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RNA promotes the formation of spatial compartments in the nucleus

Quinodoz, Sofia A. and Bhat, Prashant and Ollikainen, Noah and Jachowicz, Joanna W. and Banerjee, Abhik K. and Chovanec, Peter and Blanco, Mario R. and Chow, Amy and Markaki, Yolanda and Plath, Kathrin and Guttman, Mitchell (2020) RNA promotes the formation of spatial compartments in the nucleus. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20200826-110847363

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Abstract

The nucleus is a highly organized arrangement of RNA, DNA, and protein molecules that are compartmentalized within three-dimensional (3D) structures involved in shared functional and regulatory processes. Although RNA has long been proposed to play a global role in organizing nuclear structure, exploring the role of RNA in shaping nuclear structure has remained a challenge because no existing methods can simultaneously measure RNA-RNA, RNA-DNA, and DNA-DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the location of all RNAs relative to DNA and other RNAs. Using this approach, we identify many RNAs that are localized near their transcriptional loci (RNA-DNA) together with other diffusible ncRNAs (RNA-RNA) within higher-order DNA structures (DNA-DNA). These RNA-chromatin compartments span three major classes of nuclear functions: RNA processing (including ribosome biogenesis, mRNA splicing, snRNA biogenesis, and histone mRNA processing), heterochromatin assembly, and gene regulation. More generally, we identify hundreds of ncRNAs that form stable nuclear compartments in spatial proximity to their transcriptional loci. We find that dozens of nuclear compartments require RNA to guide protein regulators into these 3D structures, and focusing on several ncRNAs, we show that these ncRNAs specifically regulate heterochromatin assembly and the expression of genes contained within these compartments. Together, our results demonstrate a unique mechanism by which RNA acts to shape nuclear structure by forming high concentration territories immediately upon transcription, binding to diffusible regulators, and guiding them into spatial compartments to regulate a wide range of essential nuclear functions.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2020.08.25.267435DOIDiscussion Paper
https://github.com/GuttmanLab/sprite2.0-pipelineRelated ItemData/Code
ORCID:
AuthorORCID
Quinodoz, Sofia A.0000-0003-1862-5204
Bhat, Prashant0000-0003-3832-4871
Ollikainen, Noah0000-0002-1174-2400
Jachowicz, Joanna W.0000-0002-1599-682X
Chovanec, Peter0000-0001-8066-1893
Plath, Kathrin0000-0001-7796-3372
Guttman, Mitchell0000-0003-4748-9352
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Posted August 25, 2020. We thank Elizabeth Soehalim for help in adapting the SPRITE method; Sam Kim, Vickie Trinh, and Jasmine Thai for help with generating and validating cell lines and cell culture; Patrick McDonel for discussions and advice on the RD-SPRITE method and helpful comments on the manuscript; Andres Collazo for microscopy help; John Rinn, Drew Honson, Mackenzie Strehle, and Drew Perez for comments on the manuscript and helpful suggestions; Aaron Lin for sequencing help and advice; Shawna Hiley for editing; Inna-Marie Strazhnik and Sigrid Knemeyer for illustrations. S.A.Q. received support from the HHMI Gilliam Fellowship and NSF GRFP Fellowship. We thank Alexander Shishkin, Ward G. Walkup IV, Parham Peyda, and Vickie Trinh for help generating the Halo-tagged FL-SHARP vector. P.B. received support from NIH 5 T32 GM 7616-40, NIH NRSA CA247447, and the UCLA-Caltech Medical Scientist Training Program. N.O. is supported by the American Cancer Society Postdoctoral Fellowship (PF-17-240-01). J.W.J. is supported by a BBE post-doctoral fellowship from Caltech. A.K.B. was funded by NHLBI F30-HL136080 and the USC MD/PhD Program. 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, NIH Director’s Early Independence Award (DP5OD012190), CZI Ben Barres Early Career Acceleration Award, Sontag Foundation, Searle Scholars Program, Pew-Steward Scholars program, and funds from the California Institute of Technology. M.G. is a NYSCF-Robertson Investigator. 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. P.B. developed and optimized the RD-SPRITE protocol, performed SPRITE experiments, analyzed and interpreted data, contributed to data visualization, figure presentation, 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. J.W.J. designed, performed, acquired, and analyzed all the RNA-FISH, DNA-FISH, IF, IF/RNA-FISH experiments and made all imaging figures; performed all LNA-related experiments and generated the figures and results; performed Flavopiridol treatments and analysis; contributed to the writing of the centromeric RNA hub section, model schematics/illustrations, and provided comments and edits on the entire manuscript. A.K.B. performed all Kcnq1ot1 biochemical and functional experiments, including CRISPRi knockdowns, TSA treatments, 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; worked with MRB to purify SHARP and map it to Kcnq1ot1. P.C. led the effort on the data processing and curation, writing scripts and constructing pipelines that enabled data interpretation; was responsible for gene, repeat, and allele annotation as well as validation and producing several QC metrics; contributed to experimental optimization of the RNA-DNA SPRITE protocol. M.R.B. 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; advised and helped to develop and optimize the RNA molecular biology of the RD-SPRITE method in this project. 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. 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; wrote the paper with S.A.Q. and P.B. Declaration of Interests: A provisional patent has been filed for the SPRITE method. Data and Code Availability: The analysis pipeline used in this study is available at https://github.com/GuttmanLab/sprite2.0-pipeline. Datasets generated during this study will be available on GEO.
Funders:
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
NIH Predoctoral Fellowship5 T32 GM 7616-40
NIHCA247447
UCLA-Caltech Medical Scientist Training ProgramUNSPECIFIED
American Cancer SocietyPF-17-240-01
Caltech Division of Biology and Biological EngineeringUNSPECIFIED
NIH Postdoctoral FellowshipF30-HL136080
University of Southern CaliforniaUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
NIHU01 DA040612
NIHU01 HL130007
New York Stem Cell FoundationUNSPECIFIED
NIHDP5OD012190
Chan-Zuckerberg InitiativeUNSPECIFIED
Sontag FoundationUNSPECIFIED
Searle Scholars ProgramUNSPECIFIED
Pew-Steward Scholars ProgramUNSPECIFIED
CaltechUNSPECIFIED
National Heart, Lung, and Blood InstituteUNSPECIFIED
Record Number:CaltechAUTHORS:20200826-110847363
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200826-110847363
Official Citation:RNA promotes the formation of spatial compartments in the nucleus. Sofia A. Quinodoz, Prashant Bhat, Noah Ollikainen, Joanna W. Jachowicz, Abhik K. Banerjee, Peter Chovanec, Mario R. Blanco, Amy Chow, Yolanda Markaki, Kathrin Plath, Mitchell Guttman. bioRxiv 2020.08.25.267435; doi: https://doi.org/10.1101/2020.08.25.267435
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:105119
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:26 Aug 2020 19:11
Last Modified:26 Aug 2020 19:11

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