(Received for publication, August 29, 1996)
From the Laboratorium für molekulare Biologie, Genzentrum der
LMU München am MPI für Biochemie, 82152 Martinsried,
Germany, PE Applied Biosystems, Brunnenweg 13, 64331 Weiterstadt, Germany, the § Seattle Biomedical Research
Institute, Seattle, Washington 98109, and the ¶ Pathobiology
Department, SC38, University of Washington,
Seattle, Washington 98195
RNA editing in Trypanosoma brucei is
a mitochondrial RNA processing reaction that results in the insertion
and deletion of uridylate residues into otherwise untranslatable
mRNAs. The process is directed by guide RNAs which function to
specify the edited sequence. RNA editing in vitro requires
mitochondrial protein extracts and guide RNAs have been identified as
part of high molecular weight ribonucleoprotein complexes. Within the
complexes, the RNAs are in close contact with several mitochondrial
proteins and here we describe the isolation and cloning of a
gRNA-interacting polypeptide from Trypanosoma brucei. The
protein was named gBP21 for uide RNA-
inding
rotein of
kDa. gBP21 shows no homology to
proteins in other organisms, it is arginine-rich and binds to gRNA
molecules with a dissociation constant in the nanomolar range. The
protein does not discriminate for differences in the primary structures
of gRNAs and thus likely binds to higher order structural features
common to all gRNA molecules. gBP21 binding does not perturb the
overall structure of gRNAs but the gRNA/gBP21 ribonucleoprotein complex
is more stable than naked guide RNAs. Although the protein is
arginine-rich, the free amino acid or an arginine-rich peptide were not
able to inhibit the association to the RNAs. In contrast, the
gRNA-gBP21 complex formation was sensitive to potassium and ammonium
cations, thus indicating a contribution of ionic contacts to the
binding.
Mitochondrial gene expression in kinetoplastid organisms requires
a RNA processing reaction series known as RNA editing. The process is
characterized by the site-specific insertion and deletion of
exclusively uridylate residues into otherwise encrypted pre-mRNAs to create full-length mRNA molecules for protein synthesis
(reviewed in Ref. 1). Key molecules in the process are small,
metabolically stable RNA molecules, known as guide RNAs
(gRNA).1 They specify the U-insertion and
deletion process in a base pairing interaction with the pre-mRNA
molecules (reviewed in Ref. 2). Guide RNAs have an average length of
50-70 nucleotides (nt) and contain post-transcriptionally added 3
oligo(U) extensions of variable length. This U-tail has been suggested
to be the reservoir for the U-addition/deletion reaction or to
transiently stabilize the interaction with the pre-mRNAs. The
mitochondrial machinery that catalyzes RNA editing is not known today,
however, there is substantial evidence that suggests the participation
of mitochondrial polypeptides in the process. Guide RNAs as well as
pre-edited mRNAs are assembled in high molecular weight
ribonucleoprotein (RNP) complexes (reviewed in Ref. 3) and RNA editing
in vitro requires mitochondrial protein extracts (4-7).
Associated with the RNP complexes are various enzymatic activities such
as endonuclease (8, 9), RNA ligase (8-10), RNA helicase (11, 12), and terminal uridylyltransferase (8, 12, 13), which have been suggested to
be catalytic components of different steps of the RNA editing process.
Corell et al. (12) determined an apparent S value of 20 S
for a mitochondrial uridylate deletion activity.
Potential candidates of the editing machinery are polypeptides that are
in close contact with gRNA molecules. Several gRNA-binding proteins
have been detected in kinetoplastid organisms, based on zero distance
cross-linking experiments. In Leishmania tarentolae, two
potential gRNA-binding proteins with molecular masses of 18 and 51 kDa
have been isolated and cloned (14) and the p51 was identified to be
homologous to aldehyde dehydrogenase. The p18 protein displayed no
homology to known polypeptides. In Crithidia fasciculata
polypeptides with apparent molecular masses of 33, 65, and 88 kDa
showed binding specificity to gRNAs, provided that the RNAs were tailed
with 3 oligo(U) extensions (15). This was interpreted as a form of
sequence specific binding to the post-transcriptionally added U-tail of
gRNAs. In Trypanosoma brucei, several proteins with apparent
molecular masses ranging from 124 to 9 kDa were found to cross-link to
gRNA molecules (15-18). The binding of the various proteins could be
blocked by increasing the monovalent cation concentration and only
three proteins with apparent molecular masses of 90, 21, and 9 kDa were
stable at
100 mM potassium chloride. This suggested a
dependence on ionic contacts in the interaction of these polypeptides
to the gRNAs. Similar to the situation in C. fasciculata,
the 90-kDa polypeptide was, in addition, reliant on the presence of an
oligo(U) extension at the gRNAs 3
ends, thus displaying features of
sequence specificity.
All the aforementioned gRNA cross-linking studies in T. brucei identified a prominent cross-linking product in the range of 21-26 kDa, appearing as a broad radioactive signal in SDS containing polyacrylamide gels (15-18). Despite the differences in the molecular mass estimation and also some differences in the experimental conditions, the identified characteristics of that cross-link were very similar with one obvious difference: Leegwater et al. (15) reported a dependence on a long U-tail on the gRNAs whereas Köller et al. (16) found cross-linking even with a tail-less gRNA substrate. There is no apparent explanation for this difference but all other features support the interpretation that this cross-link is based on the interaction of gRNAs to the same protein or the same set of proteins. A stimulating new aspect concerning this cross-link was only recently provided by Corell et al. (12). They identified the cross-link in the same gradient fractions of partial purified mitochondrial extracts that contained a gRNA-dependent uridylate deletion activity. Although a co-localization is no direct evidence, the result might hint a potential role of this gRNA-binding protein during RNA editing.
In this paper we describe the isolation, cloning, and first
characterization of the 21-26-kDa gRNA cross-linking product. The
cross-link was identified to be due to a single polypeptide with a
calculated molecular mass of 21 kDa. It was named gBP21 for guide
RNA-binding protein of 21 kDa. gBP21 is arginine-rich and shows no
homology to known proteins. It is nuclear-encoded and presumably is
imported into the mitochondrion via a cleavable presequence of 19 amino
acids. The protein binds specifically to gRNAs with an affinity in the
nanomolar range. Binding stabilizes the gRNA structure without altering
its gross structural conformation and is only marginally dependent on a
3-terminal U-tail.
The procyclic life cycle stage of
T. brucei brucei strain IsTaR 1 (19) was grown in
SDM-79 as described (20). Bloodstream trypanosomes (clone MITat 1.2)
were grown in HMI-9 medium according to Ref. 21. Genomic DNA was
prepared as described in Ref. 22 and poly(A)+ mRNA was
purified from whole cell RNA preparations (23) using paramagnetic
oligo(dT)25 beads (Dynabeads). Mitochondrial vesicles were
isolated essentially as described by Harris et al. (24). Vesicle preparations were stored at 80 °C in 20 mM
Tris-HCl, pH 8, 2 mM Na2EDTA, 250 mM sucrose, and 50% (v/v) glycerol.
The polypeptide was isolated
from mitochondrial extracts prepared at low ionic strength in the
presence of Nonidet P-40 (3.3 mM) as described (25).
Extracts were cleared from insoluble material by centrifugation at
16,000 × g for 5 min at 4 °C and protein
concentrations were determined by dye-binding using bovine plasma
-globulin as a standard (26). The purification of gBP21 was followed
by monitoring the binding activity to T. brucei gRNA gA6-14
(16). Cleared extracts were precipitated with ammonium sulfate at
4 °C and the protein was enriched in the 50-60% (w/v) saturation
fraction. The precipitate was dissolved in 10 mM Hepes pH
7.5, 1 M (NH4)2SO4 and
loaded onto a phenyl-Sepharose column equilibrated in the same buffer.
Proteins were eluted with a linear gradient of 1-0 M
(NH4)2SO4 and the gRNA binding
activity was recovered in fractions from 0.5 to 0.3 M
(NH4)2SO4. These fractions were
concentrated by ultrafiltration and adjusted to 6 mM Hepes pH 7.5, 50 mM KCl, 0.5 mM dithiothreitol.
Samples were separated by two-dimensional gel electrophoresis.
Separation in the first dimension was based on isoelectric focusing
using a linear pH gradient from pH 3-10 followed by a second dimension
12% (w/v) SDS-polyacrylamide gel electrophoresis in the presence of
0.1 mM thioglycolate. Gels were blotted onto polyvinylidene
difluoride membranes in 25 mM Tris, 192 mM
glycine, 20% (v/v) methanol, 0.02% (w/v) SDS, and 0.1 mM
thioglycolate. Membranes were briefly stained (0.1% (w/v) Coomassie
Brilliant Blue, 50% (v/v) methanol), washed, and air dried. Protein
spots were excised and N-terminally sequenced by automated Edmann
degradation.
Both terminal ends of the gBP21
gene were cloned by rapid amplification of cDNA ends (27)) using
primers deduced from the N-terminal amino acid sequence as well as
primers directed against the spliced leader (SL) sequence and a
universal amplification primer (UAP, Life Technologies Inc.):
5-rapid amplification of cDNA ends: 1) round PCR, T.b.SL-Sal:
GTCGTCGACAACTAACGCTATTATTAGAACAG; 25kpep3
-2:
TTNCCRTCNACNGCNACNCKNGTCATNGTNCC. 2) Round PCR, T.b.SL-5
-2: GAACAGTTTCTGTACTATATTG; 25kpep3
-1: TCRTCNCKNACRTCRTGDA-TYTCRAAYTTNGG. 3
-Rapid amplification of cDNA ends. 1) Round PCR, 25kpep5
-216: GCCTGGTCTCGTGGCGCTTCTAC; UAP: GGCCACGCGTCGACTAGTAC. 2) Round PCR, 25kpep5
-244: CCGGCGTTCAGTCACTGCCC; UAP: GGCCACGCGTCGACTAGTAC. PCR
products were ligated into plasmid pCR-Script SK(+) (Stratagene) and transformed into Escherichia coli XL1-Blue MRF
Kan
cells. Plasmid DNA was sequenced and the sequence information was used to PCR amplify the full-length gBP21 gene from T. brucei genomic DNA using primers gBP21-5
:
CCACTCTTCACTGCGTAAGAGAG and gBP21-3
: CGCAAGAAGTTCCAAAGCCGGG. The
1044-bp PCR product was cloned into pCR-Script SK(+) (Stratagene) and
the nucleotide sequence of both strands was determined by automated
sequencing using the dye terminator technology. The sequence of the
gBP21 gene has been submitted to GenBank under accession
number U61382[GenBank]. Protein sequences were analyzed using the Genetics
Computer software package (28).
The gBP21 protein coding
region lacking its putative mitochondrial import sequence was amplified
from T. brucei genomic DNA using primers 25kpep5-Nde-I:
ATAGGCATATGGCTTCTACTTTTTCC and 25kpep3
-794: GCGAAGCTTTT-AATGGTATCGCGATGT. The resulting PCR fragment of 551 base
pairs was cloned into NdeI and HindIII restricted
plasmid pT7-7 (29) resulting in plasmid pT7-7-gBP21 which was
transformed into BL21(DE3) and BL21(DE3) pLysS cells (30) for
expression. Cells were grown at 37 °C in 2 × YT medium
containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol for
plasmid selection. At an A600 of 0.7 expression
was induced by adding 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside and incubating for
additional 3 h at 24 °C. E. coli cells were broken
open by freeze/thaw lysis followed by sonication in 50 mM
NaxHyPO4, pH 7.8, 500 mM NaCl.
The cytosolic extract was dialyzed against 20 mM Tris-HCl,
pH 7.5, 100 mM KCl, 2.1 mM MgCl2,
0.1 mM Na2EDTA, 0.5 mM
dithiothreitol and purified by ion exchange chromatography on Mono Q HR
5/5 and Mono S HR 5/5 columns (Pharmacia). The N terminus of the
recombinant gBP21 was verified by microsequencing.
The
binding of gBP21 to gRNA molecules was measured by retention on
nitrocellulose filters as described by Witherell and Uhlenbeck (31).
RNA molecules (gA6-14, gA6-48, gA6-14/U, gND7-506, RNA1) were
synthesized by run-off transcription from linearized plasmid DNA
templates (16, 32) using T7 polymerase following standard procedures.
Radioactive labeling of the RNAs was achieved by either using
[-32P]UTP or [
-32P]ATP in the
transcription reaction. Transcripts were purified by denaturing gel
electrophoresis and renatured in binding buffer (6 mM HEPES
pH 7.5, 50 mM KCl, 2.1 mM MgCl2 0.1 mM Na2EDTA, 0.5 mM dithiothreitol,
1 mM ATP, and 6% (v/v) glycerol) by heating to 75 °C
for 2 min followed by a slow cooling period to room temperature. Protein-RNA complex formation was achieved by mixing the two components in 0.2 ml of binding buffer for 30 min. Incubation was performed at
27 °C, the optimal growth temperature for procyclic stage
trypanosomes. Samples were put on ice and slowly filtered
(approximately 0.5 ml/min) through pre-soaked nitrocellulose filters.
Filters were washed twice with 0.5 ml of cold (4 °C) binding buffer,
dried, and counted in scintillation fluid. All filtration data were
corrected for nonspecifically adsorbed RNA, which routinely was
2%.
Experiments at high gBP21 concentrations contained yeast tRNA in a
8-10-fold molar excess over gBP21 to block nonspecific binding sites.
At low gBP21 concentrations 750 nM bovine serum albumin
were added to the binding buffer to counteract nonspecific protein
adsorption.
The sedimentation
coefficient (s20,) of overexpressed gBP21
protein was determined by ultracentrifugation in linear 5-35% (v/v)
glycerol gradients in 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 2.1 mM MgCl2, 0.1 mM Na2EDTA, 0.5 mM dithiothreitol.
Centrifugation was carried out in a Beckman SW41 Ti rotor at 35,000 rpm
for 36 h at 4 °C using RNase A (2.0 S), chymotrypsinogen (2.5 S), ovalbumin (3.5 S), and bovine serum albumin (4.3 S) as standard
proteins.
CD spectra of gBP21, gA6-14, and the
gBP21·gA6-14 complex were measured at 27 °C in 6 mM
Hepes pH 7.5, 50 mM KCl, 2.6 mM
MgCl2, 0.1 mM Na2EDTA, and 6%
(v/v) glycerol. Spectra were recorded from 300 to 195 nm with a data
acquisition every 0.1 nm. Scans were repeated 10 times and averaged.
Mean molar residue ellipticities (m) were calculated per
mole of amino acid or nucleotide monomer and secondary structure
contents were estimated according to Provencher (33). For the CD
spectra of the gBP21·gA6-14 complexes equimolar amounts of protein
and gA6-14 RNA were incubated for 30 min at 27 °C and the spectrum
was corrected for the presence of the protein by subtracting the
curve for the free polypeptide.
Absorbance versus temperature
profiles of gA6-14·gBP21 RNP complexes were recorded at 260 nm in 50 mM potassium cacodylate, pH 7.2, 50 mM KCl, 2.6 mM MgCl2, 0.1 mM
Na2EDTA, and 6% (v/v) glycerol using a thermoelectrically
controlled Perkin Elmer Lambda 16 spectrophotometer. The temperature
was increased at a heating rate of 2 °C/min at temperatures between
10 and 95 °C. For complex formation equimolar amounts of gA6-14 and
gBP21 (70 nM) were incubated for 30 min at 27 °C in the
above described buffer. Tm values were determined
from derivative plots of absorbance versus temperature as
0.5 A260.
The protein was enriched from low salt detergent extracts of T. brucei mitochondria isolated from the procyclic life cycle stage of strain IsTaR 1 (19). As an assay system for the enrichment we used the demonstrated ability of the polypeptide to specifically bind to gRNA molecules, which was monitored in an UV light induced label transfer experiment as described in Ref. 16. The procedure, as outlined below, routinely yielded 100 pmol of purified gBP21 from 20 mg of total mitochondrial protein.
A cleared mitochondrial lysate (Fig. 1, lanes mt
extract) was first fractionated by the stepwise addition of
ammonium sulfate. The majority of the gRNA cross-linking activity
(90%) precipitated in the 50-60% (w/v) saturation fraction but
this fraction still contained the bulk of mitochondrial proteins (Fig.
1, lanes 50-60% AS). We further separated this sample by
hydrophobic interaction chromatography using a phenyl-Sepharose column.
Polypeptides were eluted with a linear ammonium sulfate gradient and
the gRNA binding activity eluted between 0.5 and 0.3 M
(NH4)2SO4. Three major polypeptides remained in this fraction (Fig. 1A, lane HIC) but only the
protein band with an apparent molecular mass of 26 kDa overlapped with a broad but strong radioactive signal when cross-linked to added radiolabeled gA6-14 RNA (Fig. 1B, lane HIC). To exclude that
more than one protein co-migrated within that protein band, we
performed two-dimensional gel electrophoresis with the 26-kDa protein
separating at a pI of 9.5 (data not shown). Gels were blotted and the
protein spot was microsequenced. Forty-three amino acids were obtained from the N terminus of the protein and this sequence information was
used to PCR amplify the corresponding gene from T. brucei genomic DNA following the procedure outlined under "Materials and
Methods."
The gene encoded for an open reading frame of 621 nt (Fig.
2A) with 5- and 3
-untranslated regions of
152 and 310 nt, respectively. The translation start codon was found 57 nt upstream of the first codon identified as the N terminus of the
isolated protein. This sequence translated into a 19-amino acid long
peptide, rich in hydroxylated and positively charged amino acids but
not containing negatively charged amino acids. It can be folded into an
amphiphilic helix thus displaying features of a mitochondrial import
sequence (Fig. 2B).
The processed protein is 187 amino acids long with a
calculated molecular mass of 21,125 Da. It was named gBP21 for guide RNA-binding protein of 21 kDa. It contains single cysteine and tryptophan residues and 18 arginines as the most abundant amino acid
(13.3 molecular mass %). The polypeptide is hydrophilic (62% of the
amino acids are hydrophilic) with a calculated pI of 9.7. We were not
able to identify membrane spanning domains (34, 35) and only three very
short hydrophobic stretches exist. The extreme C terminus (13 amino
acids) is the most hydrophilic part of the polypeptide with seven
charged and five hydroxylated amino acids separated by a single valine
residue. Sequence comparison did not show significant homologies to
other polypeptides in the various data bases and no specific protein
motif was identified using the MOTIF program of the GCG software (28).
In particular, no amino acid sequence known to confer single- or
double-stranded RNA binding specificity was identified (36), with the
possible exception of three arginine-rich sequence stretches (residues 82-89, 101-112, and 194-204) showing some similarity to
arginine-rich motifs in other RNA-binding proteins (36-38). A Northern
blot analysis revealed that gBP21 was expressed as a 1.1-kilobase
mRNA in procyclic and bloodstream forms, the major life cycle
stages of the parasite (Fig. 3).
Expression of gBP21 in E. coli Cells
A gBP21
coding sequence, equivalent to the processed protein was cloned into
expression vector pT7-7 (29) and transformed into E. coli
strain BL21 (DE3) (30). This construct allowed the expression of
recombinant (r) protein after transcription induction with
isopropyl-1-thio--D-galactopyranoside and as shown in
Fig. 4A, large amounts of r-gBP21 accumulated already after 1 h of induction. Bacterial growth was performed at 24 °C to
avoid formation of inclusion bodies containing inactive r-gBP21. Small amounts of the protein were expressed even before transcription induction (Fig. 4A, lane 0), which
subsequently was avoided by using BL21 (DE3) pLysS cells as the
parental strain (30). The protein was isolated from bacterial lysates
by ion exchange chromatography taking advantage of the cationic nature
of the polypeptide for purification (see "Materials and Methods").
One hundred ml of bacterial culture yielded approximately 1 mg of
r-gBP21 with a purity of
95% as judged from silver-stained SDS
containing polyacrylamide gels. Recombinant gBP21 preparations were
checked for their cross-linking activity with synthetic, radiolabeled
gRNAs (Fig. 4B). The cross-link properties were identical to
what we had observed using mitochondrial extracts (16). gBP21
preparations slowly degraded if kept at 4 °C as evidenced by the
appearance of low molecular weight products on SDS-polyacrylamide gels.
At a pH
11 or at protein concentrations
3 mg/ml (at KCl
concentration
250 mM in the absence of a denaturant) the
protein irreversibly aggregated analogous to what has been described
for the C5 protein of RNase P (39, 40). We also tested whether r-gBP21
within a bacterial extract was capable of interacting with gRNA
molecules. Using a cytosolic extract of E. coli cells
expressing the polypeptide, we were able to demonstrate cross-linking
to added gRNAs upon UV irradiation. This suggested that no other
trypanosome-specific factors are required for the stable association of
gBP21 to gRNAs (data not shown).
Physical Properties of r-gBP21
The protein migrated in
SDS-polyacrylamide gels with apparent molecular masses of 26.3 ± 0.6 kDa (Fig. 5A). Isokinetic centrifugation experiments in linear glycerol gradients (5-35%, v/v) in comparison to proteins of known sedimentation coefficients
(s20,) led to the estimation of a Svedberg
value of 2.6 ± 0.3 S (Fig. 5B). From circular
dichroism measurements we calculated 7%
-helical structures, 48%
-sheets, and 45% random coil conformation of the recombinant
protein (Fig. 5C). Size exclusion chromatography experiments
using Superose (agarose beads) or Superdex columns (agarose-dextran
beads) (Pharmacia) demonstrated a strongly retarded elution profile of
the polypeptide (data not shown), most likely due to an interaction of
gBP21 with the column material, thus preventing the determination of
additional hydrodynamic parameters.
Characterization of the RNA Binding Properties of r-gBP21
Throughout the purification we relied on the stable and
specific binding of gBP21 to gRNA molecules which was assayed in UV cross-linking experiments. The bacterial expression of the polypeptide enabled us to estimate the affinity of the protein to different radiolabeled gRNAs in nitrocellulose filter retention assays. In a
first set of experiments we varied the gBP21 concentration at constant
gRNA concentrations and tested three different uridylated (10 Us)
T. brucei gRNAs as ligands: gA6-14, the 3 most gRNA for the
editing of the ATPase 6 (A6) mRNA; gA6-48, another A6 specific gRNA; and gND7-506, a NADH subunit 7-specific gRNA molecule. As shown in Fig. 6A we measured typical
saturation-type binding isotherms for all three RNAs with a maximal
retention on the filters varying between 35 and 50%. Guide RNA,
gA6-14/U, a RNA construct lacking the 3
-terminal U-tail but otherwise
identical to gA6-14 (16), showed a very similar binding curve when
compared to the three uridylated RNAs, indicating that the 3
-terminal
bases of the gRNAs had no strong influence on the binding of the
protein. We also tested a "non-gRNA" transcript, termed RNA1 (16),
which was of similar length to gRNAs (56 nt). It was prepared by T7 transcription from HindIII linearized plasmid
pBS
DNA (Stratagene). RNA1 showed only
5% retention at
the same assay conditions, demonstrating the binding specificity of
gBP21 for gRNA molecules in line with previous data (16). Equilibrium Kd values for the formation of gBP21·gRNA
complexes were determined in a Scatchard analysis. In these experiments
the gBP21 concentration was held constant at 100 nM and the
RNA concentrations were varied in the neighborhood of the protein
concentration. Fig. 6B shows representative examples of two
Scatchard plots and the derived Kd values for the
tested gRNAs are listed in Fig. 6C. For the uridylated gRNAs
(all three guide RNAs contained 10 Us at their 3
end) we determined
Kd values in the range of 8-10 nM
whereas the non-uridylated gA6-14 molecule (gA6-14/U) had a slightly
weaker Kd of 16 nM.
The amount of protein in the r-gBP21 preparations that was active in RNA binding was determined in a direct titration experiment. Increasing amounts of radiolabeled gA6-14 were added to a fixed amount of gBP21 (4 µM) and the data were analyzed in a plot of free versus added total gA6-14 (Fig. 6D). Complete binding occurred until the concentration of gA6-14 reached 0.4 µM from where the concentration of free RNA increased linearly. Assuming one binding site per gBP21 molecule, the data demonstrated that only 0.4 µM binding sites could be saturated from a total of 4 µM. This indicated that only 10% of the gBP21 molecules were active in binding. For different r-gBP21 preparations we determined values between 10 and 30%.
Binding Inhibition ExperimentsBased on the observation that
the sequence of gBP21 contained three arginine-rich sequence elements
(see above) which potentially could provide the RNA binding specificity
(37), we tested whether arginine would act as a competitor of complex
formation (41). As shown in Fig. 7A, even at
a concentration of 20 mM arginine, equivalent to a 4 × 103-fold molar excess over gBP21, we were not able to
see a significant decrease in gRNA binding. Lysine as another
positively charged amino acid as well as serine (data not shown) gave
similar half-maximal inhibition values (Ki 20 mM). For the arginine-rich hexapeptide KRTLRR we measured a
Ki > 5 mM. In contrast, binding
experiments at increasing potassium chloride (10-250 mM) and NH4Cl (1-500 mM) concentrations
demonstrated the salt sensitivity of the gBP21/gRNA association.
Half-maximal inhibition (Ki) occurred at 100 mM KCl and 90 mM NH4Cl (Fig.
7B).
CD and Hyperchromicity Measurements
Circular dichroism
measurements revealed that the gRNA molecules did not change their
overall structure upon binding to the protein. Fig.
8A shows the typical A-form RNA spectrum (42) of gA6-14 in its naked form (dotted line): a positive
elipticity around 270 nm and negative elipticities at 240 and 210 nm.
When compared to the spectrum in its complexed form with gBP21, no significant changes were detected (Fig. 8A, solid line).
This indicated that the gRNA structure remained largely unperturbed within the RNP complex. However, gBP21 binding did result in a stabilization of the gRNA structures. As has been shown before (32), UV
melting curves of naked gRNAs are characterized by a main melting
transition around 38-40 °C (Fig. 8B, broken line). When
complexed with gBP21 this melting transition completely disappeared, indicating 100% binding of the gRNA. In addition, two new transitions were detected (Fig. 8B, solid line): a high temperature
Tm around 70 °C and a second transition at
52 °C. Assuming that gBP21 binds only to fully folded gRNA
molecules, the Tm at 52 °C presumably reflects
the temperature-dependent dissociation of the gRNA·gBP21 complex and
thus demonstrated that the RNP complex is more stable than the naked
gRNAs (equivalent to a melting point shift of 10 °C). The
Tm at 70 °C presumably corresponds to the
complete unfolding of dissociated gRNA or may be a consequence of
denatured protein aggregation as suggested by Xing and Draper (43).
Last, we detected a general decrease in the hyperchromicity (approximately by a factor of 10), indicative of a strong shielding effect by the protein.
Here we describe the identification and first characterization of a novel T. brucei protein which we termed gBP21, for guide RNA-binding protein with a molecular mass of 21 kDa. The molecule is a basic polypeptide, 187 amino acids in length and shows no homology to known proteins. In particular, there is no homology to the p18 gRNA-binding protein identified in L. tarentolae cells (14) although the two proteins are of identical length. gBP21 and p18 share only a 16-amino acid overlap of 37.5% identity, located at the N terminus of the mature gBP21 and the extreme C terminus of p18. Deduced from the sequence of the gBP21 gene, we identified a 19-amino acid long potential N-terminal mitochondrial import sequence. This suggests a cytosolic translation of the gBP21 mRNA to yield a precursor protein that undergoes maturation after mitochondrial import. The length of the presequence is similar to what has been reported for other putative import sequences in kinetoplastid organisms (14, 44, 45). Given the absence of membrane spanning domains or long hydrophobic stretches within the polypeptide, we propose that gBP21 is a soluble mitochondrial matrix protein.
The processed protein shows an anomalous electrophoretic mobility in
SDS containing polyacrylamide gels of 26 kDa versus a calculated 21 kDa. This is likely due to the basic nature of the polypeptide (pI 9.5) causing a reduced electrophoretic mobility at pH
8.8. Our recombinant protein preparations consisted of roughly 50%
-strand secondary structure elements and only around 10%
-helical domains. Whether these values reflect the in
vivo folding characteristics of gBP21 is doubtful. Although we
avoided denaturing conditions during the isolation of the recombinant
protein, only 10-30% of the molecules were active in gRNA binding.
This might be due to a lack of obligatory post-translational
modifications of the recombinant protein isolates, or to an incorrect
folding of r-gBP21 in the E. coli cytosol. The formation of
inclusion bodies upon expression of gBP21 at 37 °C might argue for
the latter explanation. Although the values of active protein seem to
be low, they are comparable to other studies with recombinant
RNA-binding proteins (39, 46).
The polypeptide binds to various gRNA molecules with equilibrium
dissociation constants around 10 nM, thereby not
discriminating for differences in the primary sequences of the gRNAs.
It is therefore likely that higher order folding characteristics common
to all gRNAs, as suggested (32), provide the binding site for the
protein. The hyperchromicity measurements indicated a strong shielding effect of the protein and furthermore, the main melting transition of
naked gRNAs was completely abolished. Since this transition was
interpreted to be primarily due to the melting of a hairpin structure
near the 3 end of gRNAs (32), this might indicate that this secondary
structure element is, at least in part, involved in the association
with the protein. As expected for a basic polypeptide and again similar
to what has been found for other RNA-binding proteins (46), gBP21 has
also a low affinity for other unrelated RNA molecules (see binding
curve for RNA1 in Fig. 6A). Whether this is a basal,
nonspecific affinity of the protein, or whether it reflects the
existence of a second low affinity binding mode between gBP21 and gRNAs
cannot be corroborated at this point. Nevertheless, the determined
Kd values are in the same range as values measured
for other basic proteins that interact with RNA molecules, like the
well studied E. coli ribosomal protein L18 which interacts
with ribosomal 5 S RNA (47), the M1 RNA-binding protein C5 (39, 46), or
the HIV RNA-binding polypeptides rev and tat (48).
A guide RNA lacking its 3 oligo(U) tail bound to the protein almost as
strongly as the corresponding molecule containing 10 Us at its a 3
end. This excludes the U-tail as a major determinant of the binding
site for gBP21 and supports previous results from UV cross-linking
experiments using the same 3
end truncated form of gA6-14 as a
substrate (16). However, since we have studied only one pair of gRNA
molecules with and without a U-tail, we cannot exclude that the
observed difference reflects more subtle elements in the association of
gBP21 to gRNAs. In principle, two explanations can account for the
observed effect: first, removal of the U-tail might cause a subtle
structural alteration in the gBP21-binding site, thereby weakening the
interaction with the protein. Alternatively, some bases of the U-tail
might be part of the gBP21 interaction domain and provide a small but
defined contribution to the overall Kd. Our current
knowledge on the secondary structure folding of T. brucei
gRNAs (32) suggest a single stranded, helical conformation for the 3
oligo(U) tails. Therefore, the removal of the U-tail should have only
minimal structural consequences for the rest of the molecule. This in turn favors our second explanation and might indicate that some of the
3
uridine residues add to the binding of gBP21 to gRNAs. Blum and
Simpson (49) determined an in vivo length heterogeneity of
the oligo(U) extensions in L. tarentolae cells
between 5 and 24 Us and it is conceivable that small differences in the
Kd values might be sufficient to select a
subpopulation of gRNAs out of that pool. A discrepancy still remains
regarding the work of Leegwater et al. (15). They reported a
strict requirement of a T. brucei 26-kDa gRNA-specific
cross-link on the presence of an oligo(U) extension. In line with their
view, we feel it is unlikely that this cross-link represents a
different mitochondrial protein. However, their data are not supported
by the work presented here, since gA6-14/U preparations still bound to
gBP21 with a Kd in the nanomolar range.
Interestingly, from our CD measurements we were not able to resolve any structural alteration of the complexed gRNAs when compared to the free RNA structures. Obviously, no gross structural changes are induced within the gRNA molecules to accommodate the association with gBP21. However, the formed RNP complexes have more stable characteristics than naked gRNA. UV melting experiments indicated a Tm of 52 °C for the gA6-14·gPB21 complex in contrast to only 38 °C for the naked gA6-14 molecule. Thus, gBP21 binding results in a stabilization of the gRNA molecules. The possibility that a transient interaction of the protein might have induced a structural rearrangement within the RNA molecules is not supported on two counts. First, the CD measurements did not show such a structural alteration and second, a secondary structure with a melting point of 52 °C cannot be formed for gA6-14, based on the very high A/U content of the RNA molecule (32).
At the moment we can only speculate as to the molecular nature of the
gBP21-gRNA association. None of the known protein motifs known to
mediate RNA binding (reviewed in Ref. 36) such as RGG (arginine/glycine/glycine) boxes, RRM domains (RNA recognition motif),
or KH modules (50) are present in gBP21. The protein does contain three
arginine-rich sequence stretches but free arginine and also a synthetic
arginine-rich hexapeptide (KRTLRR) were not capable of inhibiting the
association to gRNAs. This is in contrast to what has been reported for
other RNA-binding arginine-rich proteins (38, 51, 52) and therefore, it
seems unlikely that the gBP21-gRNA interaction is based on specific
arginine contacts. However, we cannot exclude that the entire sequence
context of one or even all three arginine-rich sequences are required
for gRNA binding. Support for a RNA-protein interaction, at least in
part, based on ionic contacts comes from the salt sensitivity of the
gBP21-gRNA interaction. Both, KCl and NH4Cl at
concentrations around 100 mM were able to inhibit the
formation of the RNP complex. This can be interpreted as the
presentation of a defined geometry of negative charges on the surface
of the gRNAs, which is mapped by an exact array of positive charges on
the exterior of gBP21. These features are in line with previously
published work describing the salt sensitivity of gRNA-containing
mitochondrial RNP complexes (25) and gRNA-protein cross-links (16) as
well as with the salt sensitivity of the in vitro RNA
editing activity in T. brucei mitochondrial
extracts (4). Our CD measurements of recombinant gBP21 protein
identified a high content of -strand secondary structure. Although
it seems questionable that this represents the native folding of gBP21
(see discussion above), we have to consider that a platform of
-strands, as identified in several other RNA protein interactions
(53), might be used as the basal binding surface for the gRNA
molecules.
None of the identified characteristics of gBP21 allowed us to deduce a biological function for the protein. A participation during the editing process is conceivable since the polypeptide binds specifically and with high affinity to gRNAs, key components in the editing process. Further support for an involvement during RNA editing comes from the observation that a protein-gRNA cross-link of similar apparent size co-localizes with an in vitro RNA editing activity in gradient fraction of T. brucei mitochondrial extracts (12). The ability of gBP21 to cross-link to added synthetic gRNAs within an E. coli cell extract as well as in a purified form suggests, that the binding of gRNAs to gBP21 is not dependent on the interaction of other mitochondrial proteins. Although we cannot exclude a stimulatory effect by other polypeptides, this might indicate that gBP21 is an early assembly component to gRNAs which form several high molecular weight RNP complexes in T. brucei mitochondria (8, 18). In contrast, it is equally possible that gBP21 by binding to gRNAs prevents the base pairing interaction to the pre-mRNAs. In this case the protein would exclude gRNAs from the editing reaction unless it is specifically removed by other components of the editing machinery. Preliminary experiments to determine the on and off rates for the gBP21-gRNA interaction seem to support such a scenario since the on rates vastly exceed the off rates.2 Last, gBP21 could simply be a polypeptide that binds to gRNAs to regulate their turnover within the mitochondria. Experiments to corroborate the various possibilities are currently being performed.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U61382[GenBank].
We thank all members of the Göringer laboratory for helpful comments. A. Souza is thanked for critical reading of the manuscript and for stimulating discussions. G. Nörskau is acknowledged for technical assistance and L. Moroder and E. Weyher-Stingl for their help performing the CD measurements. T. Seebeck kindly provided a plasmid encoding T. brucei tubulin genes.