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From the Departments of * Medicine and
Pediatrics, Harbor-UCLA Medical Center and
Research and Education Institute, Torrance, California;
Department of Immunology and Microbiology,
Wayne State University School of Medicine, Detroit, Michigan; and
Institute of Cancer Research, College of
Physicians and Surgeons of Columbia University, New York, New York.
| Correspondence to: Wael A. Salameh, MD, Division of Endocrinology, Harbor-UCLA Medical Center, Box 446, 1000 West Carson St, Torrance, CA 90509 (e-mail: wsalameh{at}gcrc.rei.edu). |
| Received for publication July 11, 2002; accepted for publication October 25, 2002. |
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Abstract |
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Key words: Testis, spermatogenesis, meiosis, RNA processing, RNA-binding protein
We have previously cloned a putative pre-mRNA splicing factor designated RBM7 (RNA-binding motif protein 7; Guo et al, in preparation). It is highly conserved, and its homologs are found in many species. Its RNA-binding domain is highly similar to that of splicing factors such as spliceosome-associated protein 49 (SAP49), a component of splicing factor 3b (SF3b). RBM7 bares only a weak homology within its RNA-binding domain to RBMY, but it has stronger homology in this domain to other GenBank sequences (RBM11 and RBM4) grouped in the RBM family, but with a yet-unidentified function. Aside from mapping human RBM7 to chromosome 11, we have also shown its RNA to be expressed in all tissues tested, with a unique shorter transcript expressed only in the testes. Despite its expression in a wide variety of tissues, in the rat, Rbm7 was preferentially expressed in discrete cell populations within the brain. This raised the possibility that whereas RBM7 may not be important for constitutive mRNA splicing, it may enhance the splicing of a group of mRNAs in a cell-specific manner or in a certain developmental process, such as spermatogenesis. In this paper, we detail the testicular expression of RBM7 at both the mRNA and protein levels and demonstrate that RBM7 interacts with 2 essential splicing factors, SAP145 and SRp20.
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Materials and Methods |
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26 kd) production. For in vitro
translation (IVT), the NotI insert of a full-length SAP145
clone isolated from the 2-hybrid screen was subcloned into pcDNA3 (Invitrogen,
Carlsbad, Calif). To subclone SRp20 ORF, we used reverse
transcriptase–polymerase chain re-action to amplify a fragment from
mouse testis cDNA using primers 5'-TGAGCTTGGGCCTTTTGAAC-3'
(nucleotides 30–49) and 5'-CAATTTCCGCCAGTTGCTTT-3'
(nucleotides 909–890, GenBank accession number X53824), and cloned it
into pCR3.1 (Invitrogen).
Tissue Preparation
Animal handling protocols were approved by the institutional animal care
and review committee. Adult Sprague-Dawley rats were obtained from B&K
Universal (Fremont, Calif). C57/Bl6 mice were bred in-house. After the animals
were killed, testes were either fixed in Bouins solution via intracardiac
whole-body perfusion, or snap-frozen for protein extraction. Testicular
ly-sates were prepared according to the Research Applications man-ual
from Santa Cruz Technology (Santa Cruz, Calif). Proteins were solubilized in
RIPA buffer (1x phosphate-buffered saline [PBS] supplemented with 1%
NP-40, 0.5% sodium deoxycho-late, 0.1% sodium dodecyl sulphate [SDS], 1x
complete protease inhibitors [Roche Biochemicals, Indianapolis, Ind], 10 mg/mL
sodium orthovanadate, and fresh 10 mg/mL phenylmethyl-sulphonyl fluoride).
RNA In Situ Hybridization
Sense and antisense riboprobes were generated by incubation of
SacI- or SalI-linearized pBluescript templates with
digoxigenin (DIG)-labeled uridine 5'-triphosphate in the
presence of T7 (sense) or T3 (antisense) RNA polymerases according to the
manufacturer's recommendations (Roche Biochemicals). RNA in situ hybridization
was carried out on paraffin-embedded, 5-µm-thick testis transverse sections
according to an established pro-tocol
(Millar et al, 1993), except
for the omission of dithiothre-itol (DTT) in the prehybridization buffer and
RNase A in the posthybridization washes; in addition, the hybridization
temper-ature was 46.5°C. The sections were counterstained with nuclear
fast red for 10 minutes and photomicrographed with a camera mounted on an
Olympus BH2 light microscope (Olympus Op-tical, Tokyo, Japan).
Antibodies, Immunoprecipitation, and Immunoblots
An antigenic peptide, which was not similar to other proteins in the
databases, was synthesized. Its sequence was
[Cys202]-SHPY-LADRHYSREQRYSDHGSD and corresponds to mouse RBM7
amino acids 203–224. Affinity-purified anti-RBM7 peptide rab-bit
polyclonal type G immunoglobulins (IgGs) were then pro-duced (Bethyl Labs,
Montgomery, Tex). Antibody specificity was determined by immunoprecipitation.
[35S]-labeled RBM7 was in vitro–translated in a T7
quick-coupled transcription/trans-lation system (Promega, Madison, Wis) using
pBSmRBM7 as the template. A 5-µL aliquot of the mixture was incubated on
ice for 1 hour with 5 µL of either the antibody or preimmune serum and 90
µL of 0.02 M Tris-buffered saline with 0.1% Tween-20. An equal volume of
10% protein-A-agarose (Bio-Rad, Hercules, Calif) was added and incubated at
4°C for 1 hour with end-to-end rocking. The immunocomplex was analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE). The signals were detected by
phosphor imaging (Molecular Imager FX; Bio-Rad). Tissue blotting was performed
as previously described (Yamamoto et al,
2000). Primary antibodies were diluted 1:6000. For preabsorption,
the same amount of primary antibody was preincubated for 30 minutes with
5x molar excess of the neutralizing peptide to probe a duplicate
blot.
Protein Isoform Detection by Electrospray Ion Trap Mass
Spectrometry
Mouse testicular lysate was immunoprecipitated by the anti- RBM7 antibody
overnight and the immunocomplex was resolved by SDS-PAGE and subjected to zinc
staining (Bio-Rad). Protein bands (35 kd and 45 kd) were excised and
electroeluted, and the residual SDS was removed by organic extraction
(Hwang et al, 1996). Samples
were reconstituted in 50% methanol:50% water with 1% glacial acetic acid and
analyzed in the positive, full scan data acquisition mode using a Finnegan LCQ
Deca msn (Palo Alto, Calif) mass spectrometer. The instrument is
equipped with an electrospray ionization source tandem to the ion focusing
(quadrupole)–ion trap–ion detector electron multiplier axis.
During continuous sample injection, the instrument acquired data in the mass
range of 500.00–2000.00 (m/z) in full scan mode. The acquired
spectra were analyzed using Xcalibur BIOMASS Deconvolution settings in the
automatic mass-tracking mode in order to determine the mass unit of proteins
using their acquired mass/charge ratios.
Immunohistochemistry
We followed the protocol of the Unitect rabbit immunohistochemistry system
(Oncogene Research Products, Cambridge, Mass). Primary antibody dilution was
1:400 (4 µg/mL in PBS supplemented with 1% bovine serum albumin).
Equivalent concentrations of normal rabbit IgG were used as a negative
control. A second negative control consisted of prior incubation of the
primary antibody with an excess of neutralizing peptide.
Yeast Two-Hybrid Protein Interaction Screen
The bait vector pAS2/RBM7 that expressed the Gal4 DNA binding domain
(GBD)-RBM7 fusion protein was cotransformed with the human MATCHMAKER testis
cDNA library (107 transformants) into yeast strain y190.
Transformants were screened according to the instructions provided by the
manufacturer (Clontech). Putative positive clones were identified, isolated,
and reintroduced into the Y190 host strain with pAS2/RBM7 to test for reporter
activation. To eliminate potential false positives, each was transformed into
yeast either without cotransformation with pAS2/RBM7, or cotransformed with a
nonrelated vector, pAS2/mGSK3ß.
GST Fusion Protein Production and GST Pull-Down Assay
pETGEXCT and pGEX-5X-1/RBM7 were transformed into Escherichia coli
BL21(DE3) strain and induced for GST and GST-RBM7 fusion protein expression,
respectively. Although the recombinant GST was partially soluble in 1% Triton
X-100 sonicated bacterial lysate, GST-RBM7 was solubilized only by the
sarkosyl method (Frangioni and Neel,
1993). Triton X-100 was added to this sonicated lysate prior to
overnight incubation with glutathione Sepharose 4B beads (Amersham Pharmacia)
with end-to-end rotation at 4°C. Beads were washed 6 times with ice-cold
PBS and resuspended in storage buffer (50 mM Hepes pH 7.4, 0.15 M NaCl, 5 mM
DTT, 10% v/v glycerol) at -20°C. The purity of the GST-fusion proteins was
examined with Coomassie blue staining of a 12% gel as well as by
immunoblotting a duplicate gel using the anti-RBM7 peptide antibody. For the
GST pull-down assay, aliquots of RNase A–treated IVT products of
[35S]-labeled luciferase (2 µL), RBM7 (2 µL), SAP145 (15
µL), and SRp20 (10 µL) were mixed with 12.5 µL of 5% suspension of
GST or GST-RBM7 immobilized on glutathione-Sepharose beads. The binding buffer
contained 50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 2 mM ethylenediamine tetraacetic
acid (EDTA), 0.1% NP-40, and the complete protease inhibitor cocktail (Tsui et
al, 2000). To examine weak protein-protein interaction, a low salt
concentration (0.15 M) was substituted. After overnight incubation at 4°C,
beads were pelleted and washed thrice with the binding buffer. Bound proteins
were dissociated by boiling in reducing Laemmli buffer and analyzed by
SDS-PAGE followed by phosphor imaging.
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Results |
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RBM7 Protein Expression—Western Blotting
We generated and affinity-purified a rabbit polyclonal antibody against
amino acid residues 203–224 of the mouse RBM7. This antibody
specifically immunoprecipitated in vitro translated RBM7 proteins, shown in
Figure 2a as a 35-kd band and a
minor 27-kd band. Because the antibody was directed against a region near the
C-terminus the latter band probably resulted from internal initiation of
translation instead of premature termination. This antibody also strongly
reacted with a GST-RBM7 fusion protein, which appeared as a doublet at 60
kd (Figure 2b).
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We used the antibody to study the ontogeny of RBM7 expression during rodent
spermatogenesis, with an emphasis on meiosis. We chose 2 developmental stages
in particular: immediately before meiosis I initiation, and early to mid
pachytene stage. Thus, based on previous studies
(Bellve et al, 1977;
Yang et al, 1990;
Malkov et al, 1998), we used
mice at day 7 and day 12 postpartum (dpp) and rats at 12 dpp and 20 dpp.
Western blotting (Figure 2c) of
whole testis lysates detected at all time points revealed 2 bands: 50 kd
and 35 kd in mice and 50 kd and 40 kd in rats. The rat 50
kd form was consistently the more abundant form. Because all signals were
abolished on a duplicate blot probed with the antibody preincubated with an
excess of the antigenic peptide (data not shown), these bands appeared to be
specific.
Some nuclear proteins, heavily charged RNA-binding proteins in particular,
have a high degree of altered mobility on SDS-PAGE, often as a consequence of
posttranslational modification (Query et
al, 1989; Gozani et al,
1996; Klenova et al,
1997). Because the predicted size of RBM7 is 30 kd, we
explored the possibility that the 50-kd band is a posttranslationally modified
isoform of RBM7 that exhibits aberrant mobility. Electrospray ion mass
spectrometry (ESI/MS) has been used to resolve molecular weight differences
attributed to aberrant gel migration
(Iakoucheva et al, 2001). This
has been attributed to its ability to reduce many tertiary protein
conformational modifications, except those of phosphorylation
(Roepstorff, 2000). To confirm
that these 2 immunoreactive bands represented RBM7, RBM7 immunoprecipitates
from mouse testis extracts were resolved by SDS-PAGE. Plasmid 35 (p35) and p50
were then eluted and subjected to electrospray ion trap mass specrometry.
Results indicated that both bands had 28 213 mass units, and exhibited
identical fragmentation patterns (Figure 3a
and b) upon source fragmentation, wideband activation, or
collision-induced fragmentation. These results show that the 50-kd band
detected by the RBM7 antibody is an isoform with aberrant gel migration
probably due to one or more forms of posttranslational modification.
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RBM7 Protein Expression—Immunostaining
The anti-RBM7 antibody was used in standard avidin/biotin–mediated
immunoperoxidase detection of RBM7 on rat testicular sections. Neither
nonimmune IgG nor the antibody after immunoneutralization with the
oligopeptide antigen yielded any signals
(Figure 4a and b), attesting to
the specificity of this antibody. RBM7 immunoreactivity was nuclear in type B
spermatogonia (Figure 4d); and
in preleptotene (Figure 4e and
f), leptotene (Figure 4g and
h), zygotene (Figure
4i), and early (up to stage VI) pachytene
(Figure 4c, d, and i)
spermatocytes. The staining intensity appeared strongest in active
preleptotene, leptotene, and zygotene nuclei (stages VIII–XIII). RBM7
immunoreactivity then became cytoplasmic in mid and late pachytene
(Figure 4e through h), diplotene, and dividing spermatocytes
(Figure 4i), as well as in
spermatids at steps 1 through 14. Step 11 spermatids had rather strong
staining near the nuclear/acrosomal region
(Figure 4h). Occasional type A
spermatogonial staining could also be observed. Leydig cells and Sertoli cells
were not stained. Virtually identical expression results were obtained from
mouse testes (data not shown).
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These results, summarized in Figure 4j, suggest that, consistent with the pattern of mRNA expression (Figure 1i), RBM7 expression is spatiotemporally regulated during rat spermatogenesis. The expression commences at the time of meiosis initiation at type B spermatogonia and preleptotene spermatocytes, and the protein is predominantly nuclear. As the mRNA levels peak at the leptotene stage, RBM7 protein continues to accumulate in the nucleus. During early pachytene development, when the message level becomes nearly undetectable, RBM7 immunoreactivity decreases progressively. At mid pachytene, a second and coincidental up-regulation of Rbm7 mRNA and protein occurs, but interestingly, the protein is no longer nuclear. RBM7 immunoreactivity persists in the cytoplasm of late and dividing spermatocytes as well as postmeiotic spermatids prior to their cytoplasmic elimination, consistent with the decrease in mRNA levels in late pachytene cells. Interestingly, dividing spermatocytes show strong staining around the metaphase plates.
RBM7 Interacts With Splicing Factors SAP145 and SRp20
To understand the cellular function of RBM7, we used a yeast 2-hybrid
protein assay to identify proteins that could interact with RBM7. Screening
yielded 16 different clones, 12 of which carried varying lengths of the same
gene, the essential spliceosomal SF3b subunit, SAP145 (GenBank
accession number NM_006842), with a majority of them aligned to the carboxy
half of the protein. Closer examination identified a common region (amino
acids 579–750) that could contain the minimal interaction domain.
Another clone was the full-length SRp20 (GenBank accession number
NM_003017).
We confirmed the interactions in vitro by binding 35S-labeled IVT proteins (SAP145 and SRp20) to recombinant GST-RBM7 immobilized on glutathione-Sepharose beads (Figure 5). As shown, only GST-RBM7 was able to pull down SAP145 and SRp20, whereas neither GST nor GST-RBM7 captured the luciferase control. The sizes and patterns of these IVT products were in good agreement with published reports (Wang et al, 1998; Elliott et al, 2000; Bryant et al, 2001). Of note, the SRp20 experiment was performed at physiologic salt concentration (0.15 M NaCl). In a high salt condition (0.5 M NaCl), essentially no SRp20 binding was detected. To examine the possibility of RNA-mediated protein-protein interaction, aliquots of IVT products were treated with RNase before the binding assay. There was no change in the amount of SAP145 recovered after RNase treatment, suggesting that RBM7 and SAP145 directly associate. However, compared with the input, much less SRp20 was retained by RBM7 and this interaction was sensitive to RN-ase treatment. Thus, SRp20 specifically but weakly associates with RBM7, and this interaction is enhanced by the presence of RNA.
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Discussion |
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The idea that RBM7 may be a splicing factor was initially based on its homology with other splicing factors, especially SAP49. Also known as splicing factor 3b sub-unit 4, SAP49 is essential in the Caenorhabditis elegans worm (Fujita et al, 1998) and yeast (Saccharomyces cerevisiae) (Igel et al, 1998). It directly and stably interacts with SF3b subunit 2 (SAP145) and functions to tether U2 small nuclear ribonucleoprotein particle (snRNP) with pre-mRNA at the branch site during spliceosome assembly (Champion-Arnaud and Reed, 1994). This interaction between SAPs 49 and 145 is conserved for their respective essential yeast homologues, HSH49 and CUS1 (Wells et al, 1996). The spliceosome, the complex where the splicing reaction takes place, contains the pre-mRNA, several snRNPs, and a number of other splicing factors that enable splice-site selection and catalysis of the splicing reaction (Adams et al, 1996; Chabot, 1996; Will and Luhrmann, 1997). Among the non-snRNP splicing factors are a group of SR proteins that act as splicing activators through their facilitation of the recognition, selection, and pairing of splice sites (Will and Luhrmann, 1997; Tacke and Manley, 1999).
Our earlier results showed that RBM7 could promote pre-mRNA splicing (Guo et al, in preparation). However, the luciferase reporter assay therein does not yield clues as to how RBM7 promotes the reporter expression. It is conceivable that RBM7 may increase splicing efficiency via direct interaction with other splicing factors. Alternatively, it is also possible that RBM7 may enhance the nuclear export and stability of spliced transcripts, which have been shown to be coupled with splicing (Kim and Dreyfus, 2001). To explore these possibilities, we used yeast 2-hybrid screen and GST pull-down assay and established that RBM7 directly and stably associates with SAP145, and that it also interacts with SRp20. Both SAP145 and SRp20 are important splicing factors, thus this finding supports the theory that RBM7 participates in pre-mRNA splicing. There are several possible routes by which RBM7 may fulfill this role (Figure 6).
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Route 1— Because RBM7 and SAP49 are homologous in their RNA-binding domain and both interact with SAP145, RBM7 may substitute for SAP49 in situations in which SAP49 is down-regulated during differentiation and development (Ruiz-Lozano et al, 1997). It will be interesting to determine the testicular expression of SAP49 and compare it with that of RBM7. Bouck et al (1998) have suggested that splicing efficiency could be decreased through increased binding of constitutive splicing factors such as SAP49.
Route 2— In the context of route 1, an alternative function for RBM7 would be to modulate the interaction between SAP145 and SAP49 through its binding to SAP145, theoretically increasing the splicing efficiency.
Route 3— SR proteins generally enhance splicing by facilitating recognition and pairing of 5' and 3' splice sites (Will and Luhrmann, 1997; Tacke and Manley, 1999). If the interaction of SRp20 and SAP145 is concomitant, RBM7 may act to recruit SF3b to tether U2 snRNP to the branch site near the 3' splice site selected by SR proteins. Because some SR proteins, including SRp20, can facilitate selection of an otherwise unrecognized, weak 3' splice site, this model may also be relevant in alternative splicing.
SAP145, like many splicing proteins, exhibits punctate nuclear localization (Bryant et al, 2001). It remains to be determined whether RBM7 also localizes within these speckles, the preferential sites of splicing components. On the other hand, SRp20 shuttles between the nucleus and the cytoplasm and can promote mRNA export (Huang and Steitz, 2001). Because SRp20 is abundant in the testis (Ayane et al, 1991), studying the extent and site of SRp20 and RBM7 colocalization could shed light on the functional nature of their interaction. Therefore, a detailed analysis of its expression vis-à-vis RBM7 in the germ cells is warranted.
RBM is a testis-specific spermatogenesis factor coded by RBMY genes that may be deleted in some infertile men. It is localized in human testis in punctate regions of the nucleus of spermatogonia and spermatocytes and may have a role in pre-mRNA splicing (Elliott et al, 1998). It interacts with several splicing factors. These include testis-signal transduction and activation of RNA (T-STAR; Venables et al, 1999), which is a homolog of Src-associated mitotic cell protein of 68 kd (SAM68), which is implicated in RNA splicing and is negatively regulated by signal transduction during the cell cycle. RBM also interacts along with hnRNP G proteins and Tra2ß, which is the human homologue of Tra, a splicing factor that regulates sexual differentiation, spermatogenesis, and in fly courtship behavior (Venables et al, 2000). Finally, it interacts with a number of the SR family of splicing factors, chief among them, SRp20 (Elliott et al, 2000). The interaction between RBM and SRp20 occurs through the RRM and hinge regions of RBM and is not dependent on the RS domain, which is frequently used by SR proteins for interactions among each other or with other proteins (Elliott et al, 2000). We have not mapped the area of interaction between RBM7 and SRp20, but because the RS domain in SRp20 is free while it binds RBM, a concomitant interaction of all 3 factors cannot be ruled out. It is thus conceivable that these proteins act in a common pathway to regulate testicular meiotic splicing events.
The testicular expression of RBM7 at both the mRNA and protein levels appears to be spatiotemporally correlated. Our results establish that 1) Rbm7 is expressed in germ cells at the onset of meiosis initiation and throughout meiosis; and 2) RBM7 protein level follows that of its mRNA, but the protein itself relocates from the nucleus to the cytoplasm during the second round of mRNA up-regulation at mid pachytene phase. This relocation of RBM7 can be a means of separating this protein from other components of the splicing reaction. Alternatively, its translocation may suggest an additional function such as RNA transport. There is evidence that the localization, assembly, or substrate specificity of RNA-binding proteins can be altered by phosphorylation (Watanabe et al, 1997; Gu et al, 1998; Morales et al, 1998). SRp20 function is regulated through serine phosphorylation in its SR domain, which may affect its binding specificity to splicing enhancer sequences (Prasad et al, 1999) or its nuclear compartmentalization (Caceres et al, 1998). Although we have yet to determine the phosphorylation status of RBM7, the presence of several phosphorylation consensus sites near its C-terminus raises the possibility that similar mechanisms are operative in regulating its sub-cellular localization, its substrate specificity, and protein-protein interaction.
In summary, we propose that RBM7 is a cell-specific regulator of pre-mRNA splicing through its interaction with SAP145 and SRp20. The restricted mRNA expression and the differential protein subcellular localization in rodent germ cells indicate that RBM7 has a more specific role in meiosis entry and progression. It is conceivable that RBM7 and additional testis-specific RNA-binding proteins collaborate to regulate the splicing of specific pre-mRNA species that are important in the meiotic cell cycle. Elucidation of the mechanism of action of RBM7 and identification of its mRNA targets will remain the focus of future research.
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Acknowledgments |
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Footnotes |
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-RBM7 Ab) but not
by nonimmune rabbit IgG. (b) Expression and purification of recombinant
GST and GST-RBM7 fusion proteins in E. coli. Purified proteins were separated
on SDS-PAGE and stained with Coomassie blue (left panel). In parallel,
" or after incubation
with glutathione-Sepharose beads coated with GST alone (G) or GST-RBM7 (R).
Samples with or without RNase treatment were so marked for comparison (-RNase
vs +RNase). Luciferase served as positive control for IVT and negative control
for the pull-down. Assay was performed at 0.15 M NaCl for SRp20 samples,
whereas the rest were performed at 0.5 M NaCl.