©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Signal Recognition Particle (SRP) Alu-associated Protein Also Binds Alu Interspersed Repeat Sequence RNAs
CHARACTERIZATION OF HUMAN SRP9 (*)

Karl Hsu (1)(§), Dau-Yin Chang (¶) , Richard J. Maraia (**)

From the (1) Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2753 and the HHMI/NIH Research Scholars Program, Howard Hughes Medical Institute, National Institutes of Health, Bethesda, Maryland 20817

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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. Kvalues 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 Kvalues 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.


INTRODUCTION

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)() 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).

Some pol III-synthesized Alu transcripts are converted by 3`- processing to a poly(A), 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.

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.


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 SRP143` (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.

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.


MATERIALS AND METHODS

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 ``dTadapter 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 mGTP (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 (ENHANCE; 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.


RESULTS

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).

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)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 hSRP143` 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 hSRP143` demonstrated the electrophoretic mobilities in SDS-PAGE expected based on their predicted size (see Fig. 3B, lanes 3, 4, and 6).

The ability of hSRP9 and hSRP14 translation products, alone and in combination, to bind [P]RNA was examined by EMSA (Fig. 3 A). We expected that if the C-terminal truncated construct hSRP143` would bind RNA in this assay, it would produce an RNA-protein complex whose mobility was similar to the rodent [P]RNARBP 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 hSRP143` 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.

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 [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-hSRP143` ( 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.

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 [P]RNA (18, 20) .()These observations suggested that we could compare C-terminal tail-containing (hSRP14) and C-terminal tail-lacking (hSRP143`) proteins for their RNA binding activities in the same reaction vessel. Excess hSRP9 was cotranslated with hSRP14 and hSRP143`, 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 SRP143` 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.

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 [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 Kvalues 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 Kof 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 Kof 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 Kof 0.2 nM obtained is not very different from the Kof <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.


DISCUSSION

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 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) .

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 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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank/EMBL Data Bank with accession number(s) U20998.

§
Supported by the National Institutes of Health Research Scholars Program of the Howard Hughes Medical Institute.

Supported by an interpersonnel act between NICHD and the Department of Biochemistry, University of Maryland Medical School.

**
To whom correspondence should be addressed: Bldg. 6, Rm. 416, Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2753. Tel.: 301-402-3567; Fax: 301-480-9354; E-mail: maraia@ncbi.nlm.nih.gov.

The abbreviations used are: pol, polymerase; sc, small cytoplasmic; SRP, signal recognition particle; RBP, RNA-binding protein; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; UTR, untranslated region; RACE, rapid amplification of cDNA ends; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair(s).

K. Hsu and R. J. Maraia, unpublished results.

E. Englander and D.-Y. Chang, unpublished results.


ACKNOWLEDGEMENTS

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 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.


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