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MicroRNAs (miRNAs) are 21–25-nucleotide-long, noncoding RNAs that are involved in translational regulation. Most miRNAs derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm. Sequence variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have profound affects on miRNA biogenesis and function. In the context of analyzing the roles of miRNAs in Schizophrenia and Autism, we defined at least 24 human X-linked miRNA variants. Functional assays were developed and performed on these variants. In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of processing. Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature miRNA sequence may drive the evolution of new microRNAs by altering their biological function. Finally, the miRNAs examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases.  相似文献   

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目的:旨在建立一个多重表达任意miRNA的方案,实现多个pri-miRNA表达阅读框的串联,用于miRNA簇或者miRNA家族的功能研究。方法:通过改造CRISPR/Cas9系统共表达载体42230,插入EGFP编码序列以及单个pri-miRNA序列,实现单个pri-miRNA表达载体可视化的构建。在单个pri-miRNA表达载体的基础上,通过同源重组的方式,插入下一个pri-miRNA序列,实现多个pri-miRNA表达阅读框的串联表达。结果:以miR29家族的三个miRNA为例,验证了在pri-miRNA表达阅读框串联方面的可行性;在多重的pri-miRNA表达载体中,miRNA成熟体验证结果表明,pri-miR29a和pri-miR29b能够在哺乳动物细胞中被加工成熟,其成熟体明显地过表达。在转染的293A细胞中,miR29家族的靶基因PTEN的表达水平显著降低。结论:所构建的pri-miRNA多重表达方案,能够实现多个外源miRNA在同一细胞中的共表达,对于探索miRNA家族和miRNA簇的拮抗或协同调控功能起到重要的推进作用。  相似文献   

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Common functions for diverse small RNAs of land plants   总被引:13,自引:0,他引:13  
Endogenous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), are critical components of plant gene regulation. Some abundant miRNAs involved in developmental control are conserved between anciently diverged plants, while many other less-abundant miRNAs appear to have recently emerged in the Arabidopsis thaliana lineage. Using large-scale sequencing of small RNAs, we extended the known diversity of miRNAs in basal plants to include 88 confidently annotated miRNA families in the moss Physcomitrella patens and 44 in the lycopod Selaginella moellendorffii. Cleavage of 29 targets directed by 14 distinct P. patens miRNA families and a trans-acting siRNA (ta-siRNA) was experimentally confirmed. Despite a core set of 12 miRNA families also expressed in angiosperms, weakly expressed and apparently lineage-specific miRNAs accounted for the majority of miRNA diversity in both species. Nevertheless, the molecular functions of several of these lineage-specific small RNAs matched those of angiosperms, despite dissimilarities in the small RNA sequences themselves, including small RNAs that mediated negative feedback regulation of the miRNA pathway and miR390-dependent ta-siRNAs that guided the cleavage of AUXIN RESPONSE FACTOR mRNAs. Diverse, lineage-specific, small RNAs can therefore perform common biological functions in plants.  相似文献   

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