Identifying splicing targets of CLAMP by mRNA-sequencing. Identifying splicing targets of CLAMP by mRNA-sequencing

CLAMP is a GA-repeat motif binding transcription factor that regulates gene expression. We hypothesized that CLAMP also regulates RNA processing, specifically alternative splicing that occurs co-transcriptionally because CLAMP is bound to intronic regions that are rich in polypyrimidine tracts which contain GA-rich sequences. Furthermore, GA-rich repeat sequences are thought to have evolved from polypyrimidine tracts that regulate splicing. Also, MALDI-mass spectrometry data identifying putative CLAMP interactors found association with 33 RNA binding proteins, including 6 that regulate alternative splicing. Thus, to test our hypothesis that CLAMP shapes transcriptome diversity by regulating RNA transcript splicing, we performed mRNA-sequencing in the presence and absence of CLAMP in Kc (female) and S2 (male) cell lines and used a SUPPA based pipeline called time2splice (https://github.com/ashleymaeconard/time2splice) to analyze the sequencing data. Using both cell lines helped us to identify female (Kc) and male (S2) specific splicing events. We identified 452 genes differentially spliced beetween Kc and S2 cells. There are 46 and 113 CLAMP-dependent splicing events in Kc and S2 cells, respectively. Interestingly, 45 CLAMP-dependent events (belonging to 42 genes) are specific to Kc cells, i.e female-specific, whereas 112 CLAMP dependent events (belonging to 100 genes) in S2 cells are specific to S2 cells, i.e male-specific. Therefore, we identified a new role for the transcription factor CLAMP in regulated sex-specific splicing events in Kc and S2 cells. Overall design: dsRNA for CLAMP was used to deplete CLAMP from cell lines (Kc and S2), validated using qRT-PCR for CLAMP mRNA transcript. Total RNA was extracted from control (GFPRNAi) and experimental (CLAMPRNAi) Kc and S2 cell lines to make cDNA libraries and sequenced. Sequencing data was analyzed for changes in splicing events between control and experimental groups. Three replicates for each categories was performed.

CLAMP是一类结合GA重复基序（GA-repeat motif）的转录因子，可调控基因表达。我们提出如下假说：CLAMP亦可调控RNA加工过程，具体为共转录层面发生的可变剪接（alternative splicing），依据在于CLAMP可结合于富含多嘧啶序列（polypyrimidine tracts）且包含GA富集序列的内含子区域。此外，GA富集重复序列被认为起源于调控剪接的多嘧啶序列。另有研究通过基质辅助激光解吸电离质谱（MALDI-mass spectrometry）鉴定CLAMP潜在互作蛋白的实验数据显示，CLAMP可与33种RNA结合蛋白存在相互作用，其中6种可调控可变剪接。为验证"CLAMP通过调控RNA转录剪接以塑造转录组多样性"这一假说，我们分别在Kc（雌性）与S2（雄性）细胞系中设置CLAMP正常表达与敲低两组条件，开展mRNA测序（mRNA-sequencing），并使用一款基于SUPPA的名为time2splice（https://github.com/ashleymaeconard/time2splice）的分析流程对测序数据进行解析。同时使用两种细胞系，有助于我们识别雌性（Kc细胞）与雄性（S2细胞）特异性的可变剪接事件。我们共鉴定出452个在Kc与S2细胞间存在差异可变剪接的基因。其中，Kc与S2细胞中分别存在46个和113个CLAMP依赖性可变剪接事件。值得注意的是，45个CLAMP依赖性可变剪接事件（隶属于42个基因）仅在Kc细胞中存在，即雌性特异性事件；而S2细胞中的112个CLAMP依赖性可变剪接事件（隶属于100个基因）则仅在S2细胞中存在，即雄性特异性事件。综上，我们明确了转录因子CLAMP在调控Kc与S2细胞性别特异性可变剪接事件中的全新功能。实验总体设计：本研究使用双链RNA（dsRNA）靶向CLAMP，以敲低Kc和S2细胞系中的CLAMP表达，并通过实时定量逆转录聚合酶链反应（qRT-PCR）验证CLAMP mRNA的表达水平。随后，分别从对照组（GFPRNAi，即GFP RNA干扰组）与实验组（CLAMPRNAi，即CLAMP RNA干扰组）的Kc和S2细胞中提取总RNA，构建cDNA文库并开展高通量测序。对测序数据进行分析，以比对对照组与实验组间可变剪接事件的表达差异。每个实验分组均设置3次生物学重复。