Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs.

The recent discovery that the human and other mammalian genomes produce thousands of long non-coding RNAs (lncRNAs) raises many fascinating questions. These mRNA-like molecules, which lack significant protein-coding capacity, have been implicated in a wide range of biological functions through diverse and as yet poorly understood molecular mechanisms. Despite some recent insights into how lncRNAs function in such diverse cellular processes as regulation of gene expression and assembly of cellular structures, by and large, the key questions regarding lncRNA mechanisms remain to be answered. In this review, we discuss recent advances in understanding the biology of lncRNAs and propose avenues of investigation that may lead to fundamental new insights into their functions and mechanisms of action. Finally, as numerous lncRNAs are dysregulated in human diseases and disorders, we also discuss potential roles for these molecules in human health.

Generation of a useful roX1 allele by targeted gene conversion.

Methods for altering the sequence of endogenous Drosophila melanogaster genes remain labor-intensive. We have tested a relatively simple strategy that enables the introduction of engineered mutations in the vicinity of existing P-elements. This method was used to generate useful alleles of the roX1 gene, which produces a noncoding RNA involved in dosage compensation. The desired change was first introduced into a genomic clone of roX1 and transgenic flies were generated that carry this sequence in a P-element. Targeted transposition was then used to move the P-element into roX1. Remobilization of the targeted insertion produced large numbers of offspring carrying chromosomes that had precisely introduced the engineered sequences into roX1. We postulate that this occurred by gap repair, using the P-element on the sister chromatid as template. This strategy was used to introduce six MS2 loops into the roX1 gene (roX1(MS2-6)), enabling detection of roX1 RNA by a MCP-GFP fusion protein in embryos. The roX1(MS2-6) remains under the control of the authentic promoter and within the correct genomic context, features expected to contribute to normal roX1 function. The ability to replace relatively large blocks of sequence suggests that this method will be of general use.

Co-Immunoprecipitation of long noncoding RNAs.

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.