| Research ObjectivesPost-transcriptional gene regulation plays an important role in the maturation.transport,stability and translation of encoding or non-coding ribonucleic acid(RNA),in which RNA binding protein(RBP)is the core regulatory element.Regulator of calcineurin 1(RCAN1)is a multifunctional protein which has been shown closely correlated with the neurodegenerative disorders,mitochondrial dysfunction,inflammation and glycosylation.RCAN1 is especially one of the most important causal genes of Down syndrome(DS)and Alzheimer’s disease(AD).This gene consists of seven exons and six introns and alternative slicing of the first four exons generates four isoforms that differ only in their N-terminal.The RCAN1 isoform 1(RCNA1.1)is abundantly expressed in the brain while the iso form 4(RCAN1.4)is mostly expressed in myocardial and kidney tissues.There are two distinct translational start codons(AUG),which generates two isoforms of RCAN1.1,a longer isoform(RCAN1.1L)with 252 amino acids and a shorter one(RCAN1.1 S)with 197 amino acidsOur previous research found that RCAN1 can increase the stability of adenine nucleotide translocator messenger RNA(ANT1 mRNA),and lead to the mitochondrial dysfunction.However,the mechanism with which RCAN1 interacts ANT1 mRNA remains to be elucidated.In this study,we demonstrated that RCAN1 is a novel RNA-binding protein,and it directly binds to ANTI mRNA transcript at 269-291 nucleotide(nt).Systematic Evolution of Ligands by Exponential Enrichment(SELEX)analysis showed an RNA aptamer of RCAN1 named as R1SR13.Furthermore,we found that by binding with the RCAN1,the aptamer R1SR13 suppresses the inhibitory effect of RCAN1 on the NFAT and NF-κB signaling pathways and reduced RCAN1-induced neuronal apoptosis.Our work demonstrated that the RNA aptamer of RCAN1 might have a protective effect on neurons.Acute ischemic stroke(AIS)threatens the lives and health of millions of people in worldwide every year,and has always been one of the leading causes of mortality and morbidity in recent years.The incidence of AIS in China has obviously increased in the past two decades.And AIS brings a heavy financial burden to the family and society.Understanding the pathophysiology of AIS is particularly relevant in studying more effective preventive or therapeutic strategies.In AIS the rapid necrosis of neurons in the central necrotic area is difficult for salvation,but the neurons in the ischemic penumbra are entering the apoptotic process and there is still a time window for cellular repair.In this study,the neurons in the ischemic penumbra in the model mice with carotid artery ligation were used to explore the in vivo expression changes of RCAN1.And oxygen and glucose deprivation was used to in vitro mimic the AIS and the role of RNA aptamer R1SR13 was also investigated.Our study found that RCAN1.1L expression changed significantly in AIS in vitro and in vivo and we further explored how RCAN1.1L affects neuronal apoptosis.Besides,we also investigated the effects of the RNA aptamer R1SR13 on RCAN1 functions and neuronal apoptosis.Materials and methods1.Was the RCAN 1 protein an RNA binding protein to which affect the ANTI mRNA1.1 RNA immunoprecipitation assay(RIP):HEK293 cells were transfected withthe plasmids highly expressing RCAN1.1S with the Flag and Myc tags for 48hours.Protein-RNA complexes were purified from cell lysates byimmunoprecipitation(IP).After the binding RNAs were extracted by the Trizolmethod,the PCR amplification was performed to determine whether ANTI mRNA could bind to RCAN1 protein to form a protein-RNA complex.1.2 Prediction of RNA binding domain:The possible RNA binding domain of RCAN1 protein was searched in the Protein Data Bank(PDB)database.It was predicted that the amino acid sequence 14-103 of mouse RCAN1.1S protein might be an RNA binding site.1.3 RCAN1.1S protein purification:Because the RCAN1 protein has a high degree of homology,we hypothesized that the sequence of RNA binding domain of human RCAN1 protein is similar to that of the mouse protein.The BL21(DE3)competent cells were infected with the plasmid pET-28a-RCAN1.1S-1-103-6×his highly expressing human RCAN 1.1S-1-103 truncated protein.The truncated protein was then purified from total protein.1.4 Trypsin proteolysis assay:The purified RCAN1.1S-1-103 truncated protein alone or a combination of truncated protein with ANTI mRNA was digested with a suitable concentration of trypsin to observe the difference in the protein fragments produced after digestion.1.5 Ribonuclease A(RNase A)sensitivity assay:RNA binding protein is more stable when combined with RNAs to form a complex.Incubation of RNase A could be used to enzymatically decompose the RNAs in the complexes under suitable conditions.Then the stability of RNA binding proteins is reduced and the proteins are degraded.This experiment is also applied to confirm a RNA-binding protein.2.Distribution of RCAN1 protein in cells2.1 Western blotting(WB):HEK293 cells were transfected into the plasmids highly expressing RCAN1.1S or RCAN1.1S-1-103 protein,and then nucleus and cytoplasmic proteins were separated and extracted for western blot.2.2 Immunofluorescence staining(IF):HEK293 cells were transfected with the plasmids expressing RCAN1.1S or RCAN1.1S-1-103 protein and then immunofluorescent staining was performed and the subcellular localization of RCAN1 protein was observed under a confocal microscope.3.In vitro screening of RNA aptamer for RCAN1 protein and affinity measurement3.1 The Systematic Evolution of Ligands by Exponential Enrichment(SELEX)assay was used to screen the RNA aptamers with high affinity to RCAN1.1S protein in vitro3.2 Select one of the selected RNA aptamers(which was named as R1SR13)with its control RNA.synthesize R1SR13 and its control RNA and then use the Surface Plasmon Resonance(SPR)assay to measure the affinity of RNAs and ROAN 1.1S protein in vitro3.3 RNA pull-down assay:HEK293 cells were transfected with the plasmid expressing RCAN1.1S-1-103 and the purified protein was incubated with the biotinylated RNA aptamer R1SR13 or unlabeled R1SR13(10 times in concentration for competition).This experiment verified that RCAN 1.1 S-1-103 binds to R1SR13.4.Detection of RNAs binding to RCAN1 protein in vivo4.1 RIP high-throughput sequencing(RIP-seq):HEK293 cells were transfected with the plasmid highly expressing the RCAN1.1S for 48 hours and then the RCAN 1.1S protein was obtained from the cell lysate by IP for the RIP-seq to obtain the RNAs sequences4.2 SPR technology verifies the affinity of each RNA sequence obtained by sequencing with the RCAN 1.1S protein.5.Analysis of the binding site of RCAN 1 protein in ANTI mRNA5.1 In vitro transcription of RNA:The ANTI gene 1-327nt,250-327nt and 488-894nt fragments were amplified from the plasmid pCMV 10-3 × flag ANTI by PCR,and the three fragments were separately transcribed into the mRNA and purified in vitro5.2 RNA EMSA:The purified ANTI mRNA 1-327nt and 488-894nt fragments were incubated with RCAN1.1S-1-103 purified protein,respectively,and subjected to RNA gel migration electrophoresis to observe whether the two RNAs and proteins could bind to each other in vitro5.3 SPR:ANTI mRNA 1-327nt,250-327nt and 488-894nt were tested for their affinity to RCAN1.1S-1-103 purified protein.Besides,ANTI mRNA 269-291nt as well as three nearby fragments(246-268nt,292-314nt and 315-337nt)were also tested for their affinity to the to RCAN1.1S-1-103 purified protein to determine the specific binding site.6.Impact of RNA aptamers on the downstream signaling pathway of RCAN1Dual luciferase reporter gene assay was used to measure the effects of RNA aptamer R1SR13 on the activation of nuclear factor of activated T cells(NFAT)and nuclear factor kappa-B(NF-κB)7.Effect of RNA aptamer R1SR13 on the pro-apoptotic function of RCAN1 in fetal rat primary neurons;7.1 Primary rat neuron extraction and lentiviral infection in rat fetuses:Fetal rat cerebral cortical neurons were harvested and cultured in vitro at 28 days of gestation.Lentivirus infection was added at 5 days after cell culture,and the subsequent experiments were performed after 72 hours7.2 Western blot for cleaved Caspase-3 protein7.3 TUNEL assay for assessing apoptosis7.4 Luciferase assays for ATP and Caspase 3/7 protein activity8.In vitro AIS model:Oxygen and glucose deprivation model(ODD model)was established in the SH-SY5Y and fetal rat primary neurons9.Changes in the RCAN1.1 mRNA and protein levels during OGD and their effects on the apoptosis9.1 Real-time fluorescent quantitative PCR(Taqman method)was used to detect the expression of RCAN1.1 mRNA 0-10 h after OGD(every 2 h)9.2 Western blot was used to detect the RCAN1.1 protein expression changes 0-10 h after OGD(every 2 h)9.3 Adenovirus associated virus 9(AAV9)infected the rat primary neurons:rat primary neurons were cultured for 5 days,and an appropriate amount of AAV9 virus was added for 72 hours before subsequent experiments9.4 TUNEL assay,western blot for cleaved Caspase-3 protein and luciferase assays for ATPase and Caspase 3/7 protein activity were used to test the effects of overexpression and knockdown of RCAN1 on neuronal apoptosis during OGD.10.What is the influence of RNA aptamers on apoptosis during OGD?TUNEL assay,western blot for cleaved Caspase-3 protein and luciferase assays for ATPase and Caspase 3/7 protein activity were used to investigate the effect of RNA aptamer interacting with RCAN1.1 protein on the apoptosis during OGD process..11.Expression of RC AN 1.1 in the hippocampal CA1 region of the ischemic side of the unilateral common carotid artery ligation model in miceEight-week-old C57BL/6 male mice underwent right common carotid artery ligation,and the sham operation was performed on the contralateral side.The mice were divided into 4 groups according to ligation time(Oh,2h,4h and 6h)respectively.The hippocampal CA1 region of mouse brain was harvested and the expression of RC AN 1.1 mRNA and protein were detected by real-time PCR and western blot.Research results1.RCAN1 protein binds to ANTI mRNAHEK293 cells were transiently transfected with RCAN1.1S expression plasmid(flag-RCAN 1.1 S-myc)for RNA immunoprecipitation assay(RIP)and the results showed ANTI mRNA was detected in the co-precipitated complex.Trypsin digestion assay showed that trypsin degraded the recombinant RCAN1.1S 1-103 protein,However,the presence of ANTI mRNA transcript efficiently protected RCAN1 proteins from the digestion by trypsin.The above results indicated the direct binding of RCAN1.1S to ANTI mRNA.2.RCAN1 protein is mainly located in the nucleusThe results of western blot and immunofluorescence staining after subcellular fractionation assay both showed that the RCAN1.1S protein and its truncated protein RCAN1.1S-1-103 were mainly expressed in the nucleus.3.R1SR13 is an RNA aptamer of RCAN1 proteinSeveral potential RNA recognition sequences were identified by SELEX.A representative motif named R1SR13 was chosen for SPR and RNA pull-down experiments,which both confirmed that R1SR13 has a very high affinity with RCAN1 protein.4.There were several RNA sequences that could bind to RCAN1 protein.4.1 The RNase protection assay demonstrates that the protein level of RCAN1.1S 1-103 was greatly reduced in the cells treated with RNase A compared with the non-treated cells.The results clearly revealed that RCAN1 is a RNA-binding protein.4.2 RIP-Seq revealed five consensus motifs which could bind with RCAN1 protein.The five consensus RNA motifs as well as their reverse complementary strands were synthesized and subjected to SPR assay to examine their bindings with RCAN1 proteins.The results showed that RCAN1 bound to all of these RNAs5.Binding site of RCAN1 protein ANTI mRNATo map the exact binding motif in ANT1 mRN A of RCAN 1,the SELEX motif was aligned with ANTI mRNA sequence by using Clustal Omega alignment software.A putative motif was identified in the region of 269-291nt(Figure 3D,ATG is designated as+1).To examine if the putative motif bound with RCAN1,two ANTI mRNA truncations of 1-327nt and 488-894nt were transcribed using SP6 RNA polymerase in vitro for the RNA electrophoretic motility shift assay.The result indicated RCAN1 binding motif is in the ANTI mRNA 1-327ntTo determine the location of this motif three ANTI mRNA fragments spanning 1-327nt,250-327nt and 488-894nt were transcribed using SP6 RNA polymerase in vitro.SPR assay was used to measure the binding affinity of these three ANTI mRNA with RCAN1 proteins.The dissociation constant showed the binding motif is in the region of 250-327ntTo map out the binding motif of RCAN1 in the ANTI mRNA,four RNAs of 246-268nt,269-291nt,292-314nt and 315-337nt were synthesized for subsequent SPR assay.The data of dissociation constant demonstrated that 269-291nt is the binding motif of RCAN1 in ANT1 mRNA6.Effect of RNA aptamer R1SR13 on RCAN1 protein function6.1 Previous studies have found that RCAN1 protein could inhibit the transcriptional activity of NFAT and NF-κB pathways.The results of dual luciferase reporter assay indicated the R1SR13 aptamer suppressed the RCAN1-mediated inhibition of NFAT and NF-κB signaling pathways.6.2 RCAN1 induced apoptosis in primary neurons by activating the Caspase-3 pathway.The results of western blot of cleaved Caspase-3,TUNEL staining,luciferase assay for ATP level and Caspase 3/7 activity all showed that R1SR13 could inhibit the RCAN1-induced neuronal apoptosis.7.The increased RCAN1 mRNA and protein levels by OGD treatment in SH-SY5Y and rat primary neuronThe mRNA levels of RCAN 1.1 peaked in SH-SY5Y and rat primary neurons at OGD 4h or 6h respectively.And the highest levels of RCAN1.1L protein were reached at OGD 6 or 8h respectively.8.The elevated RCAN1 expression under OGD condition in SH-SY5Y cells and rat primary neurons promotes apoptosisTUNEL staining,western blot for cleaved Caspase-3 protein and luciferase assay for ATPase and Caspase-3/7 activity confirmed that the elevated expression of RCAN1.1L in SH-SY5Y cells and rat primary neurons promoted the apoptosis under OGD 6h,an effect which was reverted by the knockdown of RCAN1 via siRNA transfection.9.The RNA aptamer R1SR13 attenuates the apoptotic activity of SH-SY5Y cells and primary neurons by inhibiting RCAN1 function under OGD condition TUNEL staining,western blot for cleaved Caspase-3 protein and luciferase assay for ATPase and Caspase-3/7 activity confirmed that the apoptosis in SH-SY5Y cells and primary neurons under OGD condition were alleviated by adding R1SR13.10.The expression of RCAN1.1 in the hippocampal CA1 region of C57BL/6 mice after unilateral common carotid artery ligation was increased compared to the contralateral side.Real-time quantitative PCR and western blot showed that the mRNA and protein levels of RCAN1.1 in hippocampal CA1 region of C57BL/6 male mice after unilateral common carotid artery ligation were higher than those of contralateral side.Conclusions1.The RCAN1 protein is an novel RNA-binding protein with a binding site for ANTI mRNA at 269-291 nt.2.RCAN1 expression was elevated and it promoted the apoptosis in the neurons under OGD condition.RCAN1 mRNA and protein levels were increased in the hippocampal CA1 region of mice model after unilateral common carotid artery ligation.3.R1SR13 is a RNA aptamer of RCAN 1 protein with high-affinity in vitro,and the function of RCAN1 protein is inhibited after binding with R1SR13.Specifically,R1SR13 reduced the inhibitory effect of RCAN1 on NFAT and NF-κB transcriptional activity.And R1SR13 suppressed the neuronal apoptosis mediated by RCAN1.Significance1.This study demonstrates for the first time that RCAN1 protein is an RNA-binding protein,which provides a broader horizon for RCAN1-related research.And it explains the interaction between RCAN1 protein and ANTI mRNA.2.This study enriched the understanding of mechanism of neuronal apoptosis by accounting for the expression and function of RCAN1 in AIS.3.The RNA aptamer R1SR13 could inhibit the function of RCAN1 protein and it will be the target of therapeutic research in a variety of diseases closely related to RCAN1. |
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