Biochemical Methods To Investigate lncRNA and the Influence of lncRNA:Protein Complexes on Chromatin

Abstract

Long noncoding RNAs (lncRNAs), defined as non-translated transcripts greater than 200 nucleotides in length, are often differentially expressed throughout developmental stages, tissue types, and disease states. The identification, visualization, and suppression/overexpression of these sequences have revealed impacts on a wide range of biological processes, including epigenetic regulation. Biochemical investigations on select systems have revealed striking insight into the biological roles of lncRNAs and lncRNA:protein complexes, which in turn prompt even more unanswered questions. To begin, multiple protein-and RNA-centric technologies have been employed to isolate lncRNA:protein and lncRNA:chromatin complexes. LncRNA interactions with the multi-subunit protein complex PRC2, which acts as a transcriptional silencer, represent some of the few cases where the binding affinity, selectivity, and activity of a lncRNA:protein complex have been investigated. At the same time, recent reports of full-length lncRNA secondary structures suggest the formation of complex structures with multiple independent folding domains and pave the way for more detailed structural investigations and predictions of lncRNA threedimensional structure. This review will provide an overview of the methods and progress made to date as well as highlight new methods that promise to further inform the molecular recognition, specificity, and function of lncRNAs. Graphical Abstract *Corresponding Author: amanda.hargrove@duke.edu. Notes The authors declare no competing financial interest. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe "noncoding RNA revolution" (Cech, Stetiz) has revealed myriad functional RNA molecules with roles extending far beyond that of a messenger between DNA and protein. 1 The world of noncoding RNAs (ncRNAs), or RNAs that are not usually translated to proteins, came to light in large part as a result of the Encyclopedia of DNA Elements (ENCODE) project. 2 This consortium found that while up to 90% of the genome was transcribed only 1.2% was translated to protein. Furthermore, this large pool of untranslated transcripts demonstrated biochemical indices of function traditionally ascribed solely to proteins. 3 Research exploring the biological activity of these ncRNA transcripts promptly grew. Among the many newly discovered functions of noncoding RNAs, several classes are now known to play critical roles in the regulation of gene expression 1 as well as disease progression. 4 NcRNAs are classified based on size, with small ncRNAs less than 200 n.t. and long noncoding RNAs (lncRNAs) greater than 200 n.t. Several small ncRNA classes, including microRNAs (miRNAs) and small-interfering RNAs (siRNA), regulate gene expression by forming partially complementary duplexes with mRNAs, which in turn promote mRNA degradation or inhibit mRNA translation into peptides. [5][6][7][8] LncRNAs, on the other hand, have been found to exhibit a wide range of regulatory roles, including traffick...

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