The Preliminary Study Of Function Of Rat's RSA14-44 Gene

RSA14-44 gene was obtained from the cDNA library of rat prirnay by the method of laser capture microdissection (LCM) combined wmVsuppressive subtractive hybridization (SSH). The full-length of RSA-14-44 is 1124 bp, and it contains a 582 bp open reading frame, encoding a 194 aa protein. The sequence has been submitted to Genbank, the accession number is AY149343. The transcription patterns of RSA-14-44 in multiple tissues were analysedbvRT-PCR and the results showed that RSA-14-44 is transcribed in most organs and high eranscrib testis, epididymis and brain. RSA-14-44 was cloned into the prokaryotic expression vector pET30a(+) and the protein of interest was expressed in E.Coli BL21 with a high quantity. The fusion protein was purified by affinity chromatography. Immunohistochemical analysis was performed for the localization of RSA-14-44 protein in rat testis tissues. It was showed that RSA-14-44 protein mainly appears in primary spermatid and sertoli cell, and some signals also existed in the mature sperms For further understanding the biological function of RSA-14-44, RSA-14-44 cDNA (cloned into pAS2-l plasmid) was chosen as the "bait" to screen human testis cDNA library using yeast two-hybrid system and the results showed that the 5 subunit of human 20S proteasome (PSMB5) is capable of binding to RSA-14-44. To confirm the interaction between RSA-14-44 and PSMB5, the protein was expressed as fusion proteins. The recombinant protein was purified with affinity chromatography and the polyclonarbodies of PSMB5 were raised by immunizing the abbit. In turn, the interaction of RSA-14-44 and PSMB5 was confirmed by the in vitro binding and GST-pull down experiments. Then, their interaction in the in vivo system was verified by coimmunoprecipatation. To confirm their interaction in the cell, RSA-14-44 and PSMB5 cDNAs were correspondingly cloned into pEGFP-Nl and pDsRedl-Nl vectors. The colocalization for RSA-14-44 and PSMB5 in CHO cells was also observed. The results demonstrated that RSA-14-44 and PSMB5 were overlay perfectly, suggesting that RSA-14-44 and PSMB5 can interact with each other to function in eukaryotic cells.Previous studies have shown that PSMB5 is related with the enzyme activity of 20S proteasome, so we investlgated the effect of RSA 14-44 and PSMB5 on the activity of 20S and 26S proteasome. It is interesting that the activityof both 20S and 26S proteasome were highly improved in the hela cell trasfected with RSA 14-44 and PSMB5. In addition, the activityof both 20S and 26S proteasome were significatly improved when the cells were cotransfected with RSA 14-44 and PSMB5. It is Wuggested that the overexpression of both RSA 14-44 and PSMB5 can increase the activityof 20S and 26Sproteasome, moreover the activityof both 20S and 26S proteasome were more significantly improved when their cooperation.To find the with their signi ficance of the interaction between RSA14-44 and PSMB5, immunohistpchemical analyst was performed for PSMB5 protein. It was shown that PSMB5 existed in the spermatogonia and Sertoli cells. This is consistent with the results of RS A-14-44. In conclusion, it seems that the interaction betwe there two proteins may play a role in spermatogenesis. It is speculated that the interaction between them may promote the proliferation and division in germ cells, and regulate protein degomeration in sretoli cells. Future experiments need be made to confirm the hypothesis.