HuR And TIA1/TIAL1Are Involved In Regulation Of Alternative Splicing Of SIRT1Pre-mRNA

HuR and TIA1/TIAL1regulate the alternative splicing of SIRT1pre-mRNAAlternative splicing (AS) is a eukaryote-specific cellular mechanism of creating diverse mRNA structures to economically make one gene produces different proteins at different time and different circumstances. AS is extremely widespread in human genes, there are approximately more than95%genes involved in AS, especially in the nervous system. The alternative splicing is a complex dynamic process and the regulation mechanism of AS is related to the integrated effect of multiple factors in multiple levels. The alternative splicing regulation of biologically important genes is expected to provide a new perspective for disease prevention and treatment.SIRT1, known as III histone deacetylases (HDACs), plays critical and multifunctional roles in metabolism, senescence, cancer, longevity, stress-responses, and has become an important therapeutic target across a range of age-related diseases. Recently researches demonstrated that SIRT1pre-mRNA was undergone alternative splicing to produce different isoforms, such as SIRT1full-length (SIRT1-FL) and SIRT1-△Exon8variants. In SIRT1-△Exon8, precisely the558bp of Exon8was excluded and partially depleted the deacetylase domain as result. The SIRT1-△Exon8isoform possesses distinct properties compared to classical SIRT1-FL, including RNA/protein stability, protein-protein interactions and deacetylase activity. So that changes of SIRT1alternative splicing pattern may cause a series of biological effects and even lead to disease. However, the mechanism of alternative splicing of SIRT1is still a mystery. Owing to the consequential biological function of SIRT1, thus exploring the mechanisms underlying the alternative splicing of SIRT1would be particularly imperative.Based on the analysis of SIRT1gene sequence, we noticed there are AT/T rich domains around the exon8of SIRT1gene which might be possibly the target of Hu/TIA. Hu and TIA are probably most extensively investigated RNA banding proteins (RBPs) which participate almost every aspect of RNA metabolism (from pre-mRNAs transcription to their translation into proteins). In recent years, more and more attentions focus on the RNA alternative splicing regulation of Hu and TIA protein. It was reported that HuR is involved in regulation of alternative splicing, in which HuR usually promotes variable exon skipping by inhibiting the association of U2snRNP auxiliary factor65kDa (U2AF65) with the upstream3’splice site or preventing the binding of U1and U6snRNPs to the downstream5’splice site, or accelerating the local transcriptional elongation rate upon binding to their target sequence. Whereas TIA1/TIAL1were also reported to act as alternative pre-mRNA splicing regulators, in which TIA1/TIAL1promote variable exon inclusion through binding to U-rich sequences and advancing the combination of U1and U6snRNPs to the5’splice site. Hu and TIA1/TIAL1, therefore, usually act sequentially on shared target mRNAs by competing to one same combining site (U/AU-rich motifs), cooperating to regulate mRNA metabolism including the process of pre-mRNA alternative splicing. Thus, we presumed that HuR or TIA1/TIAL1might combine to the sites around SIRT1exon8and regulate the alternative splicing of SIRT1pre-mRNA. To test this hypothesis, we choose the ubiquitously expressed HuR in Hu protein family and TIA1and TIAL1, two members of the TIA family, constructed HuR, TIA1and TIAL1over-expression or interference plasmids, respectively, to manipulate HuR, TIA1and TIAL1levels in293T and U251cell lines and study the alternative splicing of SIRT1exon8. We utilized co-transfection with the SIRT1minigene (contained alternatively spliceable exon8flanked by introns and7/9exons) and HuR or TIA1/TIAL1to confirm their roles in the regulation of alternative splicing of SIRT1. After that, we investigated the genetic mechanisms of HuR and TIA1/TIAL1on the regulation of SIRT1exon8. Then we use UV exposure and NMDA injury to study the regulation of HuR, TIA1and TIAL1on SIRT1exon8in some injury cases, and further to observe apoptosis and cells toxicity after these regulations. The results demonstrate that HuR, TIA1and TIAL1can regulate the alternative splicing of SIRT1pre-mRNA. HuR promoted exon8exclusion by influence the histone acetylation (acetylation increase) or methylating (methylating decrease) to accelerate RNA polymeraseⅡ (RNAPⅡ) elongation rate. On the contrary, TIA1/TIAL1through retroregulation of these histone modification (acetylation decrease or methylating increase) to step down RNAPⅡ elongation rate around SIRT1exon8and promote this exon inclusion. This study consists four parts as follows:Part Ⅰ HuR and TIA1/TIAL1involve in regulation of SIRT1pre-mRNA alternative splicingObjectiveConstructing HuR or TIA1/TIAL1over-expression or depletion plasmids and manipulate the levels of HuR or TIA1/TIAL1and observe their influences on endogenous and exogenous SIRT1exon8alternative splicing.Methods1. Constructed HuR or TIA1/TIAL1over-expression or depletion plasmids by enzyme digestion and conjugation procedure method, transfected HuR or TIA1/TIAL1plasmids into293T or U251cells by Attractene Transfection Reagent plasmid transfection technique, established blank groups and experimental groups, to test the SIRT1-FL and SIRT1-△Exon8mRNA levels in the293T and U251cells by PCR and Real-timePCR.2. Constructed SIRT1minigene which only contained alternatively spliceable exon8flanked by introns and7/9exons. Co-transfected the minigene with HuR or TIA1/TIAL1constructs or with vehicle into293T cells. The percentage of exon exclusion was calculated as the ratio of signal for the lower band (the shorter one, only exon7and exon9) and upper one (the longer one, contain exon7,8and9). The results showed a significant increase in exon8exclusion in HuR over-expression cells.Results1. Constructed the HuR or TIA1/TIAL1overexpression or shRNA interference plasmids, and successfully overexpressed or interfered HuR or TIA1/TIAL1in293T and U251cell lines, screened out HuR-shRNA①was the highest efficiency of HuR interference plasmid.2. In293T and U251cells, overexpression of HuR increased the level of endogenous SIRT1-△Exon8, depletion of HuR decreased SIRT1-△Exon8; The interference of TIA1or TIAL1individually, had little effects on SIRT1-FL and SIRT1-△Exon8, depleted both of them increased the endogenous SIRT1-△Exon8.3. The over-expression of HuR apparently increased exogenous SIRT1-△Exon8, whereas TIA1/TIAL1decreased it.Summary1. HuR or TIA1/TIAL1can regulate endogenous or exogenous SIRT1exon8alternative splicing.2. HuR promoted SIRT1exon8exclusion, whereas TIA1/TIAL1promoted this exon inclusion.Part Ⅱ The epigenetic mechanisms of HuR or TIA1/TIAL1on the regulation of SIRT1pre-mRNA alternative splicingObjective1. To observe the relationships between HuR or TIA1/TIAL1levels and the RNAPⅡ elongation rate around SIRT1exon8.2. To observe the influences of HuR or TIA1/TIAL1on the histone modifications (H3Ace, H4Ace, H3K9Ace, H3K9me3, H3K27me3, H3K36me3) around SIRT1exon8.MethodsTo manipulate the level of HuR or TIA1/TIAL1:1. Detected RNAP Ⅱ elongation rates by the two methods as follow: i) By detection the distribution of phosphorylation RNAP II along SIRT1gene using RNAP II subunit C-terminal domain (CTD) antibodies specific for serine phosphorylation of-2(H5) in chromatin co-immunoprecipitation (ChIP) to reflect the transcription elongation rate.ii) By detection and calculation the ratio of the regeneration pre-mRNA products on distal end (SIRT1exon9, transcriptional start site) and proximal end (SIRT1exon1, transcriptional start site) to reflect the transcription elongation rate.2. Detected the H3Ace, H4Ace, H3K9Ace, H3K9me3, H3K27me3and H3K36me3enrichment around SIRT1gene. Results1. The accumulation of phosphorylated RNAP Ⅱ around SIRT1exon8decreased in HuR over-expression or TIA1/TIAL1interference cells, and increased in HuR depletion or TIA1/TIAL1over-expression cells;2. The value of D/P which indirectly reflected the RNAP Ⅱ elongation rate was comparatively larger in HuR overexpression or TIA1/TIAL1interference cells, and diminutive in HuR depletion or TIA1/TIAL1overexpression cells;3. The over-expression of HuR increased the levels of H3Ace, H4Ace, H3K9Ace, and decreased the levels of H3K9me3, H3K27me3, H3K36me3around SIRT1exon8. On the contrary, the over-expression of TIA1/TIAL1resulted in H3Ace, H4Ace, H3K9Ace levels decreasing and H3K9me3%H3K27me3、H3K36me3increasing around SIRT1exon8. Summary1. HuR or TIA1/TIAL1can alter the RNAP Ⅱ elongation rate around SIRT1exon8by affecting the histone modification so as to regulate the alternative splicing of SIRT1pre-mRNA.2. HuR can obviously increase the abundance of H3Ace, H4Ace, H3K9Ace, and decreased the abundance of H3K9me3, H3K27me3, H3K36me3, which can accelerate the elongation rate of local RNAP Ⅱ.3. TIA1/TIAL1decreased the H3Ace, H4Ace, H3K9Ace, and increased H3K9me3, H3K27me3, H3K36me3, thereby to slow down the local RNAP Ⅱ around SIRT1exon8.Part Ⅲ HuR and TIA1/TIAL1participated to regulate SIRT1pre-mRNA alternative splicingObjectiveTo observe the relationships between HuR or TIA1/TIAL1and the alternative splicing of SIRT1pre-mRNA under Ultraviolet exposure and NMDA injury in293T cells. Methods1. To establish UV and NMDA injury models, then detected HuR or TIA1/TIAL1mRNA levels by qRT-PCR in293T cells.2. Over-expressed or depleted HuR or TIA1/TIAL1by plasmids transfection, meanwhile, established UV and NMDA injury models, then tested SIRT1-FL and SIRT1-△Exon8mRNA levels in293T cells.Results1. HuR mRNA level increased whereas TIA1and TIAL1mRNA levels decreased in NMDA and UV injury models.2. The over-expression of HuR or depletion of TIA1/TIAL1increased SIRT1exon8exlusion, while HuR depletion or TIA1/TIAL1over-expression result this exon8inclution.Summary1. HuR or TIA1/TIAL1can regulate SIRT1exon8alternative splicing in NMDA and UV injury models.2. HuR promoted SIRT1exon8skipping, TIA1/TIAL1promoted its inclusion as the same as in nomal condition.Part IV HuR or TIA1/TIAL1influence cell functions through regulating SIRT1pre-mRNA alternative splicingObjectiveTo detect the apoptosis and cytotoxicity after the regulation of HuR or TIA1/TIAL1on SIRT1pre-mRNA alternative splicing in UV injury cells.MethodsOver-expression or depletion of HuR or TIA1/TIAL1by plasmids transfection, to test cytotoxicity by CCK-8analysis, LDH release analysis and Calcein-AM/PI detection, then detected apoptosis by Annexin V-APC/7-ADD apoptosis assay and Caspase-3viability assay.Results1. In UV injury model, the SIRT1-△Exon8increased by the over-expression of HuR or depletion of TIA1/TIAL1raised cytotoxicity and as a result reducing cell viability and cell survival ratio. While the TIA1/TIAL1over-expression or HuR depletion had no statistics distinction.2. The regulation of HuR or TIA1/TIAL1on SIRT1exon8alternative splicing had no significant differences to apoptosis in UV injury cells, which hinted that, on one hand, the mechanisms of cellular apoptosis regulation were more complex than can be imagined; on the other hand, the influence of SIRT1-△Exon8on apoptosis may be different from the other aspect of HuR or TIA1/TIAL1, so as to generated crosslink.Summary HuR or TIA1/TIAL1can influence apoptosis and cytotoxicity through regulating SIRT1exon8alternative splicing in UV injury model. The protection of SIRT1was weakening as the SIRT1-△Exon8increased, so that the injury was became more serious.Conclution1. HuR and TIA1/TIAL1were involved in the regulation of alternative splicing of SIRT1pre-mRNA.2. Through affecting the histone acetylation or methylation patterns near the SIRT1exon8, HuR and TIA1/TIAL1can accelerate or slow down the RNAP Ⅱ elongation rate, so as to promote or inhibit the exclusion of exon8.3. In some damage cases, HuR and TIA1/TIAL1were also involved in alternative splicing regulation of SIRT1, and this control can affect the cell stress response in injury conditions. If the products was mainly SIRT1-FL, the cell would avoid damage or the damage was lessen; If the SIRT1-△Exon8increased, would resulted in more serious injury.4. In view of the important neuroprotective effects of SIRT1, and the two variants resulted from the alternative splicing of SIRT1possess different biological effecys, thus exploring the underline mechanisms of SIRT1pre-mRNA to inhibit or reduce the production of SIRT1-△Exon8will help us to formulate a more reasonable prevention or treatment options target to this gene.