Moran Victoria A, Niland Courtney N, Khalil Ahmad M
Department of Genetics, Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
Methods Mol Biol. 2012;925:219-28. doi: 10.1007/978-1-62703-011-3_15.
It is now estimated that the human genome encodes thousands of long noncoding (lnc)RNAs. These novel molecules are causing a paradigm shift in the field of molecular biology as a number of lncRNAs have been shown to be involved in a wide range of biological functions including regulation of gene expression. Also, misregulation of lncRNAs has been observed in human diseases such as cancer and neurological disorders. These findings have spurred a huge interest in elucidating the functions and mechanisms of lncRNAs; and therefore, the need for new methods to do so. In this chapter, we discuss RIP-Seq, a method that is utilized to discover the lncRNA partners of a specific protein. This procedure involves immunoprecipitation of a protein from cross-linked cell lysate followed by reverse-cross-linking, isolation, and deep sequencing of RNAs, leading to the identification of all lncRNAs that are associated with a specific protein complex.
目前估计人类基因组编码数千种长链非编码（lnc）RNA。这些新型分子正在引发分子生物学领域的范式转变，因为许多lncRNA已被证明参与广泛的生物学功能，包括基因表达调控。此外，在癌症和神经疾病等人类疾病中也观察到lncRNA的调控异常。这些发现激发了人们对阐明lncRNA功能和机制的巨大兴趣；因此，需要新的方法来实现这一目标。在本章中，我们将讨论RIP-Seq，这是一种用于发现特定蛋白质的lncRNA伴侣的方法。该过程包括从交联的细胞裂解物中免疫沉淀蛋白质，然后进行RNA的反向交联、分离和深度测序，从而鉴定与特定蛋白质复合物相关的所有lncRNA。
