Splicing Factor Srsf5 Maintains Cardiac Function In Mice By Regulating Alterative Splicing Of Myom1

Processing of precursor mRNAs is a fundamental step which is essential for gene expression and function in most postnatal animals.The serine/arginine-rich(SR)splicing factors,as key regulators of RNA processing,play important roles in m RNA splicing activation,splicing inhibition,export,stabilization,and translation.There are12 members of the SR protein family with functional overlap and redundancy.In recent years,studies on Srsf1,Srsf2,Srsf3 and Srsf10 knockout mice models have shown that members of the SR protein family are specific for the regulation of alternative splicing of their target genes during development.Previous experimental results of the group showed that Srsf5 was highly expressed in the hearts of newborn mice and decreased with increasing age.Bioinformatics analysis revealed that SRSF5 was under-expressed in adult cardiomyocytes with low ejection fraction and dysfunction compared to those with normal function.It is hypothesized that Srsf5 is an important regulator in maintaining normal cardiac function in mice.However,the function of Srsf5 in tissue development and multiple diseases is unknown.To investigate the role of Srsf5 in maintaining cardiac function and its regulatory mechanisms in mice,we constructed a Srsf5 knockout(Srsf5-/-)mouse model using CRISPR/Cas9 technology.Srsf5-/-mice died perinatally and exhibited cardiac dysfunction with incomplete ventricular myocardial densification.Transcriptomic analysis identified that Myom1,a major component of the myogenic fiber M-band,is a potential substrate for Srsf5.Myom1 is a protein that crosslinks myosin filaments to sarcomere M-band and maintains the structural integrity of sarcomere during contraction.Myom1 is one of the key regulators of muscle contraction.Myom1 in the heart undergoes isoform switching before and after birth,with the long form of Myom1 containing the EH fragment(EH-myomesin)predominantly expressed during embryonic life and the short form of the mature isoform generated after birth by alternative splicing of the EH fragment.In Srsf5-deficient hearts,the EH fragment was unable to make exon skipping and Myom1 was unable to complete the switch between embryonic and mature isoforms,thereby affecting the contractile function of the heart.Thus,it is suggested that the regulation of alternative splicing of Myom1 by Srsf5 plays a key role in heart development.This study is intended to investigate the role of Srsf5 on the developmental process of mouse heart by using Srsf5 knockout mice as experimental animals,and to explore the effect of Srsf5 regulation of Myom1 alternative splicing on the heart function of mice,which provides theoretical and experimental basis for the research and diagnosis of developmental defective heart diseases.Methods:1.Effects of systemic knockout of Srsf5 on growth and development of mice(1)Generation of Srsf5 knockout mouse modelThe sg RNA sequences were designed and segments of the Srsf5 gene exon 3 to exon 6 were knocked out using CRISPER/Cas9 technology to obtain genetically competent Srsf5 knockout heterozygous mice.Heterozygous mice were mated to obtain Srsf5 knockout mice(Srsf5-/-).The genotypes of the mice were identified by PCR after extraction of tail genomic DNA.RT-q PCR,Western blot and immunohistochemical staining were used to detect the expression of Srsf5 in various organs of Srsf5-/-mice to validate the knockout effect of Srsf5 gene.(2)Survival of Srsf5 knockout miceThe number of survival and death of WT,Srsf5+/-and Srsf5-/-mice of three genotypes in the offspring of heterozygous mice from E18.5 to P7 was analyzed and Kaplan-Meier survival curves were plotted to analyze the effect of Srsf5 knockout on the survival of mice.(3)General properties and histopathological changes in Srsf5 knockout miceThe appearance of Srsf5-/-mice around birth was observed.Body weight and the weight of heart,liver,lung,brain,kidney and spleen of Srsf5-/-mice at P1 were measured.The whole-mount skeletal staining was used to observe the skeletal morphology of Srsf5 deficient mice at P1.H&E staining was performed to detect histopathological changes in the heart,liver,lung,brain,kidney,and spleen of Srsf5-/-mice.2.Effect of Srsf5 deletion on the function of mice heart(1)Effect of Srsf5 deletion on the contractile function and electrical activity of the heart in miceSrsf5-/-mice surviving at P1 were selected for the following experiments.The cardiac systolic function of Srsf5-/-mice was assessed by small animal echocardiography.The cardiac electrical activity of Srsf5-/-mice was monitored by electrocardiography.The expression of ANF and BNP,markers of myocardial injury,in the heart was detected by RT-q PCR assay to assist in the diagnosis of changes in cardiac function in Srsf5-/-mice.(2)Effect of Srsf5 deletion on the myocardial compaction in miceEndomucin and c Tn T immunofluorescence staining were used to label the endocardial contour and myocardial layer,respectively.The thickness of myocardial trabeculae and myocardial compact layer were measured in Srsf5-/-mice,and the degree of myocardial compaction in Srsf5-/-mice hearts was analyzed by calculating the ratio of myocardial trabeculae thickness to myocardial compact layer thickness.(3)Effect of Srsf5 deletion on proliferation and apoptosis of mice cardiomyocytesImmunohistochemical staining was performed to detect the expression of Ki67 and p H3 in Srsf5-/-mice cardiomyocytes at P1 for analysis of the effect of Srsf5 deletion on the proliferation of mice cardiomyocytes.TUNEL assay was performed to detect the apoptosis degree of Srsf5-/-mice cardiomyocytes.3.Mechanism of cardiac dysfunction in mice induced by Srsf5 knockout(1)Transcriptome sequencing to select differentially alternative splicing events in the Srsf5-/-heartThe hearts of P0 WT and Srsf5-/-mice were selected for RNA-Sequence,the alternative splicing events of genes were analyzed by r MATS software,and the functions of differentially spliced genes were analyzed by GO enrichment.(2)Validation of differentially alternative splicing eventsExpression of different isoforms of candidate genes in the heart of Srsf5-/-mice hearts was examined by RT-PCR to verify differential alternative splicing events.(3)Srsf5 regulates alternative splicing of Myom1 in the heartWestern blot examined the isoform switch of Myom1 in mid-embryonic development to perinatal WT and Srsf5-/-mice hearts.The Myom1 minigene was generated,and cotransfected with different doses of Srsf5 plasmid in HEK293 T cell line,and the expression of different isoforms of Myom1 was detected by RT-PCR assay.RIP assay was used to detect whether Srsf5 protein binds directly to Myom1 precursor m RNA.Results:1.Effects of systemic knockout of Srsf5 on the growth and development of mice(1)Generation of Srsf5 knockout mouse modelA total of four heterozygous mice,two females and two males,were obtained.PCR results showed that the mated offspring of heterozygous mice included three genotypes,WT,Srsf5+/-,and Srsf5-/-.None of the m RNA and protein of Srsf5 were expressed in the organs of Srsf5-/-mice.This indicates that a mouse model with systemic knockout of Srsf5 was successfully obtained and can be stably inherited to offspring.(2)Survival of Srsf5 knockout miceSrsf5-/-embryos survived at E18.5 and all died within 2 days after birth,suggesting that Srsf5 deletion causes perinatal lethality in mice.(3)Morphological and histopathological changes in Srsf5 knockout miceThe body weight of Srsf5-/-mice was significantly lower than that of WT mice in the same litter.The ratios of organ weights and body weights of heart,liver,lung,kidney,and brain of Srsf5-/-mice were not significantly different from those of WT mice,while the ratio of spleen weight and body weight of Srsf5-/-mice was significantly lower than those of WT mice.No abnormal cartilage development was observed in Srsf5-/-mice.It is suggested that Srsf5 deletion can cause developmental delay in mice.H&E staining suggested no obvious structural pathological changes in the intestine,lung,liver,both kidneys,brain,and spleen of Srsf5-/-mice.H&E staining of heart showed thinning of the myocardial compact layer,long and dense myocardial trabeculae,and vacuolar degeneration in some cardiac myocytes of Srsf5-/-mice at E18.5 and P1.It suggests that Srsf5-/-hearts have abnormal development.2.Effect of Srsf5 deletion on the function of the mice heart(1)Effect of Srsf5 deletion on the contractile function and electrical activity of the heart in miceBoth ejection fraction and LV short-axis shortening were significantly downregulated in Srsf5-/-mice,suggesting reduced LV systolic function.Srsf5 deficiency slowed heart rate,prolonged PR and QT intervals,suggesting mild AV block in the Srsf5-/-heart.The marker of myocardial injury,brain natriuretic peptide(BNP),was significantly upregulated in Srsf5-/-hearts,suggesting that Srsf5 deletion caused myocardial injury in mice.(2)Effect of Srsf5 deletion on the myocardial compaction in miceEndomucin and c Tn T immunofluorescence staining showed that the ratio of the thickness of the noncompaction layer to the thickness of the compaction layer of the ventricular wall was significantly higher than that of WT hearts,which exhibit noncompaction of ventricular myocardium.It is suggested that Srsf5 deficiency can lead to cardiomyopathy with non-compaction of ventricular myocardium in mice.(3)Effect of Srsf5 deletion on proliferation and apoptosis of mice cardiomyocytesImmunohistochemical staining showed that the expression of Ki67 and p H3 was significantly reduced in the myocardial tissue of Srsf5-/-mice,indicating that Srsf5 deficiency slowed down the proliferation of mouse cardiomyocytes.TUNEL results showed that Srsf5 did not affect apoptosis of cardiomyocytes.3.Mechanism of cardiac dysfunction in mice induced by Srsf5 knockout(1)Transcriptome sequencing to select differentially alternative splicing events in the Srsf5-/-heartA total of 583 differential alternative splicing events were screened by transcriptome sequencing combined with r MATS software analysis,involving five variable splice types,of which Skipped Exon(SE)was the major differential variable splicing event.GO analysis revealed that differentially spliced genes were mainly enriched in muscle contraction and cardiac development-related functions.(2)Validation of differentially alternative splicing eventsRT-PCR validation of five cardiac disease-associated differential alternative splicing events showed that Srsf5-/-hearts decreased the expression of transcripts containing associated exons of Myom1 and Ap1b1 and increased the expression of transcripts with associated exons removed in Col4a3 bp,Tgfb2,and Cnot2.It demonstrated that Srsf5 has a dual function of promoting both exon retention and skipping.(3)Srsf5 regulates alternative splicing of Myom1 in the heartWestern blot results showed that isoform switch of Myom1 could not occur in Srsf5-/-hearts from E13.5 to P1,suggesting that Srsf5 is a key regulator of Myom1 isoform switch during different periods of heart development.The expression of Myom1 transcripts with exon 18 removed gradually improved with increasing transfection of Srsf5,suggesting that Srsf5 promotes the skipping of Myom1 exon 18 in a dose-dependent manner.RIP experiments showed that Srsf5 bound directly to Myom1 precursor m RNA,indicating direct regulation of Myom1 alternative splicing by Srsf5.Conclusions:1.Srsf5 deletion causes perinatal death as well as abnormal growth and development in mice,suggesting that Srsf5 plays a critical role in the early postnatal survival of mice.2.Srsf5 deficiency leads to noncompaction of ventricular myocardium and cardiac dysfunction in mice,indicating the importance of Srsf5 for mouse heart development.3.Srsf5 maintains cardiac function in mice by regulating the isoform switch of Myom1 in the heart before and after birth.