From the
Nearly 1 million interspersed Alu elements reside in the human
genome. Alu retrotransposition is presumably mediated by full-length
Alu transcripts synthesized by RNA polymerase III, while some
polymerase III-synthesized Alu transcripts undergo 3`-processing and
accumulate as small cytoplasmic (sc) RNAs of unknown function.
Interspersed Alu sequences also reside in the untranslated regions of
some mRNAs. The Alu sequence is related to a portion of the 7SL RNA
component of signal recognition particle (SRP). This region of 7SL RNA
together with 9- and 14-kDa polypeptides (SRP9/14) regulates
translational elongation of ribosomes engaged by SRP. Here we
characterize human (h) SRP9 and show that it, together with hSRP14
(SRP9/14), forms the activity previously identified as Alu RNA-binding
protein (RBP). The primate-specific C-terminal tail of hSRP14 does not
appreciably affect binding to scAlu RNA. K
Alu sequences are short interspersed elements endogenous to the
human genome. Over 500,000 Alu repeats comprise about 5% of human DNA
(1, 2) . Both by Alu-Alu recombination
(3, 4, 5, 6) and by de novo insertion
(7, 8, 9, 10) Alu
sequences have caused genetic variability in humans (for review see
Ref. 11). The primate Alu element is dimeric, composed of two
contiguous, non-identical monomers followed by A-rich or poly(A)
tracts. Structural evidence suggest that Alu repeats were
retrotransposed through nascent transcript intermediates synthesized by
RNA polymerase (pol)
Some pol III-synthesized
Alu transcripts are converted by 3`- processing to a
poly(A)
Both of
the Alu monomers that comprise Alu repeats are evolutionarily related
to the 7SL RNA component of the signal recognition particle (SRP), a
ribonucleoprotein that targets a subset of nascent polypeptides to the
endoplasmic reticulum
(25, 26, 27, 28, 29) . The
Alu-homologous region of 7SL RNA
(30, 31, 32) together with a heterodimeric protein known as SRP9/14
comprise the ``Alu domain'' of SRP, which mediates pausing of
synthesis of ribosome-associated nascent polypeptides that have been
engaged by the targeting domain of SRP
(29) . SRP9/14 exists as
a stable complex composed of 9- and 14-kDa polypeptide subunits in the
absence of RNA
(25, 33, 34, 35, 36) . Detailed
structures of SRP9 and SRP14 subunits have been deduced from cognate
cDNAs isolated from canine and mouse species
(33, 34, 35, 36) .
The absence of
poly(A)-oligo(U) tracts in processed scAlu and scB1 RNAs indicates that
they are not transposition intermediaries. Rather, their precise and
conserved secondary structures as well as efficient cytoplasmic
compartmentalization suggest that these RNAs interact with Alu-specific
SRP proteins and that they might be involved in gene expression
(17, 18, 19, 20, 21, 22, 23) .
Our laboratory previously characterized an Alu RNA-binding protein (Alu
RBP) by following its activity in the scAlu RNA-mediated
electrophoretic mobility shift assay (EMSA)
(18, 20) .
Interestingly, HeLa cells contained more of this activity than did
rodent cells, and the scAlu or scB1 RNA-protein complexes reconstituted
from human cell extracts exhibited slower electrophoretic mobility than
the corresponding complexes reconstituted from mouse cells
(18) . This allowed human Alu RBP to be distinguished from the
rodent RBP using extracts prepared from rodent X human somatic cell
hybrids and the gene responsible for its activity was mapped to human
chromosome 15q22
(20) . Intriguingly, multiple independent
hybrids that expressed both human and mouse Alu RBPs contained
substantially more human Alu RBP activity as compared to the rodent RBP
activity
(20) . More importantly, the high level expression of
human Alu RBP in these cells was associated with a corresponding
increase in the amount of processed scAlu and scB1 RNAs at the apparent
expense of full-length Alu and B1 transcripts
(20) (see also
chromosome 15 cells in Fig. 3 A of Ref. 19). This
suggested that (i) human Alu RBP and scAlu RNA are associated in
vivo, and (ii) that overexpression of Alu RBP might affect nascent
Alu RNA turnover in vivo, and according to the model of Alu
mobility discussed above, in a manner that might be relevant to Alu
transposition.
Here we examine the effects of the human-specific C-terminal tail of
hSRP14 on RNA binding in vitro. In addition, since hSRP14
exhibits a variant C-terminal structure, we wanted to examine the
structure of its heterodimeric partner hSRP9, which had not previously
been characterized. We found that recombinant hSRP9 and recombinant
hSRP14 translated in vitro together reconstituted Alu RBP
activity. We then used highly purified human Alu RBP from HeLa cells to
determine the relative affinities of this protein for 7SL, scAlu and
scB1 RNA ligands. Finally, although Alu sequences interspersed in mRNAs
represent potential binding sites for SRP9/14 Alu RBP, it remained
untested whether Alu RNA sequences embedded within larger transcripts
could interact with this protein. Therefore, we also used the EMSA-RNA
competition assay to examine the ability of human SRP9/14 Alu RBP to
interact with an Alu motif embedded in a larger RNA sequence such as
those found in untranslated regions of mRNAs.
At the time of this submission, the only SRP9
sequence that has been documented is that for canine
(34) . The
open reading frames encoding human and canine SRP9 are highly
homologous (Fig. 1). The human SRP9 coding region was 96%
identical to the canine sequence at the amino acid level and 94%
identical at the nucleotide level. A GenBank data base search using
human SRP9 coding sequence yielded canine SRP9 and a Caenorhabditis
elegans genomic DNA sequence presumed to be a homologue of SRP9,
while a search using hSRP9's 3` UTR yielded only canine SRP9 3`
UTR (not shown). Despite the relatively extensive length of hSRP9 cDNA,
the longest open reading frame in all three reading frames of the mRNA
strand was that predicted to encode the SRP9 polypeptide (not shown).
To assess expression of human SRP9 mRNA, Northern blots containing
poly(A)
The ability of hSRP9 and hSRP14 translation products,
alone and in combination, to bind [
The deletion construct
confirmed the expectations that (i) the 3` trinucleotide
repeat-containing region that encodes the C-terminal tail of hSRP14
accounts for the difference in mobility between mouse and human
RNA-protein complexes and (ii) that the C-terminal tail was not
necessary for binding to RNA
(20, 36) . In the reactions
shown in lanes 5 and 6 of Fig. 3the
SRP9 protein was present in nearly equal amounts and in excess over
that of the SRP14 polypeptides, which were present in unequal amounts
(Fig. 3 B, compare lanes 5 and
6). This suggested that in these reactions the amount of SRP14
might be the limiting component for [
As discussed in the
Introduction, in experiments performed with somatic cell hybrid
extracts, more human-specific RNA-protein complex is reconstituted than
rodent complex; this is demonstrable by EMSA in the presence of excess
RNA probe and whether the probe is scB1 or scAlu
[
First, we demonstrated direct binding of Alu RBP to
7SL-Alu RNA (Fig. 5 A, lane 6). For
this experiment, we used highly purified human Alu RBP (+ lanes)
or no added protein (- lanes) and purified
[
We have shown that human SRP14 and SRP9 polypeptides together
form the RNA binding activity previously described as Alu RBP. These
results are in agreement with previous data, which demonstrated that
the SRP9 and SRP14 kDa polypeptides heterodimerize before binding to
the Alu-homologous region of 7SL RNA
(34, 36) . The
results reported here in conjunction with Western blot and
immunoprecipitation analyses using antisera made to recombinant hSRP9
and hSRP14 (not shown) leave no doubt that Alu RBP identified in rodent
and human-derived cell extracts
(18) and purified from HeLa
cells
(20) is actually SRP9/14.
The
Results obtained from this and previous studies shed
light on RNA sequence requirements for SRP9/14 binding. A comparison of
the 5` Alu-homologous regions of the RNA sequences used in this study
is shown in Fig. 7. Also included in Fig. 7are the
previously determined SRP9/14 footprints ( asterisk regions) on
7SL RNA
(60) ; these include a sequence that is highly conserved
in the SRP RNAs (indicated by letters above the 7SL sequence)
of all organisms examined
(44, 60) . Within the regions
corresponding to the footprints of SRP9/14 as determined on 7SL RNA
(60) , scAlu and 7SL share 83% identity, while scB1 and 7SL
share 72% identity
(19, 23, 60) . A non-variant
G residue (indicated by a caret corresponding to position 24
of human 7SL RNA) of all known SRP RNA sequences
(60) is
preserved in scAlu but is replaced by U in scB1. Therefore, since scB1
binds with 10-fold lower affinity than 7SL, this non-variant G residue
would appear to contribute, at most, a 10-fold effect on RNA binding
affinity. scB1 RNA interacts with Alu RBP with high specificity
(18) , further indicating that this G residue at the conserved
position is not the sole determinant of RNA binding specificity. More
importantly, intracellular scB1 RNA levels increase substantially in
response to corresponding increases in Alu RBP levels
(20) ,
suggesting that the G
We wish to emphasize that
the synthetic test mRNAs used here (Fig. 6) contained
artificially inserted Alu motifs derived from a recently transposed Alu
whose sequence matches its Alu subfamily consensus well, whereas most
Alus interspersed in mRNAs vary in sequence (although not as much as B1
and 7SL differ) and may display a range of affinities for SRP9/14 Alu
RBP. However, the fact that scB1 represents a moderately degenerate 7SL
RNA sequence yet interacts specifically and responds to increased
levels of human Alu RBP in vivo (18, 20) suggests that individual Alus which diverge from their
consensus sequence will also be able to interact specifically with this
protein. Experiments are currently under way to determine the identity
of Alu-containing mRNAs that may be recognized by Alu RBP.
The
observation that pol III-synthesized Alu transcripts increase nearly
100-fold in the cytoplasm of virus-infected cells
(62, 63) suggests that these might effect formidable competition
with 7SL RNA for Alu RBP. Consequences of Alu RNA induction on protein
synthesis could conceivably have direct effects via an SRP-like Alu RNP
and indirect effects via disruption of SRP action; exploration of
either of these putative models would be interesting given our
knowledge that remodeling of the translational apparatus usually
accompanies viral infection.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U20998.
We thank E. Ullu for the 7SL S-deletion mutant, E.
Englander for the NheI-adapted NF1 Alu, P. Munson for LIGAND,
W. Makalowski for mRNA sequence analysis, and P. Zelenka for suggesting
paper to block
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
values for three Alu-homologous scRNAs were determined using Alu
RBP (SRP9/14) purified from HeLa cells. The Alu region of 7SL, scAlu,
and scB1 RNAs exhibited K
values of 203
pM, 318 pM, and 1.8 nM, respectively.
Finally, Alu RBP can bind with high affinity to synthetic mRNAs that
contain interspersed Alus in their untranslated regions.
(
)
III
(12, 13) (reviewed in Ref. 14). According to the current model of
Alu retroposition, the 3` oligo(U) residues of a pol III-terminated Alu
transcript form intramolecular base pairs with internal poly(A)
residues of the same transcript to produce a ``self-primed
template'' for reverse transcription
(15, 16) (reviewed in Refs. 11 and 14).
, monomeric Alu RNA species of unknown
function referred to as small cytoplasmic (sc) Alu RNA
(17, 18, 19, 20) . Similarly, B1 short
interspersed elements (rodent Alu equivalents) encode
poly(A)
-containing primary transcripts, which undergo
3`-processing to produce poly(A)
scB1 transcripts
that accumulate in vivo (18, 21, 22, 23) . Based on these
observations and according to the Alu retroposition model, 3` RNA
processing would eliminate the potential for reverse cDNA synthesis
from B1 and Alu nascent transcripts. Furthermore, because active Alu
sequences in humans transpose as dimeric elements, it was suggested
that conversion of Alu nascent transcripts to monomeric scAlu RNA might
be a non-productive pathway for Alu transposition and therefore serve
to limit Alu mobility in man
(14, 19, 23, 24) . Characterization of
factors that interact with Alu RNA and affect its metabolism may
provide insights into Alu transposition as well as function.
Figure 3:
RNA binding properties of in vitro translated human SRP9 and SRP14 polypeptides. A, EMSA.
Lane R, scB1 [P]RNA probe
alone; lane 1, [
P]RNA-protein
reconstitution using extract from rodent X human somatic cell hybrid
GM10500, a positive control that produces both human- and
rodent-specific RNA-protein complexes (20); lanes 2-6,
[
P]RNA-protein reconstitution reactions using
in vitro translated proteins as indicated below the lanes and
in panel B. In vitro translations were
programmed with the synthetic human SRP mRNAs as indicated: SRP9, SRP14
(
18 kDa), and SRP14
3` (14 kDa), the latter of which
represents a deletion mutant that truncated the alanine-rich C-terminal
tail of hSRP14. Lanes shown were coelectrophoresed in the same gel.
B, aliquots of the
[
S]methionine-labeled proteins equal to that
used in the EMSA reactions above as indicated were analyzed by
SDS-PAGE/fluorography. Lanes 2-6 in B represent
lanes 2-6 in A. Lanes shown were
coelectrophoresed in the same gel.
Human Alu RBP activity copurifies with two
polypeptides in stoichiometric amounts: an 18-kDa polypeptide that
is highly homologous to mouse SRP14 and a
9-kDa protein
(20) . Based on the work of others, which indicated that SRP9/14
is a stable heterodimer in the absence of RNA
(25, 34, 37) , the
9-kDa polypeptide of Alu
RBP was suspected to be SRP9, but this remained uncertain
(20) (see below and ``Discussion''). The human
(h)SRP14-homologous component of Alu RBP is actually
18 kDa
because its mRNA contains a 3` GCA-rich trinucleotide repeat, which
encodes an
4-kDa alanine-rich C-terminal tail that is unique to
higher primates
(20, 24) . Excluding this C-terminal
tail, the human protein and mouse SRP14 are 90% identical
(20) .
Although the C-terminal tail of hSRP14 is associated with increased Alu
RBP activity
(20, 24) , it was unknown whether this is
due to an increase in the accumulation of Alu RBP or its intrinsic
affinity for RNA
(18, 20) . However, the fact that
regions of the C terminus of mouse SRP14 have been found to be
dispensable for specific RNA binding in vitro (36) suggests that the former possibility is the more likely.
Human (h) SRP9 cDNA
Partially degenerate 19`-mer
and 18`-mer oligodeoxynucleotide primers corresponding to the extreme
5` and 3` termini of canine SRP9 coding region
(34) were used
for PCR amplification of human SRP9 coding region from oligo(dT)-primed
HeLa cell cDNA. The PCR product obtained was cloned into the
EcoRI/ XbaI sites of pBAT
(38) and designated
pB-hSRP9. This construct was used as template for in vitro transcription/translation (see below). pBAT is a pSK derivative,
which contains the rabbit -globin 5` UTR subcloned into the
KpnI/ HindIII site
(38) . The integrity of
pB-hSRP9 was confirmed by sequencing. Internal sequence information
from the HeLa-derived cDNA allowed design of human-specific primers for
RACE (rapid amplification of cDNA ends)-mediated
(39) isolation
of full-length human SRP9 cDNA that contained 5` and 3` UTRs. For this
we used the Amplifinder RACE kit (Clontech, Palo Alto, CA) according to
the manufacturer's protocols. For 5` RACE, first strand cDNA was
synthesized from HeLa cell poly(A)
RNA using a
hSRP9-specific antisense primer 5`-CTTGGCTACCATAAGTCGCATTA-3`, and a
second (nested) antisense hSRP9-specific primer
5`-GACTGTGGAATTTCTCAATCTTC-3` was used for PCR amplification. To obtain
the 3` UTR of hSRP9 cDNA, we PCR-amplified HeLa oligo(dT)-primed first
strand cDNA using the hSRP9-specific sense primer
5`-CTCAAATATAGGCATTCTGATGG-3` and the ``dT
adapter
primer'' described previously
(39) . Finally, we used
primers to the 5` and 3` termini to amplify a contiguous full-length
cDNA; these primers were nested by about 25 base pairs with respect to
the absolute termini shown in Fig. 1.
Figure 1:
Nucleotide sequence and predicted amino
acid sequence of human SRP9 cDNA. Nucleotide positions are numbered to the left of the sequence. Arrows indicate the
positions of the human-specific primers used for RACE-mediated
isolation of 5` and 3` UTR-containing cDNA (see ``Materials and
Methods''). Lowercase letters above the sequence
indicate nucleotide differences in canine SRP9 cDNA (34). The
single-letter amino acid code is indicated below the
nucleotide sequence. Letters below the human amino acid
sequence indicate differences found in canine SRP9. Asterisk denotes the position of the poly(A) addition
signal.
Northern (RNA) Blotting
The hSRP9 probe was
generated from the 260-base pair coding region of hSRP9 cDNA.
Human multiple tissue Northern blots and the
-actin probe were
obtained from Clontech. Probes were labeled by random priming in the
presence of [
-
P]dCTP. Hybridization was
carried out in 5
SSPE, 10
Denhardt's solution,
50% deionized formamide, 100 µg/ml salmon sperm DNA, 1% SDS at 42
°C. The final two washes were in 0.1
SSC (1
SSC is
0.15 M NaCl plus 0.015 M sodium citrate), 0.1% SDS at
50 °C for 20 min each.
In Vitro Transcription and Translation of hSRP9 and
hSRP14 cDNAs
hSRP14 cDNA (pG-18K)
(20) was subcloned
into the HindIII/ XbaI sites of pBAT (see above) and
designated pB-hSRP14. T7 RNA polymerase-dependent templates for mRNA
synthesis were generated by PCR. A 3`-deletion mutant of hSRP14 cDNA
(designated hSRP143`) was generated from pB-hSRP14 by PCR using
the antisense primer 5`-GCTCATGCTTTGGTCTTC-3`, which changed the first
GCA repeat of wild type hSRP14 cDNA to a stop codon, thereby deleting
the coding region for the
4-kDa alanine-rich C-terminal tail.
Transcription reactions contained 40 mM Tris-HCl, pH 8.1, 16
mM MgCl
, 5 mM dithiothreitol, 0.01%
Triton X-100, 0.1 mM spermidine, 50 mg/ml bovine serum
albumin, 2 mM each of ATP, CTP, UTP, GTP (pH 8.1), 20 units of
RNasin (Promega), 2 mM m
GTP (Pharmacia Biotech
Inc.), 100 ng of DNA template, and 400 units of T7 RNA polymerase
(Promega). mRNA products were purified by phenol chloroform extraction
and ethanol precipitation. The integrity and quantity of the mRNA
products were determined by 8 M urea, 6% PAGE and ethidium
staining. The mRNAs were translated by wheat germ extract (Boehringer
Mannheim). Each translation reaction contained 250-500 ng of
mRNA. A 25-µl reaction consisted of 7.5 µl of extract, 100
mM potassium acetate, 1 mM magnesium acetate, 25
µM amino acids minus methionine, 1 mM ATP, 0.02
mM GTP, 8 mM creatine phosphate, 3.75 µg of
spermidine, 2 mM dithiothreitol, 14 mM HEPES, pH 7.6,
1 µg of creatine kinase, and 2 µl of
[
S]methionine (800 Ci/mmol) (DuPont NEN).
Incubation was for 60 min at 30 °C, after which aliquots were
frozen at -80 °C. Translation products were analyzed by
SDS-PAGE/fluorography (EN
HANCE; DuPont NEN) and quantitated
by phosphor storage densitometry (Molecular Dynamics).
Electrophoretic Mobility Shift Assay (EMSA)
scAlu
derived from the left monomer of the NF1 Alu
(10) , scB1, and
7SL-Alu (below) [P]RNAs were synthesized in
vitro from T7 RNA polymerase-dependent templates
(18, 23) , gel-purified, and incubated with 1 µl of
in vitro translation products in 15-µl reactions
containing 10 mM Tris-HCl, pH 7.5, 80 mM KCl, 5
mM MgCl
, 0.1% Triton X-100, 1 mM
dithiothreitol, 1 mM EDTA, 4 units of RNasin, 5% glycerol, and
100 ng of poly(rG)
(18, 20, 40) ; after a 40-min
incubation, samples were analyzed on nondenaturing 6% polyacrylamide
gels as described previously
(18, 20) . Two pieces of 12
bond paper between the dried gel and x-ray film blocked the
S background carried over from the in vitro translation reactions.
Determination of Alu RBP-RNA Dissociation
Constants
scB1 [P]RNA, scAlu
[
P]RNA and
[
P]Alu-homologous region of 7SL (7SL-Alu) RNA
(41) were synthesized in vitro (18) . The Alu
homologous region of 7SL RNA was synthesized from an S-region deletion
mutant of the 7SL 30.1 gene
(41) ; a T7 promoter was attached by
PCR to generate a 145 nucleotide transcript whose 5` and 3` termini
matched cellular 7SL RNA, as described previously for scB1 and scAlu
RNA synthesis
(18, 19, 23) . Alu RBP was
purified from HeLa cell cytoplasmic extract as described previously
(20) . The Alu RBP-containing faction that eluted from heparin
agarose contained 18 kDa and 9 kDa polypeptides detectable by
SDS-PAGE/silver staining
(20) , identified as SRP9 and SRP14 by
Western blotting (data not shown). EMSA was performed using aliquots of
this purified Alu RBP preparation incubated with a range of
[
P]RNA probe concentrations under standard EMSA
conditions
(18, 20) . Quantitation of protein-complexed
(bound) and free [
P]RNA was performed by
PhosphorImager (ImageQuant, Molecular Dynamics) and the data analyzed
by the LIGAND program package (kindly provided by P. Munson, NCBI, NIH,
Bethesda, MD) fitted to the one-binding-site model
(42) . For
each RNA species tested reconstitution reactions at each RNA
concentration were performed in triplicate. Overall, the quality of fit
for the triplicate determinations for each RNA were determined by the
LIGAND program ``test runs'' to be significant
(42) (not shown).
Synthesis of Chloramphenicol Acetyltransferase (CAT)
Alu-containing mRNAs
The left monomer of the NF1 derived Alu
repeat
(10) was adapted with flanking NheI or
XhoI sites and these were subcloned into the 5` NheI
or 3` XhoI site of pMAM neo-CAT (Clontech) each in the sense
and antisense orientation. Templates for in vitro transcription were constructed by adding a T7 promoter onto the
CAT 5` UTR by PCR using a T7-CAT chimeric primer
(19, 23) , and CAT mRNAs were synthesized by T7 RNA
polymerase as described above. For the 5` Alu-CAT mRNA this placed the
Alu left monomer sequence 50 bases downstream of the start site of
transcription. For the 3` CAT-Alu mRNA this placed the Alu100 bases
downstream of the stop codon of CAT, about 100 bases from the 3` end.
The size, integrity and concentration of these and all RNAs used in
this report were verified by ethidium bromide staining and comparison
with standard markers after polyacrylamide gel electrophoresis.
Human SRP9 cDNA
Human Alu RBP was initially
identified as a protein component, which reconstituted an RNA-protein
complex of a distinctive electrophoretic mobility
(18, 20) . To test if human (h)SRP9 might be part of
this complex as expected (see Introduction), we isolated cDNA for hSRP9
and used it to generate in vitro translated protein for RNA
binding studies. The PCR-based 5` and 3` RACE methods
(39) were
used to obtain hSRP9 cDNA UTRs (see ``Materials and
Methods''). Primers near the 5` and 3` termini were then used to
PCR-amplify a full-length cDNA, which spanned 1.46 kb and included
0.1 kb of 5` UTR, a 0.26-kb coding region and
1.1 kb of 3` UTR
(Fig. 1).
RNA isolated from various human tissues were
probed with the
260-base pair coding region of hSRP9 cDNA
(Fig. 2). This revealed a single
poly(A)
-containing transcript of
1.6 kb, which
was readily detectable in all tissues examined as was previously
reported for human SRP14 mRNA
(20) .
Figure 2:
Northern blot analysis of human SRP9
poly(A) RNA. Human multiple tissue Northern blot
(Clontech) containing poly(A)
RNA was hybridized
sequentially with two different
P-labeled probes.
Upper panel, probe was derived from the coding region
only of hSRP9 cDNA; exposure to x-ray film was for 4 h at room
temperature. Lower panel,
-actin control probe;
exposure was for 1 h at room temperature.
RNA-mediated Electrophoretic Mobility Shift Assay Using
in Vitro Translated hSRP9 and hSRP14
To further characterize the
coding capacity of the hSRP9 cDNA and to generate protein with which to
investigate Alu RNA binding, hSRP9 and hSRP14 polypeptides were
synthesized in vitro from synthetic mRNAs using wheat germ
extract. For this purpose, the coding regions of these cDNAs were
cloned independently into a vector containing a promoter for T7 RNA
polymerase, followed by the rabbit globin 5` UTR. This 5` UTR has
been shown to increase translational efficiency of synthetic mRNAs in
wheat germ extract
(38) (see also Ref. 34). In addition to wild
type SRP9 and SRP14 polypeptides, a C-terminal truncated SRP14
polypeptide designated hSRP14
3` was also synthesized. This was
achieved by deletion of the 3` GCA trinucleotide repeat-containing
region, which is specific to hSRP14
(20, 24) , by
converting the first GCA repeat to a translational stop codon. In
vitro transcription/translation of this construct was predicted to
produce a C-terminal truncated polypeptide structurally similar to
mouse SRP14, which migrates at
14 kDa. The three
S-labeled in vitro translation products SRP9,
hSRP14, and hSRP14
3` demonstrated the electrophoretic mobilities
in SDS-PAGE expected based on their predicted size (see
Fig. 3B, lanes 3, 4, and
6).
P]RNA was
examined by EMSA (Fig. 3 A). We expected that if the
C-terminal truncated construct hSRP14
3` would bind RNA in this
assay, it would produce an RNA-protein complex whose mobility was
similar to the rodent [
P]RNA
RBP complex,
while the wild type hSRP14 would produce the human-specific complex
(18, 20) . As a mobility marker we used a previously
characterized mouse X human somatic cell hybrid extract (GM 10500),
which reconstitutes both rodent and human RNA-protein complexes as a
positive control
(20) (Fig. 3 A, lane 1). Following in vitro translation, aliquots of
the reaction products were incubated with scB1
[
P]RNA and analyzed by EMSA
(18) . No
binding activity was observed when either hSRP9
(Fig. 3 A, lane 3) or hSRP14 ( lane 4) in vitro translated proteins were provided
independently. However, when hSRP9 and hSRP14 proteins were
cotranslated in wheat germ extract and then used for EMSA, the
human-specific RNA binding activity was observed
(Fig. 3 A, lane 5). The
S-labeled proteins used in the RNA binding reactions above
were also analyzed by SDS-PAGE/fluorography as shown in
Fig. 3B. Note that in lane 5 of
Fig. 3B the hSRP14 translation product is visible as a
faint band (compare lanes 4 and 5). As
expected, the C-terminal truncated hSRP14
3` cotranslated with
hSRP9 reconstituted an [
P]RNA-protein complex,
which migrated with the rodent-specific complex
(Fig. 3 A, lane 6). We note that
efficient binding activity was reproducibly reconstituted when both
SRP9 and SRP14 were cotranslated (Fig. 3; see also Ref. 34); by
contrast, translating these polypeptides independently and then mixing
them did not reconstitute RNA binding activity (not shown). In summary,
these data demonstrated that both hSRP9 and the
18-kDa hSRP14
protein were required to produce the human-specific Alu RNA binding
activity previously identified as Alu RBP.
P]RNA
binding activity. More of the faster migrating rodent-like complex was
formed as compared to the slower migrating human-specific complex
(Fig. 3 A, lanes 5 and 6,
respectively), consistent with there being more
S-hSRP14
3` ( lane 6) than wild type
S-hSRP14 ( lane 5) in the reactions.
Further examination of the effect of the human-specific SRP14
C-terminal tail on RNA binding is described below using scAlu
[
P]RNA as probe.
P]RNA
(18, 20) .(
)
These observations
suggested that we could compare C-terminal tail-containing (hSRP14) and
C-terminal tail-lacking (hSRP14
3`) proteins for their RNA binding
activities in the same reaction vessel. Excess hSRP9 was cotranslated
with hSRP14 and hSRP14
3`, and scAlu [
P]RNA
binding was analyzed by EMSA (Fig. 4 A, lane 3) and quantitated by PhosphorImager
(Fig. 4 C). Since wild type (
18 kDa) and
C-terminal-truncated (
14 kDa) hSRP14 polypeptides were each
synthesized in nearly equal amounts as determined by PhosphorImager
analysis of [
S]methionine-labeled polypeptides
(there are no methionines in the C-terminal tail of hSRP14; Ref. 20),
each should have had equal access to heterodimerize with an excess of
SRP9 (Fig. 4 B, lane 3) and bind the
scAlu [
P]RNA probe. The binding to
[
P]RNA was nearly equal for both complexes
(Fig. 4, A, lane 3, and C).
Thus, there is no indication that by this assay the human-specific
C-terminal tail of SRP14 appreciably affects its ability to
reconstitute Alu RBP and compete for the scAlu RNA probe. Thus, it
appears that the effects of the C-terminal tail on RNA binding observed
by this in vitro assay may not account for the more
substantial differences seen in HeLa versus rodent cells, and
in somatic cell hybrids that express both species' RBPs (see
``Discussion'')
(18, 20) .
Figure 4:
The trinucleotide repeat-encoded
C-terminal tail of human SRP14 does not affect binding to scAlu RNA
in vitro. A, EMSA with scAlu
[P]RNA using in vitro translated hSRP
proteins. Wheat germ extracts were programmed with the synthetic human
mRNAs for SRP9, SRP14 (
18 kDa), and SRP14
3` in the
combinations as indicated below the lanes. Lanes shown were
coelectrophoresed in the same gel. B, aliquots of the
[
S]methionine-labeled proteins equal to that
used for the EMSA reactions above were analyzed by
SDS-PAGE/fluorography; lanes 1-3 in A correspond to lanes 1-3 in B. Lanes shown
were coelectrophoresed in the same gel. C, quantitative
analysis of scAlu RNA binding activity when both full-length and
C-terminal truncated in vitro translated hSRP14 proteins
compete for scAlu [
P]RNA. The bands in lane 3 of A were quantitated by ImageQuant
PhosphorImager analysis (Molecular Dynamics), corrected for the amount
of
S-labeled protein in lane 3 of
B, and plotted.
RNA Binding Affinity Studies
scB1, scAlu, and 7SL
RNAs represent evolutionarily related mammalian transcripts, which
contain a similar tRNA-like secondary structure located within their 5`
end regions
(23, 31, 32, 43, 44, 45, 46) .
Although human Alu RBP was shown previously to bind to scAlu and scB1
RNAs with the same profile of specificity as determined by challenge
with a panel of competitor RNAs
(18) , the affinity of Alu RBP
for these RNAs had not been determined. Also, although it was inferred
from EMSA/RNA competition studies using full-length 7SL RNA that Alu
RBP associated with the Alu-homologous region of 7SL RNA, direct
binding to 7SL RNA had not been demonstrated for Alu RBP as isolated
from HeLa cells
(18) . For the present report we employed a 7SL
RNA gene construct in which the non-Alu sequence known as the S region
was deleted, thereby abutting the 5` and 3` terminal Alu-homologous
regions in a manner that resembled an Alu monomer
(47) . A
similar construct was used by Strub and co-workers
(36) to
monitor SRP9/14 binding. This construct could be used to produce a 7SL
Alu-homologous RNA (hereafter referred to as 7SL-Alu) whose size and
mobility were comparable to the scAlu and scB1 RNAs used previously.
This could then be readily resolved into free RNA and RNA-protein
complex using our EMSA conditions, and directly compared to scAlu and
scB1 RNAs.
P]RNAs. (The slowly migrating bands in
lanes 3 and 4, and less visible in lanes 1 and 2, are not specific since they were
present in the RNA probe alone in the absence (- lanes) of added
protein). The 7SL-Alu [
P]RNA produced the
expected shift in mobility (Fig. 5 A, lanes 5 and 6), similar to those produced by scAlu
( lane 4) and scB1 ( lane 2) RNAs.
For these experiments, the specific radioactivities of the three
Alu-related [
P]RNAs were nearly identical (see
``Materials and Methods''). The amount of purified protein
used in each reaction was also kept constant. Therefore, it is
noteworthy that less scB1 [
P]RNA was
reproducibly shifted to the bound complex (Fig. 5 A,
lane 2) as compared to scAlu
[
P]RNA ( lane 4) and 7SL-Alu
[
P]RNA ( lane 6). The results
suggested that under these conditions, which monitored specific
binding, scB1 RNA bound to human Alu RBP with lower affinity than did
scAlu RNA, which bound with lower affinity than did 7SL-Alu RNA.
Figure 5:
Scatchard analysis of binding to 7SL-Alu
RNA and Alu-related RNAs by purified human Alu RBP. A, EMSA
using purified human Alu RBP and a single concentration of three
different Alu-related [P]RNAs. 0.1 ng each of
[
P]scB1 ( lanes 1 and 2),
[
P]scAlu ( lanes 3 and
4), or 7SL-Alu [
P]RNA ( lanes 5 and 6) (each at 30,000 cpm/ng) was incubated
in the presence (+) or absence (-) of purified human Alu RBP
(1 µl) as described previously and under ``Materials and
Methods'' (18, 20). The slowly migrating, faint bands in lanes 3 and 4 (less visible in lanes 1 and 2) are not specific since they were present in the
RNA probe alone in the absence (-) of added protein. B,
Scatchard plots of binding of the different
[
P]RNAs to purified human Alu RBP as monitored
by EMSA and determined by the LIGAND program (42). Purified protein
(0.2 µl) was incubated with either scB1, scAlu, or 7SL Alu RNA
(synthesized to a specific activity of 30,000 cpm/ng for each RNA; see
``Materials and Methods''). RNA concentrations ranged from 20
pM to 1.3 nM for scAlu RNA and 7SL Alu RNA. For scB1
RNA concentrations ranged from 20 pM to 11.2 nM.
In vitro reconstitution, EMSA and quantitation were performed
as described under ``Materials and Methods'' using LIGAND
(42) for analysis. For each RNA, every point represents the average of
three reconstitution/EMSA reactions, the standard errors of the means
for which conformed to the recommendations of LIGAND. For scAlu, K = 318 ± 159 pM, for 7SL-RNA, K = 203 ± 36 pM, and for scB1, K = 1.83 ± 0.79 nM. All reactions contained
poly(rG) nonspecific inhibitor (40) (see
text).
To
compare binding affinities of the three Alu-related RNAs for purified
human Alu RBP, we applied Scatchard analysis of the RNA-protein
interaction as monitored by EMSA. RNA-mediated EMSA has been used by
others to quantitate RNA-protein dissociation constants
(48, 49) . Binding data obtained were analyzed by the
modeling program LIGAND
(42, 48, 49) . For the
studies reported here, the amount of protein was held constant while
the amount of RNA included in the reactions was varied over a wide
range of concentrations sufficient to reach saturation (not shown). As
noted in Fig. 5 B, the RNA concentration necessary to
saturate binding was significantly higher for scB1 RNA than for scAlu
and 7SL RNAs. Binding reached steady state levels well within the
incubation time.()
Each reaction contained only
one of the three RNA species and each reaction was performed in
triplicate. Bound and free RNA was quantitated by direct counting using
a Molecular Dynamics PhosphorImager, and the Scatchard plots were
generated by LIGAND and the K
values
calculated using the one binding site model
(42) (Fig. 5 B). The affinity of human Alu RBP for
scAlu RNA was slightly lower than the affinity for 7SL-Alu RNA
exhibiting K
of 318 ± 159
pM and 203 ± 36 pM respectively; standard
errors were calculated by LIGAND (Fig. 5 B). As expected
from the results of Fig. 5 A, scB1 RNA reproducibly bound
to human Alu RBP with approximately 10-fold lower affinity than 7SL RNA
exhibiting a K
of 1.83 ± 0.79
nM in parallel experiments under the same conditions
(Fig. 5 B, inset). In these experiments poly(rG)
was included to block nonspecific binding. The 7SL-Alu RNA
K
of 0.2 nM obtained is not very
different from the K
of <0.1
nM determined by a fluorescence spectroscopy method using
canine SRP9/14 and full-length canine 7SL RNA
(50) .
Human Alu RBP Can Bind to Larger Transcripts That Contain
Interspersed Alu Sequences
Alu sequences have been reported to
be present in the UTRs of many mRNAs and are abundant in pre-mRNAs
(51, 52, 53, 54, 55, 56, 57, 58) .
These and presumably other as yet unidentified Alu-containing mRNAs
would appear to comprise a significant subset of potential binding
sites for SRP9/14 Alu RBP. Moreover, it has been reported that B1 Alu
equivalent repetitive sequences in a subset of mRNAs confers
posttranscriptional regulation of growth-related molecules
(59) . Since human Alu RBP can bind to Alu and 7SL-Alu RNAs with
similar affinities, the ability of this protein to bind Alu-containing
RNAs was examined. We synthesized CAT mRNAs such that the scAlu motif
was inserted upstream (5` UTR) or downstream (3` UTR) of the CAT coding
region, each independently in the sense and antisense orientation. In
these constructs the Alu sequence represented about 10% of the overall
size of the CAT mRNA and was inserted such that the terminal
50-100 nucleotides of the RNA were represented by CAT UTR
sequences; this situation resembles mRNAs and pre-mRNAs in which the
Alu is interspersed in a larger sequence. We initially tested these
unlabeled chimeric RNAs for their ability to compete at 100 fold molar
excess with scAlu [P]RNA binding using HeLa
crude cytoplasmic extract by our standard EMSA
(18, 20) . Both of the CAT mRNAs that contained the
scAlu inserted in the sense orientation competed for scAlu
[
P]RNA-protein complex formation
(Fig. 6 A, lanes 4 and 5) as
effectively as did the scB1 RNA competitor ( lane 2).
In contrast, CAT mRNA in which the Alu was absent competed weakly,
reflecting a low level of nonspecific binding only ( wt,
lane 8). Weak competition was also observed for the
CAT mRNAs that contained antisense Alu in their 5` ( lane 6) or 3` ( lane 7) UTRs.
Figure 6:
Human
Alu RBP is able to bind to interspersed Alu sequences within larger
RNAs. 0.1 ng (0.002 pmol) of scAlu [P]RNA was
incubated with HeLa cytoplasmic extract in the presence of 0.2 pmol
each of the following unlabeled competitor RNAs: lane 1, no competitor RNA; lane 2, unlabeled
scB1 RNA; lane 3, B1-d40, a negative control in which
the 5`-region of scB1 RNA was deleted (
) (18). Lanes 4-7, synthetic CAT mRNAs that contain scAlu RNA
sequence inserted 5` to the CAT coding region in the sense orientation
( lane 4) or antisense orientation ( lane 6), or inserted 3` to the CAT coding region in the sense
orientation ( lane 5) or antisense orientation
( lane 7), or wild type synthetic CAT mRNA with no Alu
insert ( lane 8). Note that in order to compare
equimolar amounts of competitor RNAs, a greater mass of CAT RNAs were
required relative to the smaller scB1 RNA species. B, same as
panel A except that purified HeLa Alu RBP was used and the
molar excess of competitors (indicated below the lanes) was lower than
in A (see text).
The assays
in Fig. 6 A were performed using a large molar excess of
each competitor. We also performed EMSAs using lower concentrations of
competitors (Fig. 6 B). As anticipated from the results
of Fig. 5, scB1 RNA competed weakly at 3- and 9-fold molar excess
over the scAlu [P]RNA probe
(Fig. 6 B, lanes 8 and 9). CAT
mRNA containing the scAlu sense motif in the 3` UTR (3`; lanes 4 and 5) competed slightly less effectively than
free scAlu RNA ( lanes 2 and 3) but clearly
more effectively than scB1 RNA ( lanes 8 and
9). Wild type CAT mRNA contains no Alu motif and did not
compete in these assays ( wt; lanes 6 and
7). Quantitative analyses of the results shown in
Fig. 6B as well as additional EMSAs performed under the
same conditions revealed a
3-fold lower affinity for CAT-Alu
(sense) RNA than for scAlu RNA (not shown). Since scB1 RNA exhibits a
5-fold lower affinity for Alu RBP than scAlu RNA (Fig. 5), the
results in Fig. 6 B are consistent with this. However,
due to limitations inherent to precisely comparing low concentrations
of large and small RNAs, we are unsure of the significance of an
apparent
3-fold difference in affinity between the scAlu motif as
a free RNA and as part of a larger RNA. In any case, scAlu RNA embedded
within a larger context is recognized with high affinity by Alu RBP
(SRP9/14). The results indicated that human Alu RBP is capable of
specific binding to Alu sequences embedded within larger RNAs in a
position-independent, orientation-dependent manner.
4-kDa alanine-rich
C-terminal tail of hSRP14 is responsible for the slower electrophoretic
mobility of the human RNA-protein complex relative to the corresponding
rodent RNA-protein complex. As expected, the human-specific C-terminal
tail of hSRP14 is not necessary for RNA binding and it does not appear
to affect its interaction with scAlu RNA; however, binding efficiency
of primate-specific SRP9/14 to 7SL RNA in the presence of other SRP
proteins remains to be determined. Since the C-terminal tail appears
not to be a major determinant of scAlu RNA binding in vitro,
the in vivo results reported previously (see Introduction)
suggest that hSRP14 simply accumulates to higher intracellular levels
than does rodent SRP14. This interpretation is consistent with several
lines of evidence and is supported by recent data reported elsewhere
(24) .
U substitution does not affect binding
in vivo, even though scB1 RNA accumulates to a small fraction
(<1%) of the intracellular concentration of 7SL RNA. These
observations raise the possibility that highly conserved residues in
the Alu domain of 7SL RNA
(60) contribute a function to SRP RNA
independent of determining affinity for SRP9/14. Experiments to test
this hypothesis are presently under way.
Figure 7:
Comparison of the first 70
nucleotides of 7SL, scAlu, and scB1 RNAs. Asterisks (*) above
the 7SL sequence indicate positions of SRP9/14 hydroxyl radical
footprint determined by Strub et al. (60). Within this region,
letters indicate evolutionarily conserved bases (44, 60);
purine ( R) at position 16 ( underlined) is invariant,
and G at position 24 ( caret) is invariant in all SRP RNAs
catalogued to date (44, 60). Horizontal bars below
the scB1 sequence indicate regions whose deletion (positions
5-14, scB1) or substitution (positions 35-40, scB1) abolish
binding as reported previously (18).
Recent studies suggest the
possibility that a substantial amount of human Alu RBP exists in a form
independent of SRP
(18, 20) . Since the affinities of
Alu RBP for scAlu and 7SL-Alu RNAs are similar, the possibility exists
that cellular Alu RBP is distributed between 7SL RNA as a component of
SRP, scAlu RNA and nascent Alu transcripts as small RNPs, and
Alu-containing mRNAs in human cells. The presence of Alu-homologous
sequences in nearly 10% of cellular mRNAs
(61) suggests that
this protein may affect the stability and/or translatability of a
substantial subset of human mRNAs. Although Alu domain subparticles of
SRP do not inhibit translation when provided in soluble form
(31) , Alu sequences located in ribosome-associated mRNAs might
facilitate SRP9/14-ribosome interactions. In addition, Alu sequences
might provide stability to these mRNAs as suggested by the fact that
overexpression of Alu RBP is associated with increased accumulation of
scAlu RNA
(20) . According to either putative mechanism, mRNAs
that acquired Alu motifs during primate evolution may have become
subjected to Alu-mediated differential regulation as a result
(59) . The effects of human Alu RBP on the expression of
Alu-containing mRNAs deserves examination.
S radiation. We also thank members of the
LMGR for comments and J. Sarrowa, E. Englander, T. Kokubo, and V. J.
Hernandez for critical reading. We are grateful to M. L. Lanigan for
assistance with preparation of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.