Supplemental Data of "Systematic Discovery of Xist RNA Binding Proteins"

We develop comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) to reveal the composition and dynamics of specific long noncoding RNA- protein complexes (lncRNPs) in vivo. ChIRP-MS across four ncRNAs nominates protein interactors likely contributing to specific RNA functions. ChIRP-MS of snRNAs discovers an U1-specific link to 3’ RNA processing machinery. Xist, a key lncRNA for X- chromosome inactivation (XCI), interacts with 81 proteins in chromatin modification, nuclear matrix, and RNA remodeling pathways. Xist lncRNP is assembled in two steps coupled with transition from pluripotency to differentiation, and Xist lncRNP is nearly identical in random vs. imprinted XCI. HnrnpK participates in Xist-mediated gene silencing and histone modifications but not Xist localization. Drosophila Split ends homolog Spen interacts via the A-repeat domain of Xist and is further required for gene silencing. Thus, Xist lncRNA engages diverse protein complexes in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.

The supplemental data contain the following:
Table S1. Full list of enriched U1/U2 associated proteins and the peptide counts detected in each experiment.
Table S2. Sequences of ChIRP probes for the indicated RNAs and Usp9x intron-smFISH probes used in this study.
Table S3. Full list of enriched Xist RBPs and the peptide counts detected in each experiment (“-“ indicates respective negative controls).
Table S4. Specific vs. non-specific components in Xist lncRNP. Columns C-F indicate the ranks of peptide abundances in each of the indicated ChIRP experiments. Xist-specific interactors (highlighted in yellow) are defined as proteins with Xist ChIRP-MS rank at least twice better than in ChIRP-MS of U1, U2, and 7SK. Known gene repressors are annotated in blue. Non-specific interactors (highlighted in red) are proteins that show rank ratio <2 in Xist ChIRP vs. U1, U2, or 7SK. Many in the latter set are retrieved by all four ncRNAs and should be interpreted with caution in future ChIRP-MS experiments.
Table S5. Full list of siRNAs used in this study.