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Human RNA ligase 1 as a novel regulator of ribosome function and translation under oxidative stress

ABSTRACT: Human RNA ligase 1 (Rlig1) is a recently identified human 5’-3’ RNA ligase essential for maintaining 28S rRNA integrity and promoting cell survival under oxidative stress. Although the enzymatic activity of Rlig1 implies a role in RNA maintenance or repair, its broader molecular context remains insufficiently characterised. Here we identify cellular interactors of Rlig1 using affinity enrichment coupled to mass spectrometry in a HEK293 Rlig1-KO model. Our approach revealed several RNA and DNA surveillance, degradation and repair proteins as well as a diverse set of RNA binding and RNA processing enzymes as potential interactors. Notably, ribosomal proteins were highly enriched, suggesting a link between Rlig1 and the translational machinery.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Florian Stumpf

LAB HEAD: Florian Stengel

PROVIDER: PXD067856 | Pride | 2026-05-25

REPOSITORIES: Pride

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			Action	DRS
	20240710_145530_240710_FS_WithoutRNA_Paper.sne	Other
	20240710_145530_240710_FS_WithoutRNA_Paper_Report.csv	Csv
	250820_Rlig1AffinityEnrichment_1.6.15_Paper.sps	Other
	Frankenfield2022_Universal_Contaminants.fasta	Fasta
	Q2305_FS_230428_NoRNA_str_AMP_1_052.raw	Raw

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Project description:Background: During gut colonization, the enteric pathogen C. jejuni has to surmount the toxic effects of reactive oxygen species produced by its own metabolism, by the host immune system and by the intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. Results: The mechanisms of oxidative stress defenses in Campylobacter jejuni were characterized by transcriptional profiling, genes mutagenesis, and phenotypic analysis. The transcriptome changes, in response to H2O2, cumene hydroperoxide, or menadione exposure, were found to be oxidant specific and revealed the differential expression of genes belonging to a variety of biological pathways, from the classical oxidative stress defense systems, to the heat shock response, DNA repair and metabolism, fatty acid and capsule biosynthesis, and multidrug efflux pumps. To define the peroxide sensing regulator PerR, an isogenic mutant was constructed and its transcriptome profile compared to the wild-type strain. Sixty-six genes were found to belong to the PerR regulon. PerR appear to regulate gene expression both dependently and independently of the presence of iron and/or H2O2. The perR mutant was affected in its motility and attenuated in the chick colonization model. Mutagenic and phenotypic studies of the superoxide disumutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed their role in oxidative stress defenses and chick gut colonization. Conclusion: This study reveals the interplay between PerR, the iron metabolism and the oxidative stress defenses and highlights their role in the colonization and/or survival of C. jejuni in the chick cecum. Keywords: Transcriptional response to 3 oxidants (H2O2, menadione and cumene hydroperoxide) and characterization of the perR regulon (comparison of the transciptomes from the wild-type and perR mutant). To investigate the transcriptional responses of C. jejuni to oxidant exposure, hydrogen peroxide (H2O2), cumene hydroperoxide (CHP), or menadione sodium bisulfite (MND) was added to the 50 ml broth at a final concentration of 1 mM. The same amount of water or DMSO was added to the bacterial culture that served as reference samples for the transcriptional profile study in response to H2O2, MND or CHP. Furthermore, to investigate the transcriptional response of C. jejuni to H2O2 exposure in the presence of excess iron, ferrous sulfate was added to the bacterial culture at a final concentration of 40 µM, 15 min prior to H2O2 exposure. Ten minutes after the addition of the oxidant, total RNA was extracted and processed for microarray hybridization. To identify the PerR regulon, the wild-type strain C. jejuni NCTC 11168 and the perR mutant were grown in 500 ml flasks containing 250 ml of MEMα medium. At mid-log phase, 50 ml of the cultures were transferred to 100 ml flasks and ferrous sulfate and/or H2O2 were added . Ten minutes following the addition of H2O2 the cells were collected and the total RNA extracted.

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Human RNA ligase 1 as a novel regulator of ribosome function and translation under oxidative stress

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