GSE126715
Modular structure and function of the XIST RNP in X chromosome inactivation
Long noncoding RNAs (lncRNAs) are an important class of transcripts that regulate gene expression on many levels, yet their mechanisms of action remain poorly understood. Previous studies have indicated that these RNAs can serve as modular scaffolds to recruit a variety of protein complexes and coordinate their distinct functions. XIST, a founding member of the lncRNA family, controls the inactivation of an entire X chromosome in placental mammals. Here we develop and integrate several orthogonal structure-interaction analysis methods to demonstrate that XIST RNA-protein complex folds into a modular architecture that is conserved in evolution. The discrete XIST RNA domains interact with distinct sets of effector proteins to orchestrate the X chromosome inactivation (XCI). The modular architecture plays an essential role, in addition to the 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 establishes a paradigm for mechanistic studies of lncRNA functions. Overall design: We used MeRIP-seq to determine the m6A modification pattern on mouse Xist after relocating the A-repeat region in the Xist architecture. Detailed method is as follows. Total mES RNA was subjected to one round of Poly(A)Purist MAG treatment to enrich for polyadenylated RNAs as per manufacturer's instructions (Ambion). RNA was then fragmented to 100nt median sized fragments using RNA Fragmentation Reagents (Ambion) and subjected to one round of m6A immunoprecipitation. For immunoprecipitation of RNA, 5 ug of m6A antibody (Millipore) was coupled to 40 ul Protein A Dynabeads (Novex) in 100 ul 1X IPP Buffer (50mM Tris-HCl, pH 7.5; 150mM NaCl; 0.1% NP-40; 5mM EDTA) overnight at 4C. Beads were then washed twice in 1X IPP Buffer. Fragmented RNA was denatured at 70C for 2 min, cooled on ice, and bound to antibody-beads in 185 ul 1X IPP Buffer for 3h at 4C. Beads were then washed sequentially with (2x) 500 ul 1X IPP Buffer, (2x) 500 ul Low Salt Buffer (0.25X SSPE; 1mM EDTA; 0.05% Tween-20; 37.5mM NaCl), (2x) 500 ul High Salt Buffer (0.25X SSPE; 1mM EDTA; 0.05% Tween-20; 137.5mM NaCl), (1x) 500 ul TET Buffer (10mM Tris-HCl, pH 8.0; 1mM EDTA; 0.05% Tween-20). Beads were eluted with 50 ul RLT Buffer (Qiagen RNeasy Mini Kit) and incubated at 25C for 5 min, and recovered with RNeasy Mini Kit followed by concentrating with Zymo RNA Clean & Concentrator in 10 ul water. 10 ng of input RNA (before immunoprecipitation) and 10 ng of immunoprecipitated RNA were then used to prepare sequencing libraries using SMARTer Stranded RNA-Seq Kit - Pico Input Mammalian (Clontech #634411), as per manufacturer's instructions. Sequencing libraries were pooled and sequenced on the Illumina MiSeq (files named *mar08* and *apr26*) and NextSeq (files named *jun24*).