|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles
Youichi Sato1, Shojiro Yano, Ashraf A. Ewis and Yutaka Nakahori
Department of Human Genetics and Public Health, Institute of Health Biosciences, Graduate School of Medicine, The University of Tokushima, Tokushima, Japan
The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.
Key words: interaction, nuclear speckle, RPL13a, RPS7, sex determination, SRY
Abbreviations: GST, glutathione S-transferase, HMG, high-mobility group, HP1, heterochromatin protein 1, KAP1, KRAB-associated protein 1, KRAB, Kruppel-associated box, NLS, nuclear localization signals, RP, ribosomal protein, RPS7, ribosomal protein S7, SOX9, SRY-related HMG box 9, SRY, sex-determining region on the Y chromosome, WT1, Wilms' tumour suppressor 1
1To whom correspondence should be addressed (email firstname.lastname@example.org).
SRY (sex-determining region on the Y chromosome) possesses a DNA-binding HMG (high-mobility group)-box domain and two NLS (nuclear localization signals) are located at the N and C terminals of the HMG domain (Südbeck and Scherer, 1997); therefore, it is suggested that SRY is a transcription factor. Recently, Sekido and Lovell-Badge (2008) showed that SRY, SF1 (steroidogenic factor 1) and SOX9 (SRY-related HMG box 9) activate the expression of SOX9 in a synergistic manner. To date, several proteins that interact with SRY have also been reported, e.g. WT1 (Wilms' tumour suppressor 1), importin β, calmodulin, KRAB (Kruppel-associated box) and β-catenin etc. WT1, which was identified as a tumour-suppressor gene of Wilms' tumour (Call et al., 1990; Gessler et al., 1990), binds to the HMG-box domain of SRY and activates transcription synergistically at the SRY binding site (Matsuzawa-Watanabe et al., 2003). Calmodulin and importin β bind to the N and C terminal NLS of SRY, respectively, and import SRY into the nucleus (Harley et al., 1996; Forwood et al., 2001; Harley et al., 2003; Sim et al., 2005; Hanover et al., 2009). As an SRY-interacting protein, Oh et al. (2005) reported a novel protein containing only a KRAB-O domain. KRAB-O interacts with the bridge region outside of the HMG box of SRY and KAP1 (KRAB-associated protein 1), and SRY associates indirectly with KAP1 and HP1 (heterochromatin protein 1). Therefore, it is suggested that SRY constructs form complexes with KRAB, KAP1 and HP1 and regulate an as yet undetermined target gene(s). It was also demonstrated that β-catenin interacts with the N- or the C-terminus part of the SRY protein (Bernard et al., 2008; Lau and Li, 2009). R-spondin 1 and Wnt4 mediate ovarian development through the canonical Wnt/β-catenin pathway (Tomizuka et al., 2008). SRY interacts with β-catenin in the nucleus and represses Wnt/β-catenin signalling and ovarian development, thereby switching on testis determination. As presented above, many proteins that interact with SRY have been discovered; however, the molecular mechanism by which the SRY induces testis development is still unclear. Therefore, some SRY-interacting proteins might interact with other cellular proteins to exert certain functions and roles in male switching development.
In this study, we investigated the localization and interactions of SRY protein by using a yeast two-hybrid system to screen a human testis cDNA library.
2. Materials and methods
2.1. Plasmid construction
To construct the bait for the two-hybrid screen, the entire open reading frame of human SRY was inserted into the pGBKT7 plasmid vector (Clontech). For the GST (glutathione S-transferase) pull-down assay, the entire coding region and deletion fragment of SRY were cloned into the pGEX6P-1 plasmid vector (Amersham Biosciences) using an EcoRI site. To obtain the truncated form of SRY lacking the HMG domain, 1–59 and 129–205 aa (amino acid) fragments were PCR-cloned with an artificial EcoRI site at the 5′ end and an XhoI site at the 3′, and an XhoI site at the 5′ end and an EcoRI site at the 3′ end flanked the product, respectively. The resulting EcoRI–EcoRI-flanked PCR products were ligated to the pGEX6P-1 plasmid vector. The entire coding regions of RPS7 and RPL13A were cloned into pGADT7 plasmid vector (Clontech) using an XhoI site. The GFP (green fluorescent protein)–SRY was constructed by ligation of the SRY with the EcoRI site of the pEGFP–C2 vector (Clontech). Myc–RPS7 and Myc–RPL13a were subcloned into the pCMV–Myc plasmid vector (Clontech) using an XhoI site (Sigma).
2.2. Yeast two hybrid assay
The bait construct, pGBKT7–SRY, was transformed into the GAL4AD (GAL4 activation domain)-fused human testis cDNA library strain AH109 (Clontech) according to the manufacturer's instructions, which was then plated on selected media (SD/-leucine/-tryptophan/-Ade/-histidine). Positive clones were sequenced and identified by a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi.).
2.3. GST pull-down assay
GST fusion plasmid constructs were transformed into DH5α, and the expression of recombinant proteins was induced by 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside). The cells were harvested; sonicated in PBS containing 10% glycerol, 1 mM DTT (dithiothreitol) and 0.5 mM PMSF and then 1% Triton-X was added. The cells were then incubated for 30 min at 4°C. GST fusion proteins were purified with glutathione sepharose 4B beads (Amersham Biosciences) according to the manufacturer's directions. 35S-labelled HA-RPS7 and HA-RPL13a were synthesized using the TNT Quick Coupled Transcription/Translation System (Promega).
GST fusion protein was incubated for 2 h at 4°C with 35S-labelled protein in LSAB buffer (100 mM NaCl, 100 mM Tris pH 8.0, 0.1% Nonidet P40). After being washed with the LSAB buffer, the interacting proteins were subjected to SDS/PAGE. The gels were then dried, and autoradiography was performed using Mac BAS1500 (Fuji film).
2.4. Immunofluorescence staining
COS1 cells were plated on chamber slides and cultured using Dulbeco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum in 5% CO2 at 37°C, before being transfected with 1 μg of GFP–SRY and Myc–RPS7 or Myc–RPL13a plasmids using the FuGENE6 transfection reagent (Roche) according to the manufacturer's method (RP is ribosomal protein). After 48 h, immunofluorescence staining was carried out according to the standard protocol. The primary antibody used was rabbit anti-Myc (1:50) (Sigma), and the secondary antibody used was rhodamine-conjugated goat anti-rabbit IgG (1:100) (Millipore). To confirm the location of nuclei, Hoechst 33258 (Dojindo) was used. The immunolabelled cells were mounted with Vectashield (Vector Laboratories) and examined by confocal laser scanning-light microscopy (Model ECLIPSE TE2000, Nikon).
3. Results and discussion
3.1. Identification of RPS7 and RPL13a as SRY interacting proteins
To search for proteins that interact with SRY, we screened a cDNA library from the human testis using a yeast two-hybrid system. We found that SRY interacts with many proteins. This result suggested that SRY interacts with many undetermined proteins, some of which may be involved in the development of testis or repress the development of the ovary. Among those positive clones, we selected 48 positive cDNA clones by random selection and sequenced them; consequently, two ribosomal proteins, S7 and L13a, were identified. These ribosomal protein cDNA clones did not encode the entire coding regions of S7 and L13a. So, we cloned the entire coding regions of RPS7 and RPL13a and confirmed that entire RPS7 and RPL13a interact with SRY, using a yeast two-hybrid system (Figure 1A). Furthermore, these interactions between SRY and RPS7 or RPL13a were further confirmed by a GST pull-down assay (Figure 1B). We propose that the interaction between SRY and RPS7 or RPL13a plays a role in testis determination by an unknown mechanism.
3.2. HMG-box of SRY is crucial for interaction with RPS7 and RPL13a
To elucidate the domain of SRY that interacts with RPS7 and RPL13a, we produced a truncated SRY (Figure 2A) and performed a GST pull-down assay using the truncated SRY as bait and the full length RPS7 and RPL13a as prey. RPS7 and RPL13a specifically interacted with GST-N+HMG, GST-HMG+C and GST-HMG, all of which contain the HMG box, but not with GST-N, GST-C or GST–ΔHMG, which lack the HMG box of SRY (Figure 2B). These results indicated that the HMG box of SRY is crucial for its interaction with RPS7 and RPL13a. The HMG-box domain of SRY is conserved between mammalian species (Whitfield et al., 1993), and many SRY point mutations causing XY sex reversal have been characterized in the HMG-box (reviewed in Harley et al., 2003). Some of the previously described SRY-interacting proteins were also found to bind to the HMG-box of SRY. Therefore, it is suggested that the HMG domain is essential for the molecular function of SRY in sex determination. Our results support and confirm the importance of the HMG-box of SRY.
Mammalian ribosomes consist of 4 RNA species and 79 ribosomal proteins (Wool, 1979). Hence, using a GST pull-down assay, we investigated the interaction of SRY with several randomly selected ribosomal proteins. SRY interacts with many, but not all, ribosomal proteins (data not shown).
RPS7 is reported to interact with p53-MDM2, and it binds to MDM2, stabilizes p53 protein and activates p53, suggesting that it could play a role in cancer prevention (Chen et al., 2007). Thus, RPS7 is a subunit of ribosomal proteins that has other important functions.
3.3. Cellular localization of the SRY, RPS7 and RPL13a
To analyse the intracellular localization of SRY, RPS7 and RPL13a, GFP–SRY and Myc–RPS7 or Myc–RPL13a plasmids were transiently transfected into COS1 cells. Using a confocal laser-scanning microscope, we found that RPS7 and RPL13a were mainly localized in the nucleolus and that SRY was mainly localized in the nucleus. However, some SRY molecules were co-localized in nuclear speckles together with RPS7 and/or RPL13a (Figure 3). This co-localization supports the proposition that SRY interacts with RPS7 and RPL13a in mammalian cells.
The ribosomal proteins translated on ribosomes in the cellular cytoplasm are imported into the nucleus and assembled with rRNA as ribosomes or spliceosomes (reviewed in Scheer and Weisenberger, 1994; Mélèse and Xue, 1995). Ribosomes and spliceosomes catalyse the translation of mRNA to synthesize proteins and splice introns from pre-mRNA, respectively (reviewed in Staley and Woolford, 2009). Previous reports demonstrated that SRY localizes in nuclear splicing factor speckle domains and may be directly involved in the biochemical process of splicing (Ohe et al., 2002). Our results also showed that SRY co-localizes with RPS7 and RPL13a in nuclear speckles. It is suggested that SRY interacts with RPS7 or RPL13a in nuclear speckles and works as a splicing factor that affects testis determination; however, the mechanism of its effects on testis determination is still unknown.
In conclusion, we confirmed that the HMG box of SRY is crucial for the interaction of SRY with RPS7 and RPL13a. Neither the function of SRY–RPS7/–RPL13a nor their other interactions in nuclear speckles are clearly understandable. Therefore, additional studies are required to elucidate the function of SRY–RPS7 and/or –RPL13a interactions in the development of the human testis.
Youichi Sato carried out the GST pull-down assay, conceived the study, participated in its design and drafted the manuscript. Shojiro Yano carried out the plasmid constructs, yeast two-hybrid assay and immunofluorescence staining. Ashraf Ewis and Yutaka Nakahori helped to draft the manuscript.
We are grateful to Dr Masako Sei, of the University of Tokushima, for her helpful discussion.
This work was supported, in part, by Grants-in-Aid for Scientific Research on Priority Areas from
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Received 13 September 2009/24 May 2010; accepted 29 November 2010
Published as Cell Biology International Immediate Publication 29 November 2010, doi:10.1042/CBI20090201
© The Author(s) Journal compilation © 2011 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)