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Single electron transfer between sulfonium and tryptophan enables site-selective photo crosslinking of methyllysine reader proteins

ABSTRACT: Reader proteins play significant roles in cell signaling from site-specific lysine methylation, but progress of new reader investigations is rather slow. Photoreactive crosslinkers enable the discovery of reader proteins, but the chemical synthesis required to optimize the placement of the photoreactive group is laborious. In addition, active intermediates such as nitrene and carbene may cause significant non-specific crosslinking. Here we report dimethylsulfonium as a methyllysine mimic that binds to specific readers and can subsequently crosslink to a conserved tryptophan inside the binding pocket through single electron transfer under UV irradiation. The crosslinking requires σ-π electron-donor-acceptor interaction between sulfonium and indole as a key step, facilitating excellent tryptophan site-selectivity and orthogonality to distinct methyllysine readers. Moreover, excited tryptophan without proximate sulfonium leads to minimal non-specific crosslinking from relaxation. This method could escalate the discovery of methyllysine readers from complex cell samples. Furthermore, this photo crosslinking concept could also be expanded to develop more types of microenvironment-dependent conjugations to site-specific tryptophan.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Qingyun Yang

LAB HEAD: Mingxuan Wu

PROVIDER: PXD051693 | Pride | 2024-06-30

REPOSITORIES: Pride

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Project description:Protein-RNA interactions are integral components of nearly every aspect of biology including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions. Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTR) covering 72% of protein-coding transcripts encoded in the genome, confirming 3M-bM-^@M-^Y UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3M-bM-^@M-^Y UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression. Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo. Duplicate gPAR-CLIP and mRNA-seq libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. Additional duplicate mRNA-seq libraries were sequenced from yeast strains grown in the absence of 4-thiouracil. gPAR-CLIP libraries were used to determine regions of mRNA bound by proteins. mRNA-seq libraries served as controls for mRNA abundance. A Puf3p PAR-CLIP library was sequenced to determine how well gPAR-CLIP captured the binding signatures of a single RNA-binding protein.

			Action	DRS
	H3K4_Group1_sample1.raw	Raw
	H3K4_Group1_sample2.raw	Raw
	H3K4_Group1_sample3.raw	Raw
	H3K4_Group1_sample4.raw	Raw
	H3K4_Group2_sample1.raw	Raw

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Single electron transfer between sulfonium and tryptophan enables site-selective photo crosslinking of methyllysine reader proteins

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