Functional large non-coding RNAs in mammals

Abstract

It is now clear that RNA is more than a messenger and performs vast and diverse functions. These functional RNAs include the ribosomal, transfer, and splicing-associated RNAs along with a cast of tiny RNAs, including microRNAs and other families. In addition to these classic examples, there were a handful of known functional large ncRNAs that play important biological roles. To identify additional functional large ncRNAs we exploited a chromatin signature of actively transcribed genes to define discrete transcriptional units that do not overlap any known proteincoding genes. Using this approach we identified -3,500 transcriptional units in the human and mouse genomes that produce multi-exonic RNAs that lack any coding potential. We termed these large intergenic non-coding RNAs (lincRNAs). Importantly, these lincRNAs exhibit strong purifying selection across various mammalian genomes. To determine whether the lincRNA transcripts themselves have biological functions, we undertook systematic loss-of-function experiments on most lincRNAs defined in mouse embryonic stem cells (ESCs). We showed that knockdown of the vast majority of ESC-expressed lincRNAs has a strong effect on gene expression patterns in ESCs, of comparable magnitude to that seen for the well-known ESC regulatory proteins. We identify dozens of lincRNAs that upon loss-of-function cause an exit from the pluripotent state and dozens of additional lincRNAs that, while not essential for the maintenance of pluripotency, act to repress lineage-specific gene expression programs in ESCs. Despite their important functional roles, how lincRNAs exert their influence was unknown. We showed that many lincRNAs physically interact with the Polycomb Repressive Complex. We systematically analyzed chromatin-modifying proteins that have been shown to play critical roles in ESCs and identified 11 additional chromatin complexes that physically interact with the ESC lincRNAs. Altogether, we found that -30% of the ESC lincRNAs are associated with multiple chromatin complexes. These interactions are important for proper regulation of gene expression programs in ES cells. Our data suggests a model whereby a distinct set of lincRNAs is transcribed in a cell type and interacts with ubiquitous regulatory protein complexes to give rise to cell-type-specific RNA-protein complexes that coordinate cell-type specific gene expression programs.