Abstract
Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. XIST controls the inactivation of an entire X chromosome in female placental mammals. Here we develop and integrate several orthogonal structure-interaction methods to demonstrate that XIST RNA-protein complex folds into an evolutionarily conserved modular architecture. Chimeric RNAs and clustered protein binding in fRIP and eCLIP experiments align with long-range RNA secondary structure, revealing discrete XIST domains that interact with distinct sets of effector proteins. CRISPR-Cas9-mediated permutation of the Xist A-repeat location shows that A-repeat serves as a nucleation center for multiple Xist-associated proteins and m6A modification. Thus modular architecture plays an essential role, in addition to sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and suggests a general strategy for mechanistic studies of large ribonucleoprotein assemblies.
Highlights
Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms
The list of functional Long noncoding RNAs (lncRNAs) is growing rapidly as more studies are conducted in a wide variety of systems. lncRNAs are distinguished from mRNAs in their processing and ultimate mechanisms of action[10,11]
To determine the structural of the multi-functional XIST RNP complex, we develop and integrate several methods, including PARIS27, formaldehyde RNA immunoprecipitation sequencing[35], enhanced crosslinking and immunoprecipitation (eCLIP; ultraviolet (UV) crosslinking)[36], and PIRCh37
Summary
Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. Acylation analyzed by Primer Extension (SHAPE) and dimethyl sulfate sequencing (DMS-seq) probe nucleotide (nt) accessibility, a proxy for RNA base pairing probability[14]. These two approaches have been applied to several in vitro transcribed lncRNAs, such as XIST, HOTAIR, COOLAIR, and Braveheart[15,16,17,18]. DMS-seq on the XIST RNA suggested functional local structure elements but did not reveal high-level organization[19]. These studies reported vaguely defined domains in these long transcripts, but it remains unclear whether these domains are relevant in physiological conditions. Computational modeling based on chemical probing is prone to errors, especially for long transcripts[20,21]
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