Identifying and Characterizing Functional Sequence Elements in Long Noncoding RNAs

Abstract

In the last few years, high-resolution sequencing data has enabled the discovery of thousands of novel long noncoding RNAs (lncRNAs). Increasing numbers of mammalian lncRNAs are known to play fundamental roles in various biological processes—including aging and development—as well as in complex human diseases such as cancer. As a class, lncRNAs are enriched in the nucleus and many functionally relevant lncRNAs like XIST, NEAT1 and MALAT1 have nuclear roles regulating gene expression, epigenetic status, or higher-order nuclear architecture. While nuclear enrichment of lncRNAs appears to be important, the mechanisms for this localization remain inchoate. Previous work has hinted at a potential link between lncRNA-encoded local repeats and the nuclear localization of the transcripts that encode them. Interestingly, from a genomic perspective, local repeat elements are also enriched in lncRNA loci. However, in general, sequence elements responsible for RNA nuclear enrichment—and the broader significance of local repeats to lncRNA function—remain largely unknown. As a starting point for our experiments, we developed the massively parallel RNA assay (MPRNA), a method that simultaneously interrogates tens of thousands of unique transcripts for RNA-based function. Using an MPRNA that deciphers RNA nuclear enrichment domains, we analyzed 11,696 chimeric transcripts derived by densely tiling 38 lncRNAs. A statistical framework was developed to analyze MPRNA data and model the per-nucleotide nuclear localization of each parental lncRNA, from which we inferred 109 unique, conserved regions that appear responsible for nuclear enrichment. Two of these novel regions—and smaller sequence motifs highly represented therein—were validated using single molecule FISH. Moreover, we applied a range of bioinformatics and biochemical approaches to systematically screen for potential functions of local repeats within the lncRNA FIRRE locus, which has an important role in nuclear architectural organization. These experiments revealed that two such local repeats—R0 and RRD—serve dramatically different purposes. The intronic R0 repeat appears to function as an enhancer element for FIRRE, while the exonic RRD repeat functions as a nuclear enrichment domain. Collectively, this work helps elucidate the sequence elements that dictate lncRNA nuclear enrichment and provides insights into the function of lncRNA local repeats.