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Crosstalk between the RNA-binding proteins Regnase-1 and -3 shapes mast cell survival and cytokine expression

ABSTRACT: Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in murine mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, while both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon activation, with Tnf being a primary target in both human and mouse cells. Further, Regnase-3 directly interacts with Regnase-1 in mast cells and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Mast Cell, Bone Marrow

SUBMITTER: Matteo Pecoraro

LAB HEAD: Silvia Monticelli

PROVIDER: PXD051849 | Pride | 2024-05-31

REPOSITORIES: Pride

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			Action	DRS
	20231116_MP114_MaMa_SA_130_IgG_mouseIP.raw	Raw
	20231116_MP114_MaMa_SA_130_IgG_ratIP.raw	Raw
	20231116_MP114_MaMa_SA_130_Reg1_mouseIP.raw	Raw
	20231116_MP114_MaMa_SA_130_Reg3_ratIP.raw	Raw
	20231116_MP114_MaMa_SA_131_IgG_mouseIP.raw	Raw

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Project description:RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, understanding the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions focus on single RBPs, however there are many hundreds RBPs encoded in the human genome, and each cell type expresses a different catalog of these regulatory molecules, greatly limiting the ability of these single RBP approaches to capture the global landscape of RNA-protein interactions. We and others have developed approaches to globally catalog regions of mRNAs that are bound by proteins in an unbiased manner. Here we describe a detailed protocol for performing our RNase-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). In this protocol, RNA-protein interactions are stabilized by cross-linking, and un-bound regions are digested with RNases, leaving only the protein-bound regions intact. To control for RNase insensitive regions, proteins are first denatured and then RNP complexes are subject to RNase treatment. After high-throughput sequencing of remaining fragments, peak calling is performed to identify protein protected sites (PPSs). We describe the application of this protocol to a human embryonic kidney cell line and perform basic quality control, reproducibility and benchmarking analyses. Finally, we describe the landscape of protein-interactions in HEK293T cells, underscoring the value of this approach. Future applications of this method to study the dynamics of RNA-protein interactions in developmental processes will help to uncover the role of RBPs in post-transcriptional regulation. Protein interaction profile sequencing (PIP-seq) in HEK293T cells. Three replicates of formaldehyde cross-linked PIP-seq are described

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Crosstalk between the RNA-binding proteins Regnase-1 and -3 shapes mast cell survival and cytokine expression

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