Roberts, J. T. | bioRxiv Science

bioRxiv2020

RNA matchmaking remodels lncRNA structure and promotes PRC2 activity

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target genes can relieve RNA inhibition of PRC2. RNA matchmaking is promoted by heterogenous nuclear ribonucleoprotein (hnRNP) B1, which uses multiple protein domains to bind regions of HOTAIR via multi-valent protein-RNA interactions. Chemical probing demonstrates that RNA matchmaking changes HOTAIR RNA structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

biochemistry↗

bioRxiv2020

Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes, however accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-Seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP) based approaches that rely on inducing specific mutations during reverse transcription via UV crosslinking of the anti-m6A antibody to methylated RNA have been employed to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.

genomics↗