Tissue-specific and transcription-dependent mechanisms regulate primary microRNA processing efficiency of the human Chromosome 19 MicroRNA Cluster

One of the longest human microRNA (miRNA) clusters is located on chromosome 19 (C19MC), containing 46 miRNA genes, which were considered to be expressed simultaneously and at similar levels from a common long noncoding transcript. Investigating the two tissue types where C19MC is exclusively expressed, we could show that there is a tissue-specific and chromosomal position-dependent decrease in mature miRNA levels towards the 3ʹ end of the cluster in embryonic stem cells but not in placenta. Although C19MC transcription level is significantly lower in stem cells, this gradual decrease is not present at the primary miRNA levels, indicating that difference in posttranscriptional processing could explain this observation. By depleting Drosha, the nuclease component of the Microprocessor complex, we could further enhance the positional decrease in stem cells, demonstrating that a tissue-specific, local availability of the Microprocessor complex could lie behind the phenomenon. Moreover, we could describe a tissue-specific promoter being exclusively active in placenta, and the epigenetic mark analysis suggested the presence of several putative enhancer sequences in this region. Performing specific chromatin immunoprecipitation followed by quantitative real-time PCR experiments we could show a strong association of Drosha with selected enhancer regions in placenta, but not in embryonic stem cells. These enhancers could provide explanation for a more efficient co-transcriptional recruitment of the Microprocessor, and therefore a more efficient processing of pri-miRNAs throughout the cluster in placenta. Our results point towards a new model where tissue-specific, posttranscriptional “fine-tuning” can differentiate among miRNAs that are expressed simultaneously from a common precursor.

人类最长的微小RNA（microRNA, miRNA）基因簇之一定位于19号染色体（C19MC），该簇包含46个miRNA基因，此前认为它们可从一条共同的长链非编码转录本同步表达，且表达水平相近。通过研究C19MC特异性表达的两种组织类型，我们发现：胚胎干细胞中成熟miRNA水平随基因簇向3'端延伸呈现组织特异性且依赖染色体位置的递减趋势，而胎盘组织中无此现象。尽管干细胞中C19MC的转录水平显著更低，但初级miRNA（primary miRNA, pri-miRNA）水平并未出现这种渐进式递减，这表明转录后加工过程的差异可解释该观测结果。通过敲除微处理器复合体（Microprocessor complex）的核酸酶组分Drosha，我们可进一步加剧干细胞中的位置依赖性递减效应，这表明该现象的背后可能是微处理器复合体的组织特异性局部可及性差异。此外，我们还发现了一个仅在胎盘组织中具有活性的组织特异性启动子，表观遗传标记分析显示该区域存在多个推定的增强子序列。通过特异性染色质免疫沉淀（chromatin immunoprecipitation, ChIP）结合实时定量PCR实验，我们发现Drosha与胎盘中选定的增强子区域存在强关联，而在胚胎干细胞中则无此关联。这些增强子可解释胎盘中微处理器复合体更高效的共转录招募过程，进而使得整个基因簇内的初级miRNA加工更为高效。我们的研究结果提出了一种新模型：组织特异性的转录后“精细调控”可区分从共同前体同步表达的不同miRNA。