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A new world of RNA-binding proteins in heterocyst-forming cyanobacteria.

ABSTRACT: RNA-binding proteins (RBPs) are central components of gene regulatory networks and RNA metabolism. The differentiation of nitrogen-fixing heterocysts in some filamentous cyanobacteria and their cooperation with vegetative cells in the same filament is one of the evolutionarily oldest cases of true multicellularity. Nostoc sp. PCC 7120 (Nostoc) is a model organism for filamentous cyanobacteria that differentiate heterocysts. Although hundreds of noncoding transcripts may be involved in this process, the RBPs that could cooperate with them are poorly characterized. We have used R-DeeP/GradR, a technique based on the differential sedimentation of RNA-protein complexes after RNase treatment in density gradients. All macromolecular complexes in the cell are fractionated by a sucrose gradient followed by high-resolution proteomics. We have compared gradients of RNase-treated with untreated samples in triplicates. If a protein was part of an RNA-protein complex, the complex should be disassembled after the RNase treatment and a shift of the protein position in the gradient should be observed (RNA-dependent proteins). Our approach yielded 333 RNA-dependent proteins. The results were compared to a bioinformatic prediction of RBPs and biochemical validation. We validated in vivo the RNA-binding capacity of 6 new RBPs. Some of these proteins were previously known to be involved in essential biological processes such as photosynthesis or heterocyst differentiation, but their association with RNA was previously unknown. In other cases, the function of the validated candidates was unknown. A knock-out strain of alr1700, a gene encoding a validated RNA-binding protein of unknown function, showed a deregulation of heterocyst differentiation, confirming the role of RBPs in regulating this cell differentiation process.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Nostoc Sp. (strain Pcc 7120 / Utex 2576)

TISSUE(S): Photosynthetic Cell, Nitrogen Fixing Cell

SUBMITTER: Frank Stein

LAB HEAD: Prof. Dr. Wolfgang Hess

PROVIDER: PXD050404 | Pride | 2024-11-08

REPOSITORIES: Pride

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			Action	DRS
	Bert_221129_S3375_DP_GradR_gradient1_TMT18_R1_F1.raw	Raw
	Bert_221129_S3375_DP_GradR_gradient1_TMT18_R1_F10.raw	Raw
	Bert_221129_S3375_DP_GradR_gradient1_TMT18_R1_F11.raw	Raw
	Bert_221129_S3375_DP_GradR_gradient1_TMT18_R1_F12.raw	Raw
	Bert_221129_S3375_DP_GradR_gradient1_TMT18_R1_F2.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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A new world of RNA-binding proteins in heterocyst-forming cyanobacteria.

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