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J. Biol. Chem., Vol. 275, Issue 41, 32371-32378, October 13, 2000

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A Cellular Protein, hnRNP H, Binds to the Negative Regulator of Splicing Element from Rous Sarcoma Virus^*

Brent L. Fogel and Mark T. McNally

From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received for publication, June 8, 2000, and in revised form, July 25, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Incomplete RNA splicing is a key feature of the retroviral life cycle. This is in contrast to the processing of most cellular pre-mRNAs, which are usually spliced to completion. In Rous sarcoma virus, splicing control is achieved in part through a cis-acting RNA element termed the negative regulator of splicing (NRS). The NRS is functionally divided into two parts termed NRS5' and NRS3', which bind a number of splicing factors. The U1 and U11 small nuclear ribonucleoproteins interact with sequences in NRS3', whereas NRS5' binds several proteins including members of the family of proteins. Among the proteins that specifically bind NRS5' is a previously unidentified 55-kDa protein (p55). In this report we describe the isolation and identification of p55. The p55 binding site was localized by UV cross-linking to a 31-nucleotide segment, and a protein that binds specifically to it was isolated by RNA affinity selection and identified by mass spectrometry as hnRNP H. Antibodies against hnRNP H immunoprecipitated cross-linked p55 and induced a supershift of a p55-containing complex formed in HeLa nuclear extract. Furthermore, UV cross-linking and electrophoretic mobility shift assays indicated that recombinant hnRNP H specifically interacts with the p55 binding site, confirming that hnRNP H is p55. The possible roles of hnRNP H in NRS function are discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Most eukaryotic protein-encoding genes contain introns that must be removed from primary RNA transcripts by splicing to generate functional mRNA (1). Viruses that infect eukaryotic cells have evolved mechanisms to exploit the splicing process to expand their coding potential and regulate gene expression. Expression of certain viral genes requires that full-length viral RNAs undergo splicing to subgenomic forms. For example, HIV,¹ a complex retrovirus, produces ~40 spliced RNA forms for the expression of its multiple gene products (2, 3). In contrast, a simple retrovirus such as Rous sarcoma virus (RSV) contains only four genes (gag, pol, env, and src) and produces only two spliced subgenomic RNAs (env and src) (see Fig. 1) (4). In all cases, full-length genomic RNAs are required for the expression of the gag/pol genes and for use as genome in progeny virions. The maintenance of both spliced and unspliced viral RNA forms differs from the processing of most cellular pre-mRNAs and is accomplished through incomplete splicing of the viral RNAs. RSV and other members of the avian leukosis virus family contain a unique cis-acting element termed the negative regulator of splicing (NRS) that acts globally to repress splicing to both the env and src splice sites (5, 6). The NRS is located within the gag gene and is distinct from the viral splice sites, lying >300 nucleotides downstream of the 5' splice site and >4000 nucleotides upstream of the first 3' splice site (7). The importance of the NRS to splicing control and the viral life cycle is illustrated by observations that NRS deletions and mutations cause increased splicing and impaired replication (5, 6, 8).

The NRS can also inhibit the splicing of heterologous introns (5, 7, 9), demonstrating that the inhibition is a general property of the element itself. The minimal NRS RNA element (see Fig. 1) spans ~230 nucleotides and functionally can be divided into two parts (NRS5' and NRS3'), both of which are required for function (7). Despite its role in splicing inhibition, the NRS has been shown to associate with a number of essential cellular splicing factors (9, 10). NRS3' contains overlapping sites that bind the U1 and U11 small nuclear ribonucleoproteins (snRNPs) (8, 11), factors that recognize authentic 5' splice sites in U2- and U12-dependent introns and initiate the formation of the spliceosome (1, 12-14). NRS5' is highly purine-rich and binds members of the SR family of splicing factors, predominantly ASF/SF2 (10). SR proteins are a class of serine-arginine-rich splicing factors, which are required at many steps of the splicing process and among other activities aid in the recruitment of U1 snRNP to 5' splice sites (15-19). NRS splicing inhibition is dependent on SR proteins (10, 20) and on the binding of U1 snRNP (8, 11). It is predicted that the NRS functions as a "decoy" 5' splice site that sequesters the authentic 3' splice site in a stable but nonproductive complex, thereby preventing its interaction with the authentic 5' splice site.

Although considerable evidence supports a role for SR proteins in NRS-mediated splicing inhibition, deletion of the primary SR binding region in NRS5' does not fully abolish NRS function (7, 20), suggesting that the region downstream of the SR protein binding site may contribute to NRS activity. Studies of a recombinant avian leukosis virus (EU8) that induces rapid onset B-cell lymphomas due to the presence of a 42-nucleotide deletion within NRS5' (21) lend further support for a role of this downstream site in splicing inhibition. The deletion not only impairs NRS inhibition (21) but also correlates with increased readthrough past the viral polyadenylation site into a downstream cellular gene, c-myb (22). Overexpression of truncated Myb protein caused by increased splicing results in the observed lymphomas (21, 22). The deletion maps largely downstream of the SR protein binding site, suggesting that NRS impairment is caused by the loss of binding of some other factor(s) within this region. In previous studies, an unidentified protein of approximately 55 kDa (termed p55) that is biochemically distinct from the classical SR proteins was shown to bind specifically to NRS5' (10). Using a UV cross-linking assay, we have mapped the p55 binding site to a 31-nucleotide region of NRS5' downstream of the primary SR protein binding site and within the region deleted in the EU8 virus, suggesting a relationship between p55 binding and NRS function. To better understand the significance of p55, we sought to isolate and characterize this protein. Using RNA affinity selection, we have identified p55 as the cellular protein hnRNP H, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that are involved in the processing and transport of pre-mRNAs (23). Based on reported activities for hnRNP H, this protein may be involved in NRS-mediated splicing inhibition and/or polyadenylation of viral RNAs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Construction and in Vitro RNA Transcription-- RSV DNA fragments were obtained from the Prague C strain (24), and sequence coordinates are as described by Schwartz et al. (25). Plasmids p3ZKXMS, p3ZMS3', p3ZBB, p3ZBB5', and p3ZBB3' were described previously (10, 11). Additional NRS fragments (nucleotides 701-753, 754-797, 701-770, 740-797, and 740-770) were generated by polymerase chain reaction using primers containing KpnI and XbaI sites and subcloned into these sites in pGEM-3Z (Promega). Primer sequences are available on request. The sequence of all constructs was confirmed by DNA sequencing.

Plasmids were linearized with XbaI (with the exception of p3ZBB, which was linearized with BamHI) and transcribed in vitro using T7 RNA polymerase and [³²P]ATP (10, 26). Control RNA was transcribed from PvuII-digested pGEM-4Z (Promega). All RNAs were gel-purified before use.

Plasmid p3Z(740-797)x2 was generated by first digesting p3Z(740-797) with KpnI, blunting with T4 DNA polymerase, and then digesting with HindIII. The excised fragment was then subcloned into HincII/HindIII-digested p3Z(740-797) to generate the final construct. To produce RNA for affinity selections, this plasmid was linearized with SalI, and large scale in vitro transcription was performed using T7 RNA polymerase (27). Control RNAs for affinity selection were transcribed from PvuII-digested pGEM-4Z. All RNAs were trace-labeled with [³²P]ATP to monitor yield and coupling to agarose beads (described below).

RNA Affinity Selections-- 250 pmol of RNA was covalently coupled to agarose beads as described (28). Affinity selections were performed by adding the RNA-agarose beads to 325 µl of reactions containing ~2.65 mg of a 0-65% ammonium sulfate (AS65) fraction of HeLa total cell extract in 12.7 mM Hepes (pH 7.9), 38.5 mM KCl, 20 mM creatine phosphate, 0.4 mM ATP, 3 mM MgCl₂, 0.16 mM dithiothreitol, 1.9% glycerol, and 0.08 mM EDTA. After the reactions were incubated at 30 °C for 20 min, the beads were collected, washed four times with 1 ml of 20 mM Hepes (pH 7.9), 100 mM KCl, 5% glycerol, 0.2 mM EDTA, and 4 mM MgCl₂ at 4 °C, and resuspended in 30 µl of sample loading buffer. Bound proteins were eluted at 90 °C for 5 min, and half of the supernatant was analyzed on a 10% SDS-polyacrylamide gel. The gel was Coomassie Blue-stained, and bands of interest were excised directly from the gel and subjected to MALDI-TOF analysis (29) by John Leszyk at the Protein Microsequencing and Proteomic Mass Spectrometry Lab at the University of Massachusetts Medical School.

Extract Preparation and Purification of Recombinant Proteins-- HeLa S3 cells were grown in spinner flasks, and nuclear extracts were produced as described previously (30). The AS65 fraction used (a gift from L. McNally, Medical College of Wisconsin) was produced as described for the preparation of total HeLa SR proteins (31) and was dialyzed against 20 mM Hepes (pH 7.9), 100 mM KCl, 5% glycerol, and 0.2 mM EDTA before use.

The Q fraction was produced as follows. AS65 extract was adjusted to 2 M NaCl and centrifuged at 213,000 × g for 20 min at 4 °C to extract p55 cross-linking activity from endogenous complexes. The supernatant was collected, concentrated using a centrifugal filter device (Centriprep YM-30, Millipore), and separated on a Sephacryl S-200 HR (Amersham Pharmacia Biotech) gel filtration column. Fractions with peak cross-linking activity were pooled, loaded on a prepacked High Q ion exchange column (Econo-Pac, Bio-Rad), and eluted using a salt gradient from 100 to 600 mM KCl. The resulting fractions containing an ~55-kDa cross-linking activity were pooled, diluted with 20 mM Hepes (pH 7.9), 5% glycerol, and 0.2 mM EDTA to approximately 100 mM KCl, and concentrated by centrifugation (Centriplus YM-30, Millipore) before use.

To produce recombinant hnRNP H, BL21(DE3)pLysS bacterial cells (Novagen) were transfected with a pET15b vector containing the hnRNP H cDNA (generously provided by D. Black, University of California, Los Angeles), and the protein was purified from inclusion bodies using Ni²⁺ affinity chromatography (His-Bind Resin, Novagen) as described (32). The recombinant protein was eluted in 20 mM Hepes (pH 7.9), 100 mM KCl, 5% glycerol, and 0.2 mM EDTA containing 250 mM imidazole and dialyzed against 20 mM Hepes (pH 7.9), 100 mM KCl, 5% glycerol, and 0.2 mM EDTA. The protein was concentrated using centrifugal filter devices (Centriplus YM-30 and Centricon YM-30, Millipore) according to the manufacturer's instructions. Aliquots were stored at 80 °C until use. All protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad).

UV Cross-linking-- High specific activity RNAs (10⁵-10⁶ dpm) were incubated in 25-µl reaction mixtures under modified in vitro splicing conditions (13 mM Hepes (pH 7.9), 100 mM KCl, 20 mM creatine phosphate, 0.4 mM ATP, 3 mM MgCl₂, 0.16 mM dithiothreitol, 2% glycerol, 0.08 mM EDTA, and 0.4% mammalian cell and tissue extract Protease Inhibitor Cocktail (Sigma) with 25 µg of nuclear extract or 2-40 pmol of recombinant hnRNP H for 10-30 min at 30 °C. The samples were spotted onto parafilm at 4 °C and exposed to UV light (254 nm) for 20 min at a distance of ~4 cm, transferred to microcentrifuge tubes containing 20-25 µg of RNase A, and incubated at 37 °C for 15 min. Sample loading buffer was then added, and samples were separated on a 10 or 12% SDS-polyacrylamide gel. Gels were Coomassie Blue-stained to visualize size standards, dried, and analyzed by autoradiography or with a PhosphorImager (Molecular Dynamic Storm 860).

Immunoprecipitations-- Protein A-Sepharose beads (Amersham Pharmacia Biotech) were washed in phosphate-buffered saline and suspended as a 50% slurry. 10 µl of slurry was incubated in 500 µl of IP buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Triton X-100) in the presence or absence of 1 µl of antiserum and rotated at 4 °C for 3 h. The beads were washed three times with 500 µl of IP buffer and resuspended in 100 µl of IP buffer. UV cross-linking was performed as described above, and following RNase treatment, the beads were added to the samples, rotated at 4 °C for 1 h, washed three times with 500 µl of IP buffer, and suspended in 40 µl of sample loading buffer. Bound proteins were eluted by boiling for 5 min, and the supernatants were separated on a 10% SDS-polyacrylamide gel and visualized as described above.

Electrophoretic Mobility Shift Assays-- Labeled RNA (2.5 fmol) was incubated at 30 °C for 30 min in 25 µl of 13 mM Hepes (pH 7.9), 70 mM KCl, 20 mM creatine phosphate, 0.4 mM ATP, 3 mM MgCl₂, 0.16 mM dithiothreitol, 2% glycerol, 0.08 mM EDTA, and 1 µg of tRNA in the presence or absence of 5 µg of nuclear extract or 10-40 pmol of recombinant hnRNP H. Heparin (Sigma) was then added to a concentration of 5 mg/ml, and the reaction was incubated at 30 °C for an additional 10 min. The completed reaction was separated on a 4% polyacrylamide (29:1) nondenaturing gel. After electrophoresis, the gel was dried and visualized by autoradiography or with a PhosphorImager. For supershifts, assays were performed as described above except that the reactions were initially incubated at 30 °C for 15 min, 1 µl of antiserum was added, and the reactions were incubated an additional 15 min at 30 °C. Antisera against hnRNP H and hnRNP F were generously provided by D. Black. Control antisera (glutathione S-transferase-myelin-basic protein and affinity-purified Exo U) were gifts from R. Fritz and D. Frank, Medical College of Wisconsin.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Localization of the p55 Binding Site-- Previous studies established that a number of protein factors in HeLa nuclear extract bind specifically to the 5' portion of the NRS (nucleotides 701-797) (Fig. 1) (10). Although most of these proteins are implicated as being SR proteins (10), the identity of a 55-kDa protein that is biochemically distinct from the SR proteins has remained unclear. The binding site for the SR proteins was localized to nucleotides 715-748 of NRS5' (Fig. 1) (10), but because deletion of this region does not fully eliminate NRS activity (7, 20), other factor(s) that binds within nucleotides 748-797 may contribute to splicing inhibition. Although it was shown that p55 binds NRS5', its precise binding site was not determined. To identify the p55 binding site we performed UV cross-linking in HeLa nuclear extract with a panel of NRS RNA variants (Fig. 2A). A number of nonspecific RNA-binding proteins were detected when cross-linking was performed with vector RNA (Fig. 2B, lane 1). Consistent with previous studies (10), a specific cross-link of 55 kDa was observed using the full-length NRS and NRS5' but not with NRS3' or vector RNA (Fig. 2B, lanes 1-4). Using the NRS5' variants, we mapped the minimal p55 binding site to nucleotides 740-770, indicating that p55 binds primarily downstream of the SR proteins (Fig. 2B, lanes 5-9). In addition, this region maps within a 42-nucleotide region of the NRS (nt 735-776) previously shown to be functionally important for proper splicing control in a recombinant avian virus (21), further implicating p55 as a potentially important factor in NRS activity. Based on these observations, we sought to purify and identify p55.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   The Rous sarcoma virus genome and the negative regulator of splicing. A, the RSV genome is shown above with the relative positions of the long terminal repeats (LTR) and the gag, pol, env, and src genes indicated (not to scale). Alternative splicing is shown by lines between the 5' and 3' splice sites (ss). The location of the NRS is shown within the gag gene and expanded below including sequence coordinates. The overlapping binding sites for the U1 and U11 small nuclear ribonucleoproteins (snRNPs) in the 3' portion of the NRS (nt 913-926) are denoted by a black box. The lightly shaded region indicates the 5' portion of the NRS, and the site where SR proteins were previously shown to bind (nt 715-748) is dark shaded. B, a partial sequence of the purine-rich 5' region of the NRS is shown, and the binding site for p55, as determined in this study, is indicated by brackets.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Localization of the p55 binding site to nucleotides 740-770 of the NRS. A, schematic diagram of the RNAs used to localize the p55 binding site within the NRS. 4Z (open box) refers to vector sequences transcribed from pGEM-4Z, and NRS RNAs are shaded. Numbers denote the nucleotides encompassed by the RNAs. The coordinates of NRS, NRS5', and NRS3' RNAs are shown in parentheses. The minimal p55 binding site is indicated by a black box. B, UV cross-linking was performed as described under "Experimental Procedures" in 25 µg of HeLa nuclear extract under modified in vitro splicing conditions with the indicated [32P]RNAs (lanes refer to constructs in A). Cross-linked proteins were resolved on a 12% SDS-polyacrylamide gel and visualized with a PhosphorImager. Numbers on the left indicate the positions of 66- and 45-kDa size standards. The relative position of the p55 cross-link is indicated by the bracket. The figure was generated from representative phosphorimages using Adobe Photoshop 5.0.2 and Deneba Canvas 6 on a Power Macintosh 9600/233.

Identification of p55 as the Cellular Protein hnRNP H-- Because previous data indicated that p55 was present in both nuclear and cytoplasmic HeLa cell extracts (10), we chose to use HeLa total cell extracts as a starting material and follow p55 purification by UV cross-linking. A 0-65% ammonium sulfate precipitation was performed to remove SR proteins, which might interfere with or obscure p55 isolation. Initially this AS65 extract was fractionated biochemically (see "Experimental Procedures"), and enrichment of an ~55-kDa cross-link was observed (data not shown). The predominant protein in this size range was identified by mass spectrometry as a 48-kDa proteolytic fragment of nucleolin (data not shown). Importantly, this protein did not show specific binding to the NRS (data not shown), and since it has not been suggested to participate in any aspect of pre-mRNA splicing (33), we concluded that this protein was unlikely to be biologically relevant to NRS function.

Because it appeared that the specific p55 cross-link was lost in the above purification scheme, we next performed RNA affinity selection directly from AS65 HeLa extracts (Fig. 3). Several proteins were selected with vector RNA, including one that comigrated with the 48-kDa nucleolin fragment (Fig. 3, lane 2). This and other proteins were also detected using a p55-specific NRS RNA, but an additional specific protein of ~55 kDa was also selected (Fig. 3, lane 3). Because this protein was the correct size for p55, it was excised from the gel, digested with trypsin, and subjected to mass spectrometry. Nine of eleven identified masses corresponded to a single protein, and post-source decay analysis unambiguously confirmed this to be the cellular protein, hnRNP H (data not shown).

View larger version (112K): [in this window] [in a new window]   Fig. 3.   hnRNP H specifically associates with the NRS5' region. RNA affinity selection was performed as described under "Experimental Procedures" using in vitro transcribed RNAs covalently coupled to agarose beads. The beads were incubated in HeLa AS65 extract and washed extensively. Bound proteins were eluted by boiling, resolved on a 10% SDS-polyacrylamide gel and visualized by Coomassie Blue staining. Lane 1 shows ~30 µg of AS65 extract prior to selection, whereas lanes 2 and 3 show the results of selections performed using nonspecific vector RNA or an NRS-specific RNA consisting of nt 740-797 arranged in tandem. Bands in the 50-55-kDa range were detected using both RNAs, although at varying intensities, with the exception of an NRS-specific 55-kDa band indicated by an arrow. This protein was excised from the gel and identified by mass spectrometry as hnRNP H. Numbers on the left indicate the position of size standards (lane M) in kDa. Gels were photographed using an Alphaimager 2000 documentation and analysis system (Alpha Innotech Corp.), and the figure was generated as described in Fig. 2.

Antibodies against hnRNP H Specifically Immunoprecipitate the p55 Cross-link from AS65-- It was predicted that if hnRNP H is p55, antibodies against hnRNP H should immunoprecipitate the p55 cross-link. To test this, NRS RNAs were incubated in either AS65 or a fraction (Q) enriched for nucleolin but lacking hnRNP H (data not shown), the reactions were UV cross-linked, and the samples were immunoprecipitated with hnRNP H antibodies. When NRS5' was used as the target RNA, antibodies against hnRNP H specifically immunoprecipitated a 55-kDa cross-link from AS65 but not from the Q fraction (Fig. 4, compare lanes 2 and 10). The precipitated cross-link exhibited the same mobility pattern as the cross-link seen in the extract (Fig. 4, lanes 1 and 2), and no cross-link was immunoprecipitated using control antibodies or Protein A-Sepharose beads alone (Fig. 4, lanes 3 and 4). Identical results were obtained when the minimal p55 binding site (nt 740-770) RNA was used (Fig. 4, lanes 5-8 and lanes 13-16). These results are consistent with hnRNP H being p55.

View larger version (78K): [in this window] [in a new window]   Fig. 4.   Anti-hnRNP H antiserum immunoprecipitates the p55 cross-link from HeLa AS65 extract. UV cross-linking was performed as described in Fig. 2 using RNAs corresponding to either NRS5' or the minimal p55 binding site (nt 740-770) in either HeLa AS65 extract or fractionated extract (Q fraction) lacking hnRNP H but enriched for nucleolin (see "Experimental Procedures"). Following cross-linking, samples were incubated with protein A-Sepharose beads (B) either alone or coupled to antibody (Ab). Antiserum against hnRNP H (H) or the bacterial protein Exo U (U) is indicated. Samples were resolved by SDS-polyacrylamide gel electrophoresis and visualized with a PhosphorImager. Numbers on the left indicate the position of size standards in kDa, and the position of the p55 cross-link is indicated by the bracket. The figure was generated as described in Fig. 2.

Antibodies against hnRNP H Supershift NRS5'-specific Complexes-- Previous studies showed that a specific complex forms on NRS5' in HeLa nuclear extract as determined by mobility shift assays and that a 55-kDa protein is a component of this complex (10). To provide evidence that this 55-kDa protein is hnRNP H, we performed electrophoretic mobility supershift assays. The specific complex was formed on NRS5' RNA in HeLa nuclear extract (Fig. 5, lanes 1 and 2) and could be supershifted with antibodies against hnRNP H but not with control antibodies (lanes 3-6). Identical results were obtained using the minimal p55 binding site (nt 740-770) (Fig. 5, lanes 10-15). Additionally, the supershift was detected with only anti-hnRNP H and nuclear extract and not with antiserum alone (Fig. 5, compare lanes 3 to lanes 7-9, and lane 12 to lanes 16-18). Furthermore, although antibodies against hnRNP H supershifted NRS complexes, antibodies against a closely related protein, hnRNP F, did not (Fig. 5, lanes 5 and 14). Taken together, these results indicate that hnRNP H is a component of the NRS5'-specific complex observed with mobility shift assays.

View larger version (87K): [in this window] [in a new window]   Fig. 5.   Complexes assembled on NRS5' are supershifted with antibodies against hnRNP H. Electrophoretic mobility shift assays were performed using either NRS5' or p55-specific (nt 740-770) RNAs. High specific activity RNAs (2.5 fmol) were incubated under in vitro splicing conditions in 5 µg of HeLa nuclear extract (NE), treated with heparin, resolved on a 4% polyacrylamide native gel, and visualized with a PhosphorImager. For supershifts, antisera (Ab) raised against hnRNP H (H), a related protein hnRNP F (F), or control proteins Exo U (U) and glutathione S-transferase-myelin-basic protein (G) were added after the binding reaction as described under "Experimental Procedures." The positions of free RNA, the NRS-specific shifted complex (arrow), and the corresponding supershifted complex (*) are indicated. Decreases in the relative amounts of shifted complex and free RNA are caused by nucleases present in the antisera (H, F, and G) as they are not seen with affinity-purified antibodies (U). The figures were generated as described for Fig. 2.

Recombinant hnRNP H Cross-links to the NRS at the p55 Binding Site-- To determine whether hnRNP H interacts directly with the NRS, recombinant hnRNP H was produced in bacteria and utilized in UV cross-linking assays. The recombinant hnRNP H was essentially homogeneous and reacted strongly with anti-hnRNP H antibody (data not shown). Little cross-linking was detected to vector RNA with up to 40 pmol of recombinant hnRNP H (Fig. 6A, lanes 1-3). In contrast, cross-linking to NRS5' was detected with as little as 2 pmol of recombinant protein, and the cross-link was intensified with increasing hnRNP H concentration (Fig. 6A, lanes 4-7), indicating that hnRNP H binds directly to NRS5'.

View larger version (69K): [in this window] [in a new window]   Fig. 6.   Recombinant hnRNP H cross-links to the NRS at the p55 binding site. A, UV cross-linking was performed with the indicated pmol of purified recombinant hnRNP H (H). Cross-linking conditions were identical to Fig. 2 with the exception that both RNase A and RNase T1 were used for digestion. B, cross-linking was performed in either 25 µg of nuclear extract (NE) or with 10 pmol of recombinant hnRNP H (H) using selected NRS RNAs described in Fig. 2 with the exception that NRS RNA was transcribed from p3ZKXMS (nt 701-1011). Conditions were identical to Fig. 2. Numbers on the left indicate the position of size standards in kDa, and the position of cross-linked hnRNP H is indicated. The figures were generated as described for Fig. 2.

It was further predicted that if hnRNP H is p55, cross-linking to the NRS should be dependent on the presence of the p55 binding site (as determined in Fig. 2). To test this, UV cross-linking was performed with recombinant hnRNP H and selected RNAs that were used in Fig. 2 to determine the p55 binding site. Consistent with the results above, cross-linking of hnRNP H was seen with full-length NRS and NRS5' but not with vector RNA (Fig. 6B, lanes 1-6). Some cross-linking was detected using NRS3' (Fig. 6B, lanes 7 and 8), indicating a degree of promiscuity in the binding of purified hnRNP H. Cross-linking was not observed with a segment of NRS5' that does not cross-link p55 (nt 701-753) (Fig. 6B, lanes 9 and 10). In contrast, cross-linking was observed using a region of NRS5' which showed slight p55 cross-linking (nt 754-797) (Fig. 6B, lanes 11 and 12) and was clearly detected using the minimal p55 binding site (Fig. 6B, lanes 13 and 14). These results show that hnRNP H cross-linking is dependent on the presence of the p55 binding site.

Recombinant hnRNP H Induces a Mobility Shift of NRS RNAs Containing the p55 Binding Site-- Because recombinant hnRNP H binds directly to the NRS, we predicted that it would shift NRS RNAs in an electrophoretic mobility shift assay. No shift was observed with vector RNA and up to 40 pmol of recombinant hnRNP H (Fig. 7A, lanes 3-6), whereas NRS5' RNA showed an observable shift with as little as 20 pmol (Fig. 7A, lanes 9-12). To confirm that the binding of hnRNP H to the NRS requires the p55 binding site, mobility shifts were performed using selected RNAs described in Fig. 2. As expected, shifts were detected using full-length NRS and NRS 5' but not vector RNA (Fig. 7B, lanes 3, 7, and 12). The presence of secondary structure in NRS3' caused a portion of this RNA to migrate similarly to the hnRNP H-dependent complex, but this pattern was unchanged in the presence or absence of recombinant hnRNP H (Fig. 7B, compare lanes 14 and 17) indicating the protein does not form a complex with this RNA. In addition, no shift was detected using nucleotides 701-753, which do not cross-link p55 or hnRNP H (Fig. 7B, lane 21), but a shift was observed with NRS5' segments that do cross-link hnRNP H (Fig. 7B, lanes 25 and 30). Taken together, these results confirm that hnRNP H binds directly to the p55 binding site.

View larger version (68K): [in this window] [in a new window]   Fig. 7.   Recombinant hnRNP H causes a mobility shift of NRS RNAs, which is dependent on the p55 binding site. A, the indicated amounts of hnRNP H were incubated with 2.5 fmol of either 4Z RNA or NRS5' RNA, and electrophoretic mobility shift assays were performed as described in Fig. 5. The positions of free RNA and the shifted complex seen with NRS5' are indicated. B, electrophoretic mobility shift assays were performed as in Fig. 5 using either 5 µg of HeLa nuclear extract (NE) or 30 pmol of recombinant hnRNP H (H) in the presence or absence of hnRNP H antiserum (Ab). RNAs were as described in Fig. 2 with the exception that NRS3' RNA was transcribed from p3ZBB3' (nt 798-932). The positions of free RNA, shifted complexes, and the corresponding supershifted complexes (*) are indicated. Note that lanes 1-18 and 19-31 are derived from separate gels. The figures were generated as described for Fig. 2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Despite its roles in splicing inhibition, a number of essential splicing factors have been shown to interact with the NRS, including the U1 and U11 snRNPs and several SR proteins. In addition to these factors, a previously unidentified 55 kDa protein (p55) was shown to interact with the NRS by UV cross-linking (10). We became interested in this protein because of its specific binding the 5' portion of the NRS and because its biochemical properties suggested it was not an SR protein (10). We mapped the binding site for p55 to nucleotides 740-770 of the NRS, downstream of the primary SR protein binding site (nt 715-748). Because this region can support a degree of NRS activity in the absence of the predominant SR protein binding site (7, 20) and is required for maximal NRS-mediated splicing inhibition (21), we sought to identify and characterize this protein.

Through biochemical purification, we identified two proteins of approximately 50-55 kDa that cross-link to nucleotides 740-770 in HeLa cell extracts, only one of which was sequence-specific (data not shown). The nonspecific protein was determined to be a previously described proteolytic fragment of nucleolin containing the RNA binding domains (34). To purify the protein responsible for the specific p55 cross-link, we used RNA affinity and identified it as the cellular factor hnRNP H by mass spectrometry. Three observations support the view that hnRNP H is p55. First, antibodies against hnRNP H immunoprecipitated the p55 cross-link from HeLa cellular extracts. Second, hnRNP H antibodies supershifted complexes that form on NRS5' in HeLa nuclear extract. Third, recombinant hnRNP H assembled a similar complex and could be cross-linked only to NRS RNAs that contained its binding site, indicating that hnRNP H binds the NRS directly. Furthermore, these hnRNP H-induced complexes migrated identically to those observed in nuclear extract (Fig. 7B, compare lanes 7, 12, 25, and 30 to lanes 5, 10, 23, and 28) suggesting that hnRNP H is solely responsible for these shifts. Collectively, these results indicate that hnRNP H is p55.

hnRNP H is a member of a large group of proteins known as heterogeneous nuclear ribonucleoproteins that bind to RNA polymerase II transcripts and contribute to their maturation to mRNAs (23). The interaction of an hnRNP protein with a viral RNA is not unique. Recently, members of the hnRNP A/B family of proteins were found to bind to an exonic splicing silencer sequence in exon 2 of the HIV-1 tat RNA and shown to be required for splicing inhibition (28). The fact that hnRNP H may be involved in NRS activity might reflect a common role for hnRNPs in viral RNA splicing inhibition. However, the requirement for additional sequences within the NRS (i.e. the U1 site) highlights an important difference between the mechanisms of NRS and HIV tat splicing inhibition.

Although our data do not address function, the significance of hnRNP H in NRS function is suggested by studies of the EU8 recombinant avian leukosis virus (21). This virus, which induces rapid onset B-cell lymphomas through insertional activation of the c-myb gene (22), carries a gag deletion that impairs NRS function (21). This deletion removes nucleotides 735-776 of the NRS, which encompass the hnRNP H binding site. It is tempting to speculate that the loss of hnRNP H binding may result in the NRS defect seen in the EU8 virus. Although we cannot rule out the possibility that other proteins may functionally interact with this region, the specificity of hnRNP H makes it a prime candidate.

In contrast to previously identified NRS binding factors (SR proteins and snRNPs), which have defined roles in pre-mRNA splicing, the role of hnRNP H in cellular RNA metabolism is not clear. Recent reports have implicated hnRNP H in the regulated splicing of at least two genes. Chou et al. (32) have shown hnRNP H to be a component of a neuron-specific enhancer complex that stimulates splicing of the c-src N1 exon. hnRNP H has also been shown to bind an exonic splicing silencer element in exon 7 of the rat -tropomyosin gene that represses splicing of this exon in nonmuscle cells (35). However, immunodepletion had no effect on the splicing of either an adenoviral (32) or -globin (35) pre-mRNA, suggesting that hnRNP H is not a general splicing factor. Although hnRNP H is required in both these cases, it is unclear how this protein precisely contributes to either positive or negative splicing regulation. In addition, although these are both examples of tissue-specific alternative splicing, hnRNP H does not exhibit tissue-specific expression in humans (36), rats (37), or mice (35), suggesting that these effects may be mediated via protein-protein interactions. Determination of additional factors that functionally interact with hnRNP H will be important in understanding how this protein acts to modulate pre-mRNA splicing.

hnRNP H is a member of a subfamily of distinct but structurally similar hnRNP proteins that include hnRNP H, hnRNP H', and hnRNP F (36, 38). hnRNP F is 78% identical to hnRNP H (36) and is also a functional component of the c-src splicing enhancer complex (39). Although hnRNP H and hnRNP F may bind as a heterodimer to the c-src enhancer (32), hnRNP F was not detected in hnRNP H-containing NRS5' complexes by supershift assays, suggesting that hnRNP F is not involved in NRS function. hnRNP H', which is 96% identical to hnRNP H, binds a G-rich element downstream of the core SV40 late polyadenylation signal and stimulates 3' end processing (40). Despite their extreme homology, hnRNP H' was not detected by RNA affinity selection with NRS-specific RNAs (data not shown). However, because of the high homology between these two proteins it is unlikely that they possess distinct binding specificities, and the absence of hnRNP H' may reflect a difference in the relative levels of hnRNP H and hnRNP H' in HeLa cells.

How might hnRNP H function in NRS-mediated splicing inhibition? Recent evidence indicates that NRS5' can function as a potent splicing enhancer, which is consistent with the binding of SR proteins to NRS5' (20). However, enhancer activity is not restricted to the primary SR binding region (nt 715-748) and is diffusely localized throughout NRS5' (20). This suggests that hnRNP H may be capable of mediating splicing enhancer activity or augmenting other enhancer activities, consistent with its role in the neural c-src splicing enhancer complex (32). An alternative role for hnRNP H is suggested by the observations that hnRNP H' is involved in pre-mRNA 3' end processing (40). Based on the high identity between hnRNP H and hnRNP H', it is likely that the two proteins act similarly in vivo. As discussed previously, the EU8 virus exhibits increased readthrough of viral RNAs into the downstream c-myb gene (22). This readthrough effect is likely caused by the decreased efficiency of viral RNA 3' end processing, which is observed upon deletion of the NRS (41). The loss of the hnRNP H binding site in EU8 suggests a role in polyadenylation. Additionally, because deletion of the hnRNP H binding site appears to lead to both splicing and polyadenylation defects, it is possible that hnRNP H may participate in the coordination of the splicing and polyadenylation machinery on the viral RNAs. We are currently conducting experiments to explore these possibilities to more clearly define the role of hnRNP H in NRS-mediated splicing inhibition.

	ACKNOWLEDGEMENTS

We thank Doug Black and Min-Yuan Chou for the recombinant hnRNP H plasmid and the hnRNP H and hnRNP F antibodies and Dara Frank and Robert Fritz for control antibodies. We thank John Leszyk for mass spectrometry analysis of proteins. We thank Lisa McNally for the AS65 extract and members of the McNally laboratory for critical review of the manuscript.

	FOOTNOTES

^* This research was supported by Public Health Service Grant R01 CA78709 from the National Cancer Institute.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.

To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8749; Fax: 414-456-6535; E-mail: mtm@mcw.edu.

Published, JBC Papers in Press, August 8, 2000, DOI 10.1074/jbc.M005000200

	ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; RSV, Rous sarcoma virus; NRS, negative regulator of splicing; snRNP, small nuclear ribonucleoprotein; hnRNP, heterogeneous nuclear ribonucleoprotein; nt, nucleotide(s); SR, serine-arginine rich; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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