Genome-wide analysis of gene expression and intron retention during deveopment in a U12-type splicing-deficient zebrafish mutant [microarray]

To determine the global impact of the clbn mutation on gene expression and efficiency of U2- and U12-type splicing, we analyzed the transcriptome of 108hpf wt and clbn mutant larvae by microarrays and RNA sequencing. RNAseq data was used to characterize intron retention of U2-type and U12-type intron on a genome-wide scale to confirm that rnpc3 deficiency specifically impairs U12-type splicing. RNAseq and microarray data were combined to yield high-confidence lists of differentially expressed genes which show that impaired U12-type splicing has a wide-ranging effect on the developing transcriptome. Total RNA was prepared from pools consisting of approx. 20 individually genotyped homozygous wildtype or mutant larvae, respectively. Three biologically independent replicate pools were generated and analyzed for each condition.

为明确clbn突变对基因表达及U2型剪接（U2-type splicing）、U12型剪接（U12-type splicing）效率的全局影响，我们通过基因芯片（microarrays）与RNA测序（RNA sequencing）技术，分析了108 hpf野生型（wildtype, wt）及clbn突变型幼体的转录组。我们利用RNA测序数据在全基因组范围内表征U2型内含子与U12型内含子的内含子保留情况，以确认rnpc3缺陷会特异性损伤U12型剪接。结合RNA测序与基因芯片数据，我们得到了高置信度的差异表达基因列表，结果证实受损的U12型剪接对发育中转录组具有广泛影响。总RNA分别从约20株经独立基因型鉴定的纯合野生型或突变型幼体混合池中提取得到。每种实验条件均设置3个独立生物学重复混合池，并完成了相关分析。