ABSTRACT

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 a 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.

摘要

最长的人类 microRNA (miRNA) 簇之一位于 19 号染色体 (C19MC)，包含 46 个 miRNA 基因，这些基因被认为是从一个共同的长非编码转录本以相似的水平同时表达的。研究仅表达 C19MC 的两种组织类型，我们可以证明成熟 miRNA 水平在胚胎干细胞中朝着簇的 3ʹ 末端存在组织特异性和染色体位置依赖性降低，但在胎盘中没有。尽管 C19MC 转录水平在干细胞中显着降低，但在初级 miRNA 水平上不存在这种逐渐降低，这表明转录后加工的差异可以解释这一观察结果。通过消耗 Drosha，微处理器复合体的核酸酶成分，我们可以进一步增强干细胞的位置减少，证明微处理器复合体的组织特异性、局部可用性可能是该现象背后的原因。此外，我们可以描述一个在胎盘中完全活跃的组织特异性启动子，并且表观遗传标记分析表明该区域存在几个推定的增强子序列。进行特异性染色质免疫沉淀，然后进行实时定量 PCR 实验，我们可以显示 Drosha 与胎盘中选定的增强子区域有很强的关联，但在胚胎干细胞中则不然。这些增强子可以解释更有效的微处理器共转录募集，从而更有效地处理整个胎盘簇中的 pri-miRNA。