From the Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
Received for publication, March 2, 2001, and in revised form, April 2, 2001
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ABSTRACT |
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The 3'-terminal adenylic acid residue in
several human small RNAs including signal recognition particle (SRP)
RNA, nuclear 7SK RNA, U2 small nuclear RNA, and ribosomal 5S RNA is
caused by a post-transcriptional adenylation event (Sinha, K., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). Using the Alu portion of the SRP RNA as a substrate
in an in vitro adenylation assay, we purified an
adenylating enzyme that adds adenylic acid residues to SRP/Alu RNA from
the HeLa cell nuclear extract. All the peptide sequences
obtained by microsequencing of the purified enzyme matched a unique
human cDNA corresponding to a new adenylating enzyme having
homologies to the well characterized mRNA poly(A)
polymerase. The amino terminus region of the human SRP RNA
adenylating enzyme showed ~75% homology to the amino terminus of the
human mRNA poly(A) polymerase that includes the catalytic domain.
The carboxyl terminus of the human SRP RNA adenylating enzyme showed
less than 25% homology to the carboxyl terminus of poly(A) polymerase,
which interacts with other factors and provides specificity. The SRP
RNA adenylating enzyme is coded for by a gene located on chromosome 2 in contrast to the poly(A) polymerase gene, which is located on
chromosome 14. A recombinant protein for the SRP RNA adenylating enzyme
was prepared, and its activity was compared with the purified enzyme
from HeLa cells. The data indicate that in addition to the SRP RNA
adenylating enzyme, other factors may be required to carry out accurate
3'-end adenylation of SRP RNA.
Most eukaryotic RNA molecules are synthesized as precursor
molecules and are subsequently processed. These post-transcriptional processing events include 5' capping, polyadenylation on the 3' end of
mRNAs, CCA turnover on the 3' end of tRNAs, and splicing in the
case of pre-mRNAs.
Small RNAs are a diverse class of RNAs involved in a variety of
important cellular functions (1-4). We recently showed that several
small RNAs in human cells contain a single post-transcriptionally added
adenylic acid residue on their 3' ends. These RNAs include SRP1 RNA, 7SK RNA, U2 snRNA,
and ribosomal 5S RNA (5-9). SRP RNA is the RNA component of the signal
recognition particle, which plays an important role in translocation of
membrane proteins and secretory proteins (4, 10). SRP RNA is
synthesized in the nucleus by RNA polymerase III and is transported
through the nucleolus on its way to the cytoplasm (11). Data from our
laboratory showed that SRP RNA in the nucleolus is already processed
and adenylated on its 3' end (12). These data indicate that the 3'-end
processing and adenylation of SRP RNA are early events in the
biogenesis of the signal recognition particle.
The U2 snRNA is a required component for the splicing of pre-mRNA
(13, 14). 60-70% of human SRP RNA, 7SK RNA, and U2 snRNA molecules
contain post-transcriptionally added adenylic acid residues on their 3'
ends (8). In the case of ribosomal 5S RNA, ~10% of the molecules was
found to contain this post-transcriptionally added adenylic acid
residue (8). The 3'-end adenylation of small RNAs is conserved through
evolution because SRP RNA from rodents, amphibians, and insects contain
this post-transcriptionally added adenylic acid residue (5, 8, 15).
The functions of this 3'-end adenylation found in the small
RNAs are not fully understood. Because this 3' adenylation occurs in
many different RNAs with diverse functions, it is likely that the
function of this 3'-adenylic acid residue is related to the metabolism
of these RNAs. Data from our lab showed that small RNAs with
post-transcriptionally added adenylic acid residue are not substrates
for 3' extension of RNAs by polyuridylation (16). In addition, the
post-transcriptionally added 3'-adenylic acid residue has a relatively
low turnover (8). These data suggest that one of the functions of this
3' adenylation is to protect the 3' ends of RNAs from digestion by
exonucleases and also to prevent 3' extensions by uridylation (16).
Our initial studies were aimed at determining whether mRNA poly(A)
polymerase (PAP) is responsible for this 3' adenylation of small RNAs.
The data showed that neither PAP nor tRNA nucleotidyltransferase is
involved in carrying out this 3' adenylation (17). We carried out
studies to identify and purify the enzyme responsible for post-transcriptional adenylation of human SRP RNA. This study shows the
purification and characterization of a novel 3'-adenylating enzyme that
has homology to PAP but it is a product of a distinct gene. A
recombinant SRP RNA adenylating enzyme with adenylating activity was
obtained and its activity compared with that of the SRP RNA adenylating
enzyme purified from HeLa cells.
Chemicals and Isotopes--
[ Preparation of Substrate RNAs--
Plasmid DNA corresponding to
the Alu portion of canine SRP RNA (p7Alu) under the T7 promoter
(18) was a gift from Dr. Katharina Strub. For in vitro
transcription reactions, a DNA template was prepared by PCR to have the
desired sequence on the 3' end of Alu RNA. The U6 snRNA was made by T7
RNA polymerase transcription from a U6 snRNA-containing plasmid
linearized by DraI (19). The in vitro
transcription of linearized plasmid DNAs with T7 RNA polymerase was
performed according to standard protocols (New England Biolabs). All
RNA products were purified by fractionation on a 10% polyacrylamide
gel. RNAs were extracted from the gel and purified by precipitation
with ethanol. The concentration of the RNAs was determined by optical
density measurements at 260 nm.
Preparation of HeLa Cell Nuclear Extracts and in Vitro
Labeling--
Extracts from HeLa cells grown in suspension culture
were prepared by the procedure of Dignam et al. (20). The
final protein concentration of the extract was 5 mg/ml. The amount of
Alu RNA used as substrate for each adenylation assay was ~1 µg (20 pmol), and the in vitro adenylation assays were carried out
essentially as described earlier (8). In the case of other RNAs, ~20
pmol of RNA was added into each reaction. For the in vitro
labeling of RNAs, 5 µl of 10× in vitro labeling buffer (6 mM each GTP, UTP, and CTP, 250 µM ATP, 10 mM dithiothreitol, 200 mM KCl, 60 mM creatine phosphate, and 100 mM Tris-HCl, pH
8.0), 40 µl of nuclear extract, and 50 µCi of
[ Purification of the SRP RNA Adenylating Activity--
The HeLa
cell extract prepared by the method of Dignam et al. (20)
was used as the starting material. Solid ammonium sulfate was added to
40% (w/v) saturation to the HeLa cell extract. The mixture was kept on
ice, occasionally shaken for 30 min, and then centrifuged at 3,000 × g for 30 min. The pellet was discarded and the
supernatant was brought to 65% saturation with solid ammonium sulfate
and again centrifuged at 3,000 × g for 30 min. The
resulting pellet was dissolved in 10 ml of buffer A (20 mM
Tris-HCl buffer, pH 8.2, with 50 mM KCl, 5 mM
dithiothreitol, 2 mM EDTA, and 10% glycerol) and dialyzed
at 4 °C overnight against the same buffer. The resulting material
was applied to a DEAE-Sepharose CL-6B column (Amersham Pharmacia
Biotech) pre-equilibrated with buffer A. The elution was achieved with
a linear gradient of buffer A and buffer A containing 1 M
KCl. Samples containing SRP/Alu RNA adenylating activity were pooled
and loaded onto a hydroxyapatite (Bio-Rad) column. The elution was
carried out with a linear gradient of 10 mM sodium
phosphate buffer, pH 6.8, 0.5 mM dithiothreitol and the
same buffer containing 500 mM sodium phosphate. After
hydroxyapatite chromatography, a size-exclusion chromatography system,
Superdex 200 column from Amersham Pharmacia Biotech, was used.
Fractions were collected and assayed for SRP/Alu RNA adenylating
activity. Active fractions were pooled and loaded onto a strong anion
exchanger (Source 15Q column from Amersham Pharmacia Biotech). The
bound proteins were eluted in a linear gradient of 20 mM
Tris-HCl buffer, pH 8.2, with 10 mM NaCl and the same
buffer containing 400 mM NaCl. All column purifications
were carried out using an AKTA fast protein liquid
chromatography system from Amersham Pharmacia Biotech.
Sequencing of the Purified Protein Bands--
The purified
protein obtained after the Source 15Q column was subjected to
microsequencing at the Protein Chemistry Core Facility (Baylor College
of Medicine, Houston, TX).
Reverse Transcription-PCR and Cloning into the Expression
Vector--
Using partial cDNA sequences from
GenBankTM (accession numbers BE781551 and AK024249),
primers were designed with 5' overhangs containing restriction enzyme
sites (NcoI on the 5' end and HindIII on the 3'
end). Using the Qiagen One-Step reverse transcription-PCR kit, a
>2-kilobase product was obtained from a HeLa cell total RNA
preparation. This product was digested with NcoI and
HindIII and inserted into the pPROEX-HTa vector (Life
Technologies, Inc.). Positive clones were checked for the insert, and
then the DNA sequence was determined to ensure that the full-length
cDNA had been obtained.
Purification of the Recombinant Protein--
Competent DH5 SRP/Alu RNA 3'-Adenylation Activity--
Unlabeled SRP/Alu RNA
was added to an adenylation reaction containing 50 mM
Tris-HCl, pH 7.9, 0.6 mM MgCl2, 0.15 mM dithiothreitol, 0.5% polyethylene glycol, 2 mM ATP, and 20 mM creatine phosphate. 10 µCi
of [ Accurate in Vitro Adenylation of Alu RNA--
To study the
adenylation of the 3' end of small RNAs, HeLa cell extracts prepared by
the method of Dignam et al. (20) were used (Fig.
1A). Upon incubation with
labeled ATP, in addition to tRNAs that get labeled because of the CCA
turnover, endogenous SRP RNA was also adenylated on its 3' end (Fig.
1A, lane 1). The Alu RNA is a domain in
the SRP RNA corresponding to its 5' and 3' ends (9, 21). When the HeLa
cell extract was supplemented with wild-type Alu RNA, the 3' end of Alu
RNA was adenylated (Fig. 1A, lane 2). However,
mutant Alu RNA that cannot bind SRP 9/14 proteins (18, 22) was not
adenylated in vitro (Fig. 1A, lane 3).
The 3'-end analysis of labeled SRP RNA and Alu RNA showed that a single
adenylic acid residue is added to the 3' end of these RNAs, which is
identical to the single A addition on the 3' end of SRP RNA in
vivo (8, 16). This 3' adenylation of exogenously added Alu RNA by
the HeLa cell extract and by purified fractions was used as an assay to
purify the SRP/Alu RNA adenylating enzyme.
Purification of the Adenylating Enzyme--
To purify and obtain
the adenylating enzyme that adds a single A residue to the 3' end of
SRP RNA, the HeLa cell nuclear extract was fractionated through several
columns. The adenylating enzyme was sequentially fractionated through
ammonium sulfate precipitation and chromatography through
DEAE-Sepharose, hydroxyapatite, Superdex 200, and Source 15Q columns
(Fig. 1B and Table I). The
fractions containing peak adenylation activity were pooled and
fractionated on a 10% polyacrylamide/SDS gel. The pooled fraction from
the Source 15Q column contained a major protein of ~80 kDa (Fig.
1B, lane 7). The SRP RNA adenylating activity
eluted on the Superdex 200 column with an apparent molecular mass of
190-220 kDa. This suggests that the native enzyme functions in a
multisubunit complex, which is larger than the 80-kDa monomer. Table I
shows the enrichment of SRP RNA adenylation activity and yield of human
SRP RNA adenylating enzyme at different steps of purification.
The specificity of the purified adenylating enzyme was checked at
various stages of enzyme purification (Fig.
2). All the fractions up to the final
purification were able to adenylate the Alu RNA. However, the pooled
fractions from the hydroxyapatite column adenylated not only the
wild-type Alu RNA (Fig. 2, lanes 4 and 5) but
also adenylated the mutant Alu RNA (Fig. 2, lane 6). This is
in contrast to the specificity observed in nuclear extracts where
mutant Alu RNA was not adenylated (Fig. 1A, lane 3). These data show that some of the specificity observed in
nuclear extracts was lost as the purification progressed through
various chromatography steps. However, some RNAs such as U4 and U6 RNAs were not adenylated by this partially purified enzyme (Fig. 2, lanes 7 and 8), indicating that this enzyme is
not adenylating RNAs nonspecifically.
Isolation of Human cDNA for SRP RNA Adenylating Enzyme--
To
obtain the cDNA sequence for the SRP RNA adenylating enzyme,
several partial peptide sequences were obtained from the purified 80-kDa enzyme from the Source 15Q column fraction (Fig. 1B,
lane 7). A search of the available protein/expressed
sequence tag data banks yielded a perfect match to a unique cDNA
with a reading frame for an ~80-kDa protein. One of the proteins that
copurified with the SRP RNA adenylating enzyme up to the Superdex 200 column (Fig. 1B, lane 6), upon microsequencing,
turned out to be a previously characterized 78, kDa protein, GRP-78/BiP
(23).
The cDNA sequence corresponding to this new adenylating enzyme has
been submitted to GenBankTM with the accession number
AY029162. This cDNA sequence contains a 2,211-nucleotide
open reading frame followed by an ~1,800-nucleotide 3'-untranslated
region. The consensus polyadenylation signal AAUAAA and the cleavage
site are not found in the currently characterized expressed sequence
tag data bank clones. This indicates that this probably is a partial
cDNA sequence and this mRNA contains a longer, as yet
uncharacterized, 3'-untranslated region. There is a 166-nucleotide long
5'-untranslated region, which contains an in-frame upstream terminator
codon corresponding to
This new SRP RNA adenylating enzyme is 736 amino acids in length, and
comparison with human PAP (Fig. 3) shows
that the amino terminus (amino acids 17-490) has ~75% identity, and
the carboxyl terminus is highly divergent with only ~25% identity.
Although the catalytic domain and the nuclear localization signals are conserved between these two enzymes, the regulatory domains seem to be
different (Fig. 4C). The three
consensus and four nonconsensus cyclin-dependent kinase
phosphorylation sites (underlined in red in Fig. 3)
identified in human PAP (24) are not well conserved in the SRP
adenylating enzyme. But there are potential
cyclin-dependent kinase phosphorylation sites in the SRP
RNA adenylating enzyme in the same vicinity that conform to the
consensus S/TPXK/R and the nonconsensus S/TPXX
sequence motifs. The cyclin recognition motif (SKIRILVG) present in PAP
(25) is also present in the SRP RNA adenylating enzyme (Fig. 4).
Unique Gene for SRP RNA Adenylating Enzyme--
A search of the
available human genome sequences using the partial cDNA sequence
identified two overlapping high throughput genomic sequencing phase
entries that contained the entire gene for the SRP RNA adenylating
enzyme. The gene for this enzyme is located on chromosome 2, whereas
the gene for PAP is on chromosome 14 (26). The exon-intron organization
(Fig. 5) was identified using a BLAST
algorithm to align the cDNA sequence against the genomic sequence.
The SRP RNA adenylating enzyme gene has 21 exons encoding the
protein sequence. Exons coding for the 5'- and 3'-untranslated regions
have not been included because the untranslated regions are incomplete.
The exons 1-15 between PAP and the SRP adenylating enzyme are highly
conserved (Fig. 5), and these data indicate that both of these genes
originated from a common ancestral gene. Because Saccharomyces
cerevisiae has only the PAP gene (27) and contains no distinct
gene for SRP RNA adenylating enzyme, it is likely that this new gene
for the adenylating enzyme arose from duplication of the PAP gene and
acquired divergence in the regulatory domains while keeping the
catalytic domain largely intact (see Figs. 3, 4, and 5). It is also
worth noting that 3' adenylation is observed in only ~2% of the SRP
RNA molecules in S. cerevisiae (15), whereas 70% of the
human SRP RNA molecules contain a post-transcriptionally added single A
residue (8). These data indicate coevolution of 3' adenylation of some
small RNAs and the necessary enzymatic machinery to accomplish this single A addition.
Recombinant Protein for SRP RNA Adenylating Enzyme--
Using HeLa
cell total RNA, a cDNA clone was obtained by reverse
transcription-PCR, and a cDNA sequence corresponding to amino acids
1-736 (Fig. 3) was inserted into a pPROEX-HTa expression vector. The
cDNA was sequenced and was found to be in frame with the histidine
tag and corresponded to the full-length cDNA. The recombinant
protein was expressed in E. coli DH5 Recombinant Enzyme Shows Adenylation Activity in Vitro--
The
recombinant protein was assayed for in vitro adenylation
activity using the conditions recommended for E. coli PAP
(Fig. 7). Alu RNA, U6 RNA, and yeast tRNA
were used as substrate RNAs. As expected, the endogenous SRP RNA and
supplemented Alu RNA were adenylated by the 3' addition of a single
adenylic acid residue in the HeLa nuclear extract (lane 1).
Both E. coli PAP (lane 7) and the recombinant
human SRP RNA adenylating enzyme (lanes 2-6) showed
polyadenylation activity. The recombinant SRP RNA adenylating enzyme
added multiple adenylic acid residues to all of the RNA substrates
tested. This apparent lack of specificity and regulation in limiting
the number of adenylic acid residues added to the 3' end of Alu RNA
show that accurate and regulated adenylation of human SRP/Alu RNA
in vitro may require other components. Although the most
purified preparation of the SRP RNA adenylating enzyme from HeLa cells
contained an ~80-kDa protein as the major band, other proteins were
also present when more sample was loaded on the gel and stained (data
not shown). It is worth noting that the substrate for the accurate
adenylation in vitro is an RNA-protein complex (17).
This study presents data on the purification of a new
3'-adenylating enzyme from HeLa cells. This purified enzyme is capable of adding adenylic acid residues to SRP/Alu RNA. The cDNA for this
enzyme has been isolated, and interestingly, this enzyme is highly
homologous to the well characterized mRNA PAP (Figs. 3 and 4).
However, the SRP RNA adenylating enzyme is the product of a
distinct gene located on chromosome 2.
The homology between these two enzymes is ~75% in the
amino-terminal region (Figs. 3 and 4). However, the carboxyl
terminus is highly divergent and shows only 25% homology between these two enzymes. It is not surprising that the amino terminus is conserved because this region contains the catalytic domain. The carboxyl terminus of the PAP contains the domain that interacts with other factors including the cleavage and polyadenylation
specificity factor, which confers the specificity for the enzyme (28).
Therefore, it is reasonable to expect divergence in the carboxyl
terminus of the SRP RNA adenylating enzyme.
The recombinant SRP RNA adenylating enzyme expressed in E. coli was capable of adenylating RNAs in vitro. However,
there was no substrate specificity, and it added multiple adenylic acid residues (Fig. 7). This is not very surprising in light of what we
already know about other purified enzymes. PAP in vivo and in nuclear extracts exhibits substrate specificity and adenylates only
mRNAs containing the polyadenylation signal. However, purified PAP
adenylates any RNA with a 3'-OH group (29, 30). Similarly, the capping
enzyme guanylyltransferase caps only RNAs transcribed by RNA polymerase
II in vivo. However, purified guanylyltransferase caps any
RNA with appropriate 5' phosphates (31, 32). In all these cases, the
specificity and regulation of enzymatic activity is caused by a
multiprotein complex involved in these biological reactions. From the
results obtained with the recombinant SRP RNA adenylating enzyme, it is
very likely that other factors are necessary to carry out accurate and
regulated adenylation in vitro. In this context, it is worth
noting that the ribonucleoprotein complex consisting of Alu RNA bound
to SRP 9/14 proteins is the required substrate for adenylation in
vitro (17). Therefore, it may be necessary to use Alu RNA
complexes with SRP 9/14 proteins to obtain accurate 3'-end adenylation
in vitro. It is also possible that in addition to
adenylating SRP RNA, this new adenylating enzyme is an mRNA poly(A)
polymerase for some cellular mRNAs.
The enzymatic activity of the SRP RNA adenylating enzyme may be
regulated by other post-translational modifications such as phosphorylation. The PAP, for example, is highly phosphorylated and
regulated through its phosphorylation (33). Hyperphosphorylation of PAP
inhibits adenylation activity, and the dephosphorylated enzyme is more
active (33). Several consensus and nonconsensus phosphorylation sites
are present in the S/T-rich domain at the carboxyl terminus of the SRP
RNA adenylating enzyme, and the corresponding region in PAP is highly
phosphorylated. Therefore, it is very likely that the SRP RNA
adenylating enzyme is phosphorylated in vivo, and its
activity may be regulated by phosphorylation. Further work is needed to
experimentally verify this possibility.
The cDNA sequence for the SRP RNA adenylating enzyme is a partial
sequence, and further work is needed to characterize the complete
cDNA sequence in the 5'- and 3'-untranslated regions. The available
1,768 nucleotides of the 3'-untranslated region show no AAUAAA
polyadenylation signal. Although the human PAP cDNA was isolated
several years ago, the complete 5'-untranslated region has not been
characterized, and the transcription initiation site is not yet known.
Our unpublished data indicate that there is a long 5'-untranslated
region in the case of the SRP RNA adenylating enzyme. It will be
interesting to characterize the complete cDNA sequence of the PAP
and SRP RNA adenylating enzyme because genomic organization (Fig. 5)
suggests that the SRP RNA adenylating enzyme may have arisen by
duplication of the PAP gene and subsequent divergence in the carboxyl
terminus region.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was
purchased from ICN Biochemicals, Inc. Prepacked columns and
chromatography media were purchased from Amersham Pharmacia Biotech.
The hydroxyapatite media was from Bio-Rad. The reverse
transcription-PCR kit was from Qiagen. Restriction enzymes were from
Life Technologies, Inc. The T7 RNA polymerase was from New England Biolabs.
-32P]ATP were mixed in a total reaction volume of 50 µl and incubated at 30 °C for 3 h. After incubation, labeled
RNAs were extracted using the phenol-chloroform procedure,
precipitated, and fractionated on 10% polyacrylamide/7 M
urea gels. Radiolabeled bands were quantitated with a PhosphorImager
densitometer (Molecular Dynamics) using IMAGE QUANT software. The gels
were also analyzed by routine autoradiographic methods.
host cells were transformed with the pPROEX-HTa plasmid containing the
full-length insert. A single colony was picked from the transformation
plate and inoculated into 25 ml of LB medium containing 100 µg/ml of
ampicillin. After the culture grew to an A600 of
0.5-0.7, 0.6 mM
isopropyl-
-D-thiogalactopyranoside was added to induce
the synthesis of protein from the lac promoter and grown further at
37 °C for 4 h. The cells were harvested and resuspended in 15 ml of lysis buffer (20 mM Tris-HCl, pH 8.5, 10 mM 2-mercaptoethanol, and 1 mM
phenylmethylsulfonyl fluoride). The cell suspension was sonicated and
then centrifuged at 12,000 × g for 30 min. To the
clear supernatant, 400 µl of a 50% nickel-nitrilotriacetic acid
resin pre-equilibrated with buffer B (20 mM Tris-HCl, pH 8.5, 100 mM KCl, 10 mM 2-mercaptoethanol, 10%
glycerol, and 20 mM imidazole) was added. The suspension
was mixed for 45 min at 4 °C. Then the suspension was centrifuged
for 1 min, and the supernatant was removed. The resin was washed with
10 ml of buffer C (20 mM Tris-HCl, pH 8.5, 1 M
KCl, 10 mM 2-mercaptoethanol, and 10% glycerol) and packed
into a small column. The bound proteins were eluted as 500-µl
fractions with buffer D (20 mM Tris-HCl, pH 8.5, 100 mM KCl, 10 mM 2-mercaptoethanol, 10% glycerol,
and 250 mM imidazole). Protein-containing fractions were
analyzed by fractionation on an SDS-polyacrylamide gel. Peak fractions
were pooled and dialyzed against 20 mM Tris-HCl buffer, pH
8.5, containing 10% glycerol. After dialysis, the protein was assayed
for the adenylation activity.
-32P]ATP was added to monitor the extent of 3'
adenylation. Escherichia coli PAP was used as the positive
control in the reaction. The reaction mixture was incubated at
30 °C for 1 h. Labeled RNAs were extracted using the
phenol-chloroform procedure, purified, and fractionated on 10%
polyacrylamide/7 M urea gels. The gels were dried and
subjected to PhosphorImager (Molecular Dynamics) analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
In vitro adenylation assay and
purification of the human SRP RNA 3'-adenylating enzyme. A,
the 3'-end adenylation of human RNAs in vitro was carried
out as described earlier (8, 12). The HeLa cell nuclear extract was
incubated in the presence of [ -32P]ATP (lane
1). In addition to the labeled ATP, 1 µg of Alu RNA (lane
2) or mutant (mt) Alu RNA (lane 3) was added
to the reactions. The labeled RNAs were purified, fractionated on a
polyacrylamide gel, and visualized by PhosphorImager. B,
HeLa cell nuclear extract was fractionated consecutively by ammonium
sulfate (AS) precipitation, DEAE-Sepharose, hydroxyapatite,
gel filtration (Superdex 200), and finally on Source 15Q columns.
Fractions containing SRP RNA adenylating activity (assayed as in
A) were pooled and subsequently loaded onto the next column.
20 µg each from nuclear extract and ammonium sulfate, 10 µg each
from peak activities of DEAE-Sepharose and hydroxyapatite, and 100 ng
each from Superdex 200 and Source 15Q fractions were loaded on a 10%
SDS-polyacrylamide gel and subjected to electrophoresis. The
proteins were visualized by silver staining. Standard refers
to standard molecular mass markers.
Purification of the human SRP/Alu RNA adenylating enzyme starting from
HeLa cell nuclear extract
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Fig. 2.
Substrate specificity of SRP RNA adenylating
enzyme in nuclear extract and in partially purified fractions. The
HeLa nuclear extract was precipitated by ammonium sulfate and
subsequently fractionated on DEAE-Sepharose and hydroxyapatite
(HTP) column chromatography (Fig. 1B). Nuclear
extract and partially purified fractions were assayed for their ability
to adenylate Alu RNA (lanes 1-5). The hydroxyapatite
fraction was tested further for substrate specificity using different
RNAs as indicated above lanes 6-8. The labeled RNAs
were purified, fractionated on a polyacrylamide gel, and visualized by
PhosphorImager. mt, mutant; NE, nuclear extract;
AS, ammonium sulfate.
94 to
96 and a purine at the
3 position of
the initiator ATG codon, constituting an adequate Kozak consensus
sequence. The transcription initiation site of this mRNA has not
yet been determined.
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Fig. 3.
Comparison of the cDNA-derived
amino acid sequences of human SRP RNA 3'-adenylating enzyme and human
PAP. The amino acid sequences of the human PAP (34) and the
human SRP RNA adenylating enzyme were aligned using the ClustalW 1.8 multiple alignment program, and the identical amino acids were shaded
yellow using the BOXSHADE 3.21 program. The amino
acids obtained by peptide sequencing are indicated with a blue
line below the SRP adenylating enzyme (SRP RNA
AE). The consensus and the nonconsensus
cyclin-dependent kinase phosphorylation sites of human PAP
(24) are indicated with a red double line below the PAP
sequence.
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Fig. 4.
Schematic representation of the various
domains of human PAP (A) and the SRP RNA adenylating
enzyme (B). The domains of PAP are listed with
reference to their corresponding amino acids (C). The
designation of functional modules in SRP RNA adenylating enzyme is by
sequence homology. The data for the figure are derived from Refs. 29,
35, and 36.
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Fig. 5.
Diagrammatic representation of the exon
organization of human poly(A) polymerase and the human SRP RNA
adenylating enzyme. Blue squares indicate exons in the
two proteins that are homologous to one another. The exons shown in
pink and yellow are regions that show ~25%
homology. Introns are represented by the angled lines
connecting the exons (26).
cells, and the protein was purified using a nickel-nitrilotriacetic acid affinity column. Analysis of this recombinant protein on an SDS-polyacrylamide gel (Fig. 6) showed one major protein
band. The major band in lane 1 corresponded to the
full-length protein of about ~75 kDa in size. As expected, digestion
of this recombinant protein with tobacco etch virus protease
cleaved the histidine tag and resulted in slightly faster mobility of
the protein (Fig. 6, lane 2).
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Fig. 6.
Analysis of the recombinant SRP RNA
adenylating enzyme. The human cDNA for the SRP RNA adenylating
enzyme was cloned into the pPROEX-HTa vector in frame with the
histidine tag and expressed in E. coli. The bacterial pellet
was lysed, and the recombinant protein was purified by affinity
chromatography using nickel-nitrilotriacetic acid (Qiagen) columns. The
bound protein was eluted with 100 mM imidazole and
dialyzed, and an aliquot of the purified recombinant protein was
fractionated on a 10% SDS-polyacrylamide gel (lane
1). Lane 2, the affinity-purified protein digested with
recombinant tobacco etch virus protease to remove the histidine
tag. The proteins were stained with Coomassie Brilliant Blue.
M, molecular mass markers.
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Fig. 7.
The in vitro adenylation
activity of the recombinant human SRP RNA adenylating enzyme. The
recombinant enzyme (Fig. 6, lane 1) and E. coli
poly(A) polymerase purchased from Life Technologies, Inc. were used to
test for adenylation activity in vitro. The conditions used
were those recommended for the assay of E. coli poly(A)
polymerase. Lane 1, HeLa cell nuclear extract incubated in
the presence of [ -32P]ATP and T7-transcribed Alu RNA.
The substrate RNA and the enzyme used for each of the in
vitro adenylation assays are indicated above the lanes.
Lanes 2, 4, and 6 were incubated with
0.1 µg of recombinant enzyme and [
-32P]ATP;
lanes 3 and 5 were incubated with 0.3 µg of
recombinant enzyme and [
-32P]ATP. Incubation was at
30 °C for 60 min; lane 7 was incubated with 1 unit of
E. coli PAP. The labeled RNAs were purified and
fractionated on a 10% polyacrylamide gel and subjected to
autoradiography. NE, nuclear extract.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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) AY029162.
To whom correspondence should be addressed: Dept. of Pharmacology,
Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.:
713-798-7906; Fax: 713-798-3145; E-mail: rreddy@bcm.tmc.edu.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101905200
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ABBREVIATIONS |
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The abbreviations used are: SRP, signal recognition particle; snRNA, small nuclear RNA; PAP, poly(A) polymerase; PCR, polymerase chain reaction.
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REFERENCES |
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