Expression And Regulation Of Pre-mRNA Splicing Factor ZNF265 And Its Function

Zinc finger protein 265(ZNF265,also termed"zis" or"zfp265")is a novel RS (arginine/serine-rich)domain-containing protein that was first identified in rat renal juxtaglomerular(JG)cells.JG cells are specialized myoepithelioid cells located in the afferent arteriole at the entrance to the glomerulus.Their main function and distinctive feature is the synthesis and release of renin,the key hormone-enzyme of the renin-angiotensin system that regulates arterial blood pressure.Despite their relevance to health and disease,not much is known about factors that confer or maintain JG cell identity.ZNF265 transcript was found in differentiated renin-expressing cells,but not in cells that had dedifferentiated and lost their ability to express renin.Researches on the structural basis for ZNF265 showed that ZNF265 had two zinc finger domains,a Ser-Arg-rich domain,a glutamic acid-rich region,and a nuclear localization signal (NLS),features consistent with a role in the splicing of primary RNA transcripts or mRNA binding.As a proof,study had shown that overexpression of ZNF265 resulted in exclusion of exons 2 and 3 from the Tra-2β1 pre-mRNA,which led to an increase in the production of the Tra-2β3 alternatively spliced isoform.Therefore,ZNF265 as a multifunctional protein in alternative splicing,may affect renin expression directly by regulating the processing of renin pre-mRNA or indirectly altering cell differentiation by regulating the expression of other genes.The main focus of the present study is to determine the expression of ZNF265 in cells,various tissues,explore the function of ZNF265 in regulating pre-mRNA alternative splicing and identify the potential genes that are regulated by ZNF265.To begin the study of the potential function of ZNF265,RT-PCR analysis was applied to detect the expression of ZNF265 mRNA in various tissues in humans and mice.The results demonstrated that ZNF265 mRNA is ubiquitously expressed in various tissues in both species.Moreover,we identified a new isoform(mZNF265-2) in mice that shares high similarity with the previously reported human ZNF265-2 (hZNF265-2).Analysis of the expression pattern of the two isoforms of ZNF265 (ZNF265-1 and ZNF265-2)in various tissues of humans and mice showed that both isoforms are expressed ubiquitously.In 20 week human fetus,the expression levels of ZNF265-1 and ZNF265-2 vary in various tissues although the expression is ubiquitous.As examples,in the cerebrum,cerebella and liver,the ratios between two transcript isoforms are around 1.0(0.9～1.1);in the heart,lungs and intestine,the expression of ZNF265-2 is higher than that of ZNF265-1 with ratios of around 0.5 (ZNF265-2/ZNF265-1,0.45～0.7);in the kidney,ZNF265-1 is much higher than ZNF265-2(ZNF-1/ZNF-2 around 1.48).In comparison,in mice,ZNF-1 is a dominant isoform in all tissues without significant variation in the ratio(around 4:1)between two isoforms among the tissues.The results suggest that ZNF265 expression is tissue-specific and seems subject to more complicated regulation in humans compared to that in mice.To further study the expression of ZNF265,we prepared antisera specific to ZNF265 or two isoforms(ZNF265-1 and ZNF265-2),respectively.After the specificity of the generated antibodies was confirmed using ELISA,recombinant GST-fusion proteins and Western blot and Immunocytochemical(ICC)analyses,we used Western blot analysis to show that ZNF265-1 was ubiquitously expressed in both human and mouse tissues,but with a development-dependent pattern in mice.In mouse neonates,variation in ZNF265 protein levels among various tissues is insignificant.However,in adult mice,ZNF265 protein levels are higher in the kidney, lung and heart than in the brain,and liver.In addition,either in human or mouse tissues,no ZNF265-2 proteins are detected.The results indicate that in humans and mice,ZNF265-1 is a dominant protein form.It is possible that ZNF265-2 may exit as transcripts only or its protein level is too low to be detected using Western blot.As an SR protein associated with alternative splicing,ZNF265 may function mostly in the nuclei.We determined the cellular distribution of endogenous ZNF265-1 or recombinant his-tagged or GFP-fused ZNF265-1 proteins using the generated rabbit anti-ZNF265 antiserum and anti-histag antibodies.The results demonstrated that the ZNF265 is localized mainly in the cell nuclei,which is consistent with their potential functions in regulation of pre-mRNA splicing.ZNF265 has been suggested to affect renin expression directly or indirectly.Thus we examined the expression,distribution and regulation of ZNF265 in mouse reproductive system where has intrinsic renin-angiotensin system using immunohistochemistry(IHC)in immunocytochemistry(ICC).The results showed that ZNF265 is specifically distributed in the nuclei of corpus luteum,zona pellucida and blood vessel in the ovary,the middle region of sperm tail and the principal cells of epididymal tubules of the epididymis in male mice.The distribution is consistent with the reported pattern of AgentensinⅡin the renin-angiotensin system.The effects of castration and testicular hormonal replacement on the expression of ZNF265 were also investigated.Results from Western blot and ICC analyses consistently showed that the expression of ZNF265 is decreased by castration whereas its expression is completely restored to control levels when the castrated mice are hormonally replaced with testosterone.Interestingly,it has been reported that the regulation of renin-angiotensin system in the epididymis is also subject to steroid hormonal regulation.This consistency to the report leads to the possibility that ZNF265 may be involved in the regulation of gene expression related to renin-angiotensin system, including renin gene.It is known that the renin gene generates multiple alternative spliced transcript products in humans,including Renin-a in the kidney,Renin-b in the brain and Renin-c in the lung.As an alternative splicing regulator,ZNF265 is possibly involved in the splicing of renin pre-mRNA thereby regulates the renin expression.To test this possibility,we constructed a renin minigene containing exons 1,2 and 3.In the minigene,the exons 1a and 1c are subject to exclusive splicing,depending on either it is spliced in the kidney or lung.Our studies with this minigene showed that the splicing pattern of the exons 1a and 1c is cell-type specific,indicating that cell-type specific splicing regulators may play important roles in regulating renin pre-mRNA splicing.In addition,we observed that the overexpression of ZNF265 did not affect the splicing pattern.It implies that ZNF265 requires extra cis-elements not existed in the minigene in regulating the splicing of renin pre-mRNA or does not directly regulate the splicing.Further studies are required for determining these possibilities.The ubiquitous expression of ZNF265 indicates that ZNF265 may function in regulating the alternative splicing of various pre-mRNAs.Therefore,we constructed several minigenes including FGF-2R,GluR-B,hZis and mZis and studied the effects of ZNF265 on those minigenes and others(SMN2 and CFTR minigenes).Among them,FGF-2R and GluR-B are neural-specific genes.SMN2 and CFTR genes are associated with spinal muscular atrophy(SMA)and congenital bilateral absence of the vas deferens(CBAVD)diseases,respectively.The hZis and mZis minigenes contain the exons 9,10 and 11 of the ZNF265 gene and used for studying the self-regulation of ZNF265 splicing.The results showed that ZNF overexpression promotes the exclusion of the exon 14 of GluR-B,the exon 7 of SNMN2 and the exon 9 of CFTR in a ZNF265 concentration-dependent pattern,but did not affect FGF-2R splicing.In addition,we also found that ZNF265-2 overexpression alters the splicing patterns of hZis and mZis minigenes,indicating ZNF265-2 functions as a self-regulator in regulating the splicing pattern of ZNF265 pre-mRNA.ZNF265 has a classic RNA domain that is suggested to bind the mRNA.Previous studies have indicated that ZNF265 may be involved in cell differentiation,in addition to regulating the expression of renin.Therefore,as an SR protein,ZNF265 may bind to the mRNAs of renin or other genes that are involved in cell differentiation.Our studies using immunoprecipitation(IP)combined with RT-PCR analysis showed that ZNF265 binds to the mRNAs of cell cycle-related factors,Cyclin B1 and Cyclin D2, but not renin mRNA.The results indicate that ZNF265 may regulate the expression of Cyclin B1 and Cyclin D2,in consistent with the previous report showing that C4SR (frog homologous)and Sfp265(rat homologous)bind to Cyclin B1 transcripts in vitro. It has been known that Cyclin B1 and Cyclin D2 are overexpressed in various tumour types,as well as in differentiating cells.Thus ZNF265 may contribute to the well-known de-differentiation(loss of identity)of JG cells during prolonged culture via interacting with Cyclins.In conclusion,the present work initiated studies of the function and mechanisms of ZNF265 in regulating pre-mRNA splicing.Based on the results from the analysis of ZNF265 expression,function in regulating pre-mRNA splicing and interaction with mRNAs,our studies for the first time show that ZNF265 is involved in alternative pre-mRNA splicing and cell differentiation.