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Mechanism of protein abundance-independent 3′UTR-regulated JMJD3 activity in human cells

ABSTRACT: The 3′UTR in the KDM6B mRNA can control the cellular activity of the produced KDM6B/JMJD3 protein, but the mechanism is unclear. To examine whether 3′UTR-dependent JMJD3 protein complexes are responsible for the observed 3′UTR-dependent protein activity, KDM6B cDNA expression constructs with or without the KDM6B 3′UTR were transfected into HeLa cells grown in SILAC media, followed by co-IP of the HA-tagged JMJD3 protein. Cells were grown in SILAC medium for 5 passages and were validated for amino acid incorporation before the experiment. We did not detect major differences in JMJD3 protein interactors, when JMJD3 was translated from mRNA templates with or without its 3′UTR.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Zhuoning Li

LAB HEAD: Christine Mayr

PROVIDER: PXD074293 | Pride | 2026-06-01

REPOSITORIES: Pride

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			Action	DRS
	2489YL_IP1_proteinGroups.xlsx	Xlsx
	2489YL_SILAC_Sample7_IP1Sec3.raw	Raw
	2489YL_SILAC_Sample8_IP1Sec2.raw	Raw
	2489YL_SILAC_Sample9_IP1Sec1.raw	Raw
	checksum.txt	Txt

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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.

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Mechanism of protein abundance-independent 3′UTR-regulated JMJD3 activity in human cells

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