Effect Of Motifs Within The CA-repeat-rich Intron4Region Of Surfactant Protein B(SFTPB) Gene On MRNA Splicing

Background and Objectives:Pulmonary surfactants are lipoprotein complexes that are produced, assembled, and synthesized by lung alveolar type II cells. The primary functions of pulmonary surfactants include lowering alveolar surface tension, improving lung compliance, promoting alveolar gas exchange, and preventing lung damage. The major functional components are the surfactant proteins A. B. C and D (e.g., surfactant protein, SP-A, B, C, D) and lipids, of which, SP-B is essential for normal lung function and abnormal expression of SP-B is associated with multiple lung diseases.Human SP-B gene contains a number of polymorphisms. In addition to point mutations (exons and introns), there are sequence insertion and deletion mutations that directly affect the structural region of the SP-B protein; therefore, these mutations may be related to the decreased expression of mature SP-B, and/or affect proSP-B post translational processing and secretion, which cause SP-B protein synthesis disorder.There are11different (CA)n motifs in the first half of intron4of the human SP-B gene. Each motif is composed of a conserved sequence (-20bp) and2-17subsequent CA repeats. In1995, the work in our laboratory demonstarted that gene polymorphisms in this region were associated with acute respiratory distress syndrome (ARDS). In2005, the work further found that (CA)n motif deletions were related to the incomplete splicing of SP-B mRNA, which was associated with lung cancer. These studies indicated that the intron4(CA)n motifs in the SP-B gene may-by influencing SP-B mRNA splicing processes-lead to the reduction of mature SP-B mRNA and influence the expression of functional SP-B protein. However, the detailed mechanisms of SP-B mRNA splicing regulation by the11(CA)n motifs are unknown.RNA splicing is an important post-transcriptional regulation process in eukaryotic gene expression. RNA sequences can determine RNA splice sites, and correct RNA splicing requires the availability of the correct splicing site. In addition, splice-regulating proteins can bind to specific RNA sequences called exon/intron splicing enhancers (ESE or ISE, exonic/intronic splicing enhancer) or exon/intron splicing silencers (ESS or ISS, exonic/intronic splicing silencer); the former enhances splicing near the splicing site, while the latter inhibits splicing. Additionally, RNA conformation, namely the alteration of RNA secondary structure and stability, also affects the availability of the splicing site and the binding ability of the enhancer/silencer sequence.To further investigate the role of the (CA)n motif on the SP-B mRNA splicing process, we conducted a systematic study of the intron4(CA)n motifs in the SP-B gene. We synthesized the intron4gene sequence from the wild-type SP-B gene (WT intron4) by PCR method. Based on this sequence, we constructed a series of (CA)n deletion mutant plasmids. These DNA plasmids were transfected into CHO cells and then Northern blot and real-time quantitative PCR analyses were used to determine the effects of the (CA)n motif on SP-B mRNA splicing. Additionally, through electrophoretic mobility shift assay (EMSA) experiments, we studied the (CA)n motif-related RNA sequences and the potential interactions with binding proteins and discussed the mechanism of (CA)n motif-regulated SP-B gene splicing.Methods Part Ⅰ:pcDNA3.1SP-B minigene and WT intron4fragment were synthesized from the SP-B gene by PCR method. By using the WT intron4sequence, we constructed a series of (CA)n motif mutated fragments (DC-A, DC-B,... DC-K) that characterized by deletion of the different motif one by one. Each of intron4(CA)n motif fragment was inserted into the pcDNA3.1SP-B minigene, so that a series of mutanted SP-B minigene plasmids contained different (CA)n motif deletion were generated. These plasmids were transfected into CHO cells and the RNA was extracted. Northern blot and real-time quantitative PCR analyses were used to determine the mRNA-splicing efficiency (completed splicing/incompleted splicing), and then the effects of the intron4(CA)n motifs on SP-B mRNA splicing were evaluated.Part Ⅱ:Online programs were used to predict the splicing site and to analyze the RNA secondary structure of the deletion mutants, which included the analysis of structural characteristics and stability. Additionally, comparative analysis among the specific motifs relative to their RNA secondary structure was performed. Part Ⅲ:Specific (CA)n motifs that potentially bind RNA-binding proteins were analyzed. To confirm RNA-binding protein interaction with specific (CA)n motif, RNA probes that contained potentially specific binding sites as well as mutated-sequence probe were synthesized. EMSA technology was used to study the correlation and interactions between (CA)n motif and potential binding proteins.Results Part Ⅰ:1) The splicing efficiencies of RNA in all SP-B minigenes that deleted different (CA)n motifs were lower than that of WT minigene.2) All minigene constructs, including WT, expressed visible variable amount of incompletely spliced RNA in addition to completely spliced RNA (or normal mRNA). The Motif8and Motif9were able to enhance and attenuate, respectively, RNA splicing.Part Ⅱ:Prediction of splice sites:The distance between the branch point site and the3’-end of the possible AG-determining regions was258bp, and4groups of potential splicing proteins have a possibility to bind in the intron4motif region. HnRNP L was found to bind the longer (CA)n repeat, while the motif8may have SRp20-specific binding site. We observed that:1) All RNA secondary structures of the minigene and mutants shared similar properties:CA-repeat regions or CA clusters tended to form stable loops by themselves or with other CA rich regions. On the other hand, the conservative sequences (GC rich) tended to form stems with varying stability.2) The stability of the secondary structures of the various constructs, and/or "microstability" of stem/loop elements may differ within constructs and these differences correlated with the observed splice ratios.3) When the stability of the secondary structures was the same in different minigene constructs, the missing motif sequence(s) with potential splicing site may be the reason of different splicing efficiency.Part III:The analysis of specific (CA)n motif demonstrated potential RNA-binding proteins in the(CA)n motifs.:(CA)n motifs may exist in the enhancers and silencers that affect RNA splicing. A specific binding site of SRp20exists in the conserved sequence of the motif8, which may enhance RNA splicing in this study.Conclusions:The (CA)n Motif8and Motif9of SP-B intron4are able to enhance and attenuate, respectively, RNA splicing. A specific RNA-binding site of SRp20exists in the conserved sequence of the motif8, which may have a role of enhancing RNA splicing. The RNA secondary structures may be the considerable element in the analysis of different splicing efficiency.