Published November 11, 2021 | Submitted + Supplemental Material + Accepted Version

Journal Article Open

RNA promotes the formation of spatial compartments in the nucleus

Creators: Quinodoz, Sofia A.; Jachowicz, Joanna W.; Bhat, Prashant; Ollikainen, Noah; Banerjee, Abhik K.; Goronzy, Isabel N.; Blanco, Mario R.; Chovanec, Peter; Chow, Amy; Markaki, Yolanda; Thai, Jasmine; Plath, Kathrin; Guttman, Mitchell

Abstract

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

Additional Information

© 2021 Elsevier Inc. Received 24 August 2020, Revised 25 August 2021, Accepted 13 October 2021, Available online 4 November 2021. We thank Elizabeth Soehalim, Sam Kim, Vickie Trinh, Alexander Shishkin, Ward G. Walkup IV, and Parham Peyda for help with experiments; Patrick McDonel for advice on the RD-SPRITE method and comments on the manuscript; Andres Collazo for microscopy help; John Rinn, Drew Honson, Mackenzie Strehle, and Drew Perez for comments on the manuscript; Aaron Lin for sequencing help and advice; Shawna Hiley for editing; and Inna-Marie Strazhnik and Sigrid Knemeyer for illustrations. This work was funded by an HHMI Gilliam Fellowship and NSF GRFP Fellowship (S.A.Q.); NIH 5 T32 GM 7616-40, NIH NRSA CA247447, and the UCLA-Caltech Medical Scientist Training Program (P.B.); American Cancer Society Fellowship (N.O.); Caltech BBE fellowship (J.W.J.); and NHLBI F30-HL136080 and USC MD/PhD Program (A.K.B.). Imaging was performed in the Biological Imaging Facility with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. This work was funded by the NIH 4DN (U01 DA040612 and U01 HL130007), the NYSCF, CZI Ben Barres Early Career Acceleration Award, Sontag Foundation, Searle Scholars Program, and funds from Caltech. Data and code availability: SPRITE datasets generated during this study have been deposited on GEO and are publicly available as of the date of publication at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE151515. Accession numbers are listed in the Key resources table. The original code for the SPRITE analysis pipeline used in this study is available on Github at https://github.com/GuttmanLab/sprite2.0-pipeline and https://github.com/GuttmanLab/sprite-pipeline. DOIs are listed in the Key resources table. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Author contributions: S.A.Q. conceived of this project with M.G., led the development and optimization of the RD-SPRITE method, performed experiments, analyzed and interpreted data, generated figures, oversaw all aspects of the project, and wrote the paper. J.W.J. designed, performed, acquired, and analyzed all the RNA-FISH, DNA-FISH, IF, and IF/RNA-FISH experiments; made all imaging figures; performed all LNA-related experiments and generated the figures and results; performed ActD and FVP treatments and analysis; performed imaging of SHARP and Kcnq1ot1/Nap1l4 localization; and contributed to the writing of the centromeric RNA hub section, model schematics/illustrations, and provided comments and edits on the entire manuscript. P.B. developed and optimized the RD-SPRITE protocol; performed SPRITE experiments; analyzed and interpreted data; contributed to data visualization, figure presentation, and model schematics/illustrations; and wrote the paper. N.O. led the effort to analyze and interpret data, wrote software, created new methods for data analysis and visualization, performed analysis and visualization on the data and contributed major findings and results, created main and supplemental figures, and contributed to the initial draft of paper, model schematics/illustrations, and reviewed and edited the manuscript. A.K.B. performed all Kcnq1ot1 biochemical and functional experiments, including CRISPRi knockdowns, TSA treatments, H3K27ac ChIP-seq, and functional characterizations; worked with A.C. to develop and characterize the inducible Kcnq1ot1 cell line and to generate homozygous deletions of the SHARP Binding Site within Kcnq1ot1; and worked with MRB to purify SHARP and map it to Kcnq1ot1. I.N.G. performed the data processing and analysis of the ActD-treated SPRITE datasets; developed statistical methods for multiway RNA analyses; created new methods for data analysis and visualization; wrote scripts and constructed pipelines that enabled data curation; and wrote and provided comments and edits for the manuscript, figures, supplemental tables, and methods. M.R.B. performed the ActD RD-SPRITE and DNA-SPRITE experiments, performed analysis on the H3K27ac ChIP-seq, developed the engineered SHARP lines for CLAP and methods for purification of SHARP, worked with A.K.B. to perform SHARP purifications for Kcnq1ot1 binding, and advised and helped to develop and optimize the RNA molecular biology of the RD-SPRITE method in this project. P.C. led the effort on the data processing and curation, writing scripts and constructing pipelines that enabled data interpretation of the RD-SPRITE dataset; was responsible for gene, repeat, and allele annotation as well as validation and producing several QC metrics; and contributed to experimental optimization of the RNA-DNA SPRITE protocol. A.C. developed all engineered cell lines used in this study, including the doxycycline inducible Xist cell lines, Kcnq1ot1 lines, SHARP binding site deletions, and dCas9 cell lines. Y.M. performed all live-cell 3D-SIM imaging and analysis of FL-SHARP and ΔRRM-SHARP localization. J.T. helped develop and maintain engineered cell lines used in this study, and aided in the preparation of samples used in this study. K.P. provided guidance and support on imaging, analysis, ideas, and discussions on the paper. M.G. conceived of this project with S.A.Q. and oversaw all experiments and analysis; performed computational analysis and generated scripts for analyzing the RD-SPRITE data; and wrote the paper. The co-second authors agree to listing their names in the second position on CVs, resumes, and presentations. Declaration of interests: S.A.Q. and M.G. are inventors on a patent covering the SPRITE method.

Attached Files

Accepted Version - nihms-1750767.pdf

Submitted - 2020.08.25.267435v1.full.pdf

Supplemental Material - 1-s2.0-S0092867421012307-mmc1.xlsx

Supplemental Material - 1-s2.0-S0092867421012307-mmc2.xlsx

Supplemental Material - 1-s2.0-S0092867421012307-mmc3.xlsx

Supplemental Material - 1-s2.0-S0092867421012307-mmc4.xlsx

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