Regulation And Function Of HnRNP-R/hnRNP-Q In Light Reponse Process In Rat Retina

The present study focuses on the expression and functions of the genes associated with neural development.To this end,we used the cDNA microarray to analyze the gene expression profile in human brain tissues at different developmental stages.Several potential genes were identified.Using Northern blot analysis,we confirmed two neural development-related genes;COⅩ-Ⅲand hnRNP-R.The results showed that the expression of COⅩ-Ⅲwas up-regulated during the neural development and the expression of hnRNP-R was down-regulated during the neural development.Along with our long-term interests in studying the function of genes related to pre-mRNA processing in neural development,the function of hnRNP-R gene was further studied in the present study because hnRNP-R is a RNA binding protein which is reported recently to be involved in pre-mRNA processing in neural tissues.To study the expression and functions of hnRNP-R in neural tissues,we produced the specific antiserum to hnRNP-R by using Keyhole Limpet Hemocyanin (KLH)-conjugated synthesized peptides as an antigen.We examined the expression of hnRNP-R in various cells.The results showed that hnRNP-R was ubiquitously expressed in those cells.Immunocytochemistry(ICC)analysis demonstrated that the subcellular localization of hnRNP-R was not altered during the retinoic acid(RA) induced P 19 neuronal differentiation.It has been known that the function of a gene can be regulated by altering the splicing pattern of the gene.To begin to study the function of hnRNP-R gene,we performed computer-aided analysis to predict the alternative splicing pattern of hnRNP-R.Based on the information from Human Alternative Splicing Database (HASDB),the result revealed seven isoform candidates.Among which the two candidates are most likely to encode proteins.We examined these two candidates using RT-PCR analysis in human fetal cerebral tissues.The sequence analysis demonstrated the existence of the two isoforms.The novel isoform hnRNP-R2 lacks the exon 5(114 bp)without amino acid frame shift compared to hnRNP-R1 which is reported as hnRNP-R previously.This exon likely contains a tyrosine phosphorylation and a N-myristoylation site.Therefore,the absence of the exon 5 may be critical in functionally distinguishing the two isoforms.As subcellular distribution of proteins is an indicator of their functions,we examined the subcellular distribution of GFP-hnRNP-R1 and GFP-hnRNP-R2 fusion proteins in R28 and HeLa cells.The Western blot results showed that R28 cells expressed two isoforms endogenously while HeLa cells expressed only the hnRNP-R1.GFP-hnRNP-R1 as well as GFP-hnRNP-R2 were located in the nuclei in either R28 or HeLa cells,indicating that both proteins are nuclear in neural and non-neural cells.The expression pattern of two isoforms in human fetal tissues at 12 weeks was characterized by using both RT-PCR and Western blot analysis.The RT-PCR results showed that hnRNP-R1 was ubiquitously expressed and its expression was strikingly higher than that of hnRNP-R2.In contrast,the expression of hnRNP-R2 was relatively higher in neural tissues.The Western blot results is consistent with the RT-PCR results.The result demonstrated that the expression pattern of hnRNP-R2 is neural-specific,suggesting that hnRNP-R2 may have special function in neural development.To address whether the neural expression of hnRNP-R isoforms is developmentally regulated,the expression of the two isoforms in the cerebrum and cerebellum at two developmental stages(12W and 18W)was studied.We found that the relative amount of hnRNP-R1 and R2 proteins toβ-actin was significantly down-regulated developmentally.In addition,the ratio of hnRNP-R2 to R1 protein levels was down-regulated developmentally as well,indicating the differential regulation of the two isoforms during the brain development.To further study the function of hnRNP-R in neural tissues,we examined its expression pattern in various rat neural tissues by using Western blot analysis.The results showed that the expression of hnRNP-R was significantly higher in the retina compared to other tissues,indicating that hnRNP-R may play important roles in rat retina.The immunohistochemistry analysis in rat retina showed that the hnRNP-R immunoreactive signal was detected only in the inner nuclear layer(INL)and ganglion cell layer(GCL).There was no hnRNP-R immunoreactive signal detected in the outer nuclear layer(ONL),which could correspond with photoreceptor cells. The results suggest that hnRNP-R may have special functions in these two layers.It is more interesting that the localization of hnRNP-R in rat retinas is consistent with that of c-fos,hnRNP-Q,80%homology protein of hnRNP-R,is reported to be involved in c-fos mRNA turnover process and able to interact with hnRNP-R.It is strongly suggested the relationship between hnRNP-R,hnRNP-Q and c-fos.It has been reported previously that c-fos is circadian regulated in the rat retina and its regulation manner is different between in GCL/INL and in ONL.Therefore,in the present study,we used an rat light/dark cycle(LD12;12)model to examine the expression pattern of hnRNP-R in circadian cycle.The results showed that the expression of hnRNP-R was differently regulated in GCL and INL in circadian cycles.The expression of hnRNP-R in GCL was down-regulated from light period to dark period compared to it in INL,suggesting the different regulation mechanisms of hnRNP-R in GCL and INL.In order to study the functions of hnRNP-R in regulating c-fos expression in rat retina,we need a suitable cell model for gain-of-function analysis.An immortalized retinal precursor cell line R28 was used as the cell model.To study the c-fos expression,we set up a bFGF-induced c-fos expression model in R28 cells.Transient transfection of R28 cells with hnRNP-R was performed in R28 cells.The RT-PCR results showed that hnRNP-R inhibited the c-fos expression induced by bFGF.Some sequence elements in c-fos mRNA are described to be involved in the regulation of c-fos mRNA stability during the past two decades.Among which AU-rich element (ARE)located at 3'-UTR of c-fos mRNA was well characterized.Former report showed that hnRNP-Q could enhance the stability of the RNA containing AU-rich element(ARE).To further study the relationship between hnRNP-R and c-fos,we constructed a green fluorenscence protein expression vector with 3' ARE of c-fos mRNA(GFP-ARE).The GFP proteins in the cells co-transfected with hnRNP-R and GFP-ARE were analyzed using Western blot analysis.The results showed that hnRNP-R dramatically inhibited the expression of GFP proteins in the cells transfected with GFP-ARE,but not the cells transfected with GFP without ARE.It suggests that hnRNP-R may down-regulate the c-fos expression via an ARE-related mechanisms.In addition,given the very high homology between hnRNP-Q and hnRNP-R, and the reported effect of hnRNP-Q on c-fos,we also studied the expression and regulation of hnRNP-Q in rat retina.We firstly produced specific hnRNP-Q antiserum using the same method of hnRNP-R antiserum production.The obtained antiserum was purified by absorption with GST recombinant hnRNP-Q antigen peptide.The circadian regulation of hnRNP-Q in rat retina was studied by both RT-PCR and Western blot analysis.The results showed that the splicing pattern of hnRNP-Q exon 8 was obviously regulated during the circadian cycle,which is consistent with the circadian regulation of c-fos mRNA expression.It suggests that different hnRNP-Q isoforms may have different effect on c-fos mRNA stability. Among the three hnRNP-Q isoforms,the exon 8 splicing pattern of hnRNP-Q2 is different from that of the other two isoforms(hnRNP-Q1 and hnRNP-Q3).However, at the protein level,the circadian expression of known hnRNP-Q isoforms in rat retina was not detected by Western blot analysis,suggesting that other potential mechanism was involved.Western blot analysis of various types of cells showed that the splicing pattern of hnRNP-Q is cell-type specific,implying the important roles of hnRNP-Q alternative splicing.To study the effect of hnRNP-Q on ARE in the regulation of c-fos mRNA stability,the co-transfection samples of hnRNP-Q isoforms and GFP-ARE in R28 cells were analyzed by Western blot analysis.The results showed that none of hnRNP-Q isoforms affect on ARE-RNA expression in R28 cells,suggesting that hnRNP-Q do not regulate the stability of ARE-RNA in retinal cells.The above results suggest that the stability of c-fos mRNA in retinal cells is regulated by hnRNP-R,but not hnRNP-Q.