fig. 3.
RBP16 nucleotide and deduced amino acid
sequence. A, the complete 1142-nucleotide cDNA
sequence predicts a 141-amino acid preprotein with a predicted
molecular mass of 13.9 kDa and a pI of 9.1 and a mature protein with a
predicted molecular mass of 13.2 kDa and a pI of 9.3. The N-terminal 17 amino acids that presumably make up a cleaved mitochondrial import
sequence are shown in italics. N-terminal sequence obtained
from the purified RBP16 protein is indicated by a double
underline. Peptide sequences obtained from trypsin cleavage
products are shown with a single underline. The N-terminal
half of the mature protein is highly homologous to a known nucleic acid
binding motif called the CSD (shaded region). The C-terminal
half of the mature protein is rich in glycine (32%) and arginine
(14%) residues. B, alignment of the RBP16 CSD
(Tb) with the E. coli CspA (Ec)
(GenBankTM accession number P15277) and the CSD of
eukaryotic Y-box proteins from Schistosoma mansoni
(Sm) (GenBankTM accession number U39883),
Xenopus laevis (Xl) (GenBankTM
accession number P21574), and Homo sapiens (Hs)
(GenBankTM accession number P16991). Identical amino acids
are indicated with an asterisk. Semiconservative and
conservative substitutions are indicated with one dot and
two dots, respectively. The RNP1 RNA binding motif,
conserved in all CSDs, is boxed.
-D-galactopyranoside, an
abundant protein corresponding to the expected size of a MBP-RBP16 fusion protein (apparent molecular mass, 59 kDa), was produced (Fig.
4A, lane In). This protein was
not present in extracts of uninduced cells (data not shown). The
expressed fusion protein was purified from bacterial cells first based
on amylose affinity (Fig. 4A, lane Am) and subsequently
based on affinity for poly(U) (Fig. 4A, lane U). After the
two-step purification, no contaminating bands were evident when 13 µg
of protein were stained with Coomassie Brilliant Blue.
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Fig. 4.
Expression of MBP-RBP16 fusion protein in
E. coli and characterization of RBP16 nucleic acid
binding properties. A, E. coli cells
harboring the pMal-C2 plasmid with the RBP16 gene in frame with MBP
were grown in the presence of 0.3 mM
isopropyl-1-thio- -D-galactopyranoside. Molecular mass
markers (M), extracts from
isopropyl-1-thio-
-D-galactopyranoside-induced cells
(In), amylose column eluant (Am), and subsequent
poly(U)-purified protein (U) were separated by SDS-PAGE on a
12.5% gel and stained with Coomassie Brilliant Blue. The 59-kDa fusion
protein is indicated with an arrow. B, native
RBP16 (nat) purified from trypanosome mitochondria by
poly(U)- and poly(A)-Sepharose chromatography (100 ng/reaction) and the
bacterially expressed MBP-RBP16 fusion (fus) (680 ng/reaction) were UV cross-linked to radiolabeled gA6[14] in the
presence of no competitor (N) or in the presence of 100-, 1000-, 5000-, or 10,000-fold mass excess unlabeled ribonucleotide
homopolymer competitors. C, UV cross-linking, as in
B except in the presence of ribonucleotide heteropolymer
competitors. D, UV cross-linking in the presence of
unlabeled poly(U), oligo-(dT)20, and double-stranded RNA
(poly(A)·(U)).
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Fig. 5.
Determination of gRNA binding affinity of
MBP-RBP16 fusion protein. Increasing concentrations of MBP-RBP16
or 10 µM MBP were incubated with radiolabeled gA6[14]
as in UV cross-linking experiments. Following electrophoresis on a 4%
nondenaturing acrylamide gel, RNA-protein complexes were detected by
autoradiography. Positions of unbound RNA and MBP-RBP16-gA6[14]
complexes are indicated by brackets.
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Fig. 6.
Co-immunoprecipitation of RNA with
RBP16. A, anti-RBP16 IgG (100 µg) was incubated with
mitochondrial extract from 3 × 109 trypanosomes. Immune complexes were purified
using protein A-Sepharose. Co-immunoprecipitating RNA was extracted and
precipitated from protein A-Sepharose pellets, as well as reaction
supernatants. RNA was either dephosphorylated and labeled with T4
kinase or capped with guanylyl transferase. The positions of RNA size
markers are shown on the left. Total RNA from 3 × 109 trypanosome mitochondria (lanes 1 and
4), unbound RNA (lanes 2 and 5) and
bound RNA (lanes 3 and 6) are shown. RNA in
lanes 1-3 was labeled with T4 kinase. RNA in lanes
4-6 was labeled with guanylyl transferase. The positions of the 9 S and 12 S rRNAs are indicated. Labeled gRNA and tRNA are shown in
brackets. Capped RNA that may correspond to the truncated
gRNAs observed by Blum and Simpson (16) is marked with an
arrow. B, same as in A except that
mitochondrial extract was incubated with 100 µg of preimmune IgG.
Unbound RNA (lanes 1 and 3) and bound RNA
(lanes 2 and 4) are shown. Lanes 1 and
2 were labeled with T4 kinase after dephosphorylation.
Lanes 3 and 4 were labeled with guanylyl
transferase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank members of the Read laboratory for helpful suggestions, Tom Melendy for advice on protein purification, and Edward Niles for his generous donation of guanylyl transferase. In addition, we thank Terry Connell, Kevin Militello, and Edward Niles for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM53502 (to L. K. R.) and by a Burroughs Wellcome Fund New Investigator Award in Molecular Parasitology (to L. K. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF042492.
To whom correspondence should be addressed: Dept. of
Microbiology, State University of New York at Buffalo School of
Medicine, 138 Farber Hall, Buffalo, NY 14214. Tel.: 716-829-3307; Fax:
716-829-2158; E-mail: lread{at}acsu.buffalo.edu.
2 K. T. Militello and L. K. Read, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: kRNA, kinetoplastid RNA; gRNA, guide RNA; CSD, cold shock domain; nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; MBP, maltose-binding protein; PCR, polymerase chain reaction.
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