A functional screen of RNA binding proteins identifies genes that promote or limit the accumulation of CD138+ plasma cells
- The Babraham Institute, United Kingdom
- Queen Mary University of London, United Kingdom
Abstract
To identify roles of RNA binding proteins (RBPs) in the differentiation or survival of antibody secreting plasma cells we performed a CRISPR/Cas9 knockout screen of 1213 mouse RBPs for their ability to affect proliferation and/or survival, and the abundance of differentiated CD138+ cells in vitro. We validated the binding partners CSDE1 and STRAP as well as the m6A binding protein YTHDF2 as promoting the accumulation of CD138+ cells in vitro. We validated the EIF3 subunits EIF3K and EIF3L and components of the CCR4-NOT complex as inhibitors of CD138+ cell accumulation in vitro. In chimeric mouse models YTHDF2-deficient plasma cells failed to accumulate.
Data availability
The sgRNA library is available upon request and from Addgene (#169082). The CRISPR/Cas9 knockout screen data and m6A-eCLIP data that support the findings of this study have been deposited in GEO with the GSE179919 accession code, and the RNA-seq data has been deposited in GEO with the GSE179281 accession code.
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The RNA m6A binding protein YTHDF2 promotes the B cell to plasma cell transitionNCBI Gene Expression Omnibus, GSE179919.
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Transcriptomic analysis of in vitro induced germinal centre-like B cells and plasmablast differentiationNCBI Gene Expression Omnibus, GSE179281.
Article and author information
Author details
Funding
Biotechnology and Biological Sciences Research Council (BBS/E/B/000C0427)
- Martin Turner
Biotechnology and Biological Sciences Research Council (BBS/E/B/000C0428)
- Martin Turner
Wellcome Trust (200823/Z/16/Z)
- Martin Turner
Biotechnology and Biological Sciences Research Council (BB/L016745/1)
- David J Turner
European Molecular Biology Organisation (ALTF 880-2018)
- Fiamma Salerno
Cancer Research UK (C29967/A26787)
- Kamil R Kranc
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All mouse experimentation was approved by the Babraham Institute Animal Welfare and Ethical Review Body and was licensed by the United Kingdom Home Office under PPL P4D4AF812.
Copyright
© 2022, Turner et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A functional screen of RNA binding proteins identifies genes that promote or limit the accumulation of CD138+ plasma cells
eLife 11:e72313.
https://doi.org/10.7554/eLife.72313
Further reading
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- Developmental Biology
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Niches are often found in specific positions in tissues relative to the stem cells they support. Consistency of niche position suggests that placement is important for niche function. However, the complexity of most niches has precluded a thorough understanding of how their proper placement is established. To address this, we investigated the formation of a genetically tractable niche, the Drosophila Posterior Signaling Center (PSC), the assembly of which had not been previously explored. This niche controls hematopoietic progenitors of the lymph gland (LG). PSC cells were previously shown to be specified laterally in the embryo, but ultimately reside dorsally, at the LG posterior. Here, using live-imaging, we show that PSC cells migrate as a tight collective and associate with multiple tissues during their trajectory to the LG posterior. We find that Slit emanating from two extrinsic sources, visceral mesoderm and cardioblasts, is required for the PSC to remain a collective, and for its attachment to cardioblasts during migration. Without proper Slit-Robo signaling, PSC cells disperse, form aberrant contacts, and ultimately fail to reach their stereotypical position near progenitors. Our work characterizes a novel example of niche formation and identifies an extrinsic signaling relay that controls precise niche positioning.
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- Computational and Systems Biology
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The Notch signaling pathway uses families of ligands and receptors to transmit signals to nearby cells. These components are expressed in diverse combinations in different cell types, interact in a many-to-many fashion, both within the same cell (in cis) and between cells (in trans), and their interactions are modulated by Fringe glycosyltransferases. A fundamental question is how the strength of Notch signaling depends on which pathway components are expressed, at what levels, and in which cells. Here, we used a quantitative, bottom-up, cell-based approach to systematically characterize trans-activation, cis-inhibition, and cis-activation signaling efficiencies across a range of ligand and Fringe expression levels in Chinese hamster and mouse cell lines. Each ligand (Dll1, Dll4, Jag1, and Jag2) and receptor variant (Notch1 and Notch2) analyzed here exhibited a unique profile of interactions, Fringe dependence, and signaling outcomes. All four ligands were able to bind receptors in cis and in trans, and all ligands trans-activated both receptors, although Jag1-Notch1 signaling was substantially weaker than other ligand-receptor combinations. Cis-interactions were predominantly inhibitory, with the exception of the Dll1- and Dll4-Notch2 pairs, which exhibited cis-activation stronger than trans-activation. Lfng strengthened Delta-mediated trans-activation and weakened Jagged-mediated trans-activation for both receptors. Finally, cis-ligands showed diverse cis-inhibition strengths, which depended on the identity of the trans-ligand as well as the receptor. The map of receptor-ligand-Fringe interaction outcomes revealed here should help guide rational perturbation and control of the Notch pathway.
