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Author:
Date:
2012-10-05
[Abstract] MicroRNAs(miRNAs) are a group of endogenously expressed 20~23 nt small noncoding RNAs, which can directly regulate mRNA stability or translation in a sequence specific manner by incomplete base pairing at the 3’UTR of target mRNA, or indirectly affect transcriptional network by regulating transcription factors. As key regulators of gene expression, miRNAs are involved in the control of diverse developmental and physiological processes, including embryogenesis, differentiation, developmental timing, organogenesis, growth control, and programmed cell death. Aberrant miRNA expression profiles have been observed in many pathological conditions, including cancers, psychiatric diseases, virus infection, etc. However, the underlying mechanisms have been difficult to study in part due to the ...
[摘要] 微小RNA（miRNA）是一组内源性表达的20〜23nt的小非编码RNA，其可以通过在靶mRNA的3'UTR处的不完全碱基配对以序列特异性方式直接调节mRNA稳定性或翻译，或间接影响转录网络调节转录因子。作为基因表达的关键调节剂，miRNA参与多种发育和生理过程的控制，包括胚胎发生，分化，发育时间，器官发生，生长控制和程序性细胞死亡。在许多病理状况，包括癌症，精神疾病，病毒感染等中已观察到异常的miRNA表达谱。然而，部分由于复杂组织的细胞异质性，潜在的机制难以研究。
为了系统地分析复杂组织中的miRNA表达，我们在这里提出了一种新的miRNA标签和亲和纯化方法，miRAP，其可以应用于小鼠中任何复杂组织中的遗传定义的细胞类型。这种方法基于这样的事实，即成熟的miRNA掺入RNA诱导的沉默复合物（RISC），其中Argonaute蛋白AGO2直接结合miRNA和它们的mRNA靶。我们证明表位标签的AGO2蛋白允许直接从组织匀浆使用针对工程分子标签的抗体的miRNA纯化。我们进一步建立Cre-loxP二元表达系统，以将表位标记的AGO2（tAGO2）递送到遗传定义的细胞类型。
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