PDF(Pubmed)
Abstract：
Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.
摘要：
Mirtron代表microRNAs（miRNAs）的一个亚类，其成熟依赖于剪接机制。然而,这种与Drosha无关的处理的分子细节仍然没有完全理解；作为一个例子，即使存在剪接位点突变，微处理器复合物也无法处理转录物中的mirtronicpre-miRNA。为了研究可变剪接位点对mirtron形成的影响，我们生成了含有人工内含子的增强型绿色荧光蛋白（EGFP）报告基因，以比较经典miRNAs和mirtro子的处理。尽管两个剪接位点的突变产生了复杂的替代转录本模式，与规范的hsa-mir-33bmiRNA的正常加工相反，mirtron的形成总是受到严重影响。然而,我们还发现，虽然它的形成也受到了阻碍，mirtron来源的hsa-mir-877-3pmiRNA比hsa-mir-877-5p受某些突变的影响更小.通过击倒Drosha,我们表明，这种现象不依赖于微处理器的活性，而是指向来自不同臂的miRNA之间的潜在稳定性差异。我们的结果表明，当主要剪接位点突变时，mirtron的形成不能通过附近的替代剪接位点来拯救，5p和3p物种之间的稳定性差异也应考虑到mirtros的功能研究。
