Many intercellular signals are synthesised as transmembrane precursors that are released by proteolytic cleavage ('shedding') from the cell surface. ADAM17, a membrane-tethered metalloprotease, is the primary shedding enzyme responsible for the release of the inflammatory cytokine TNFα and several EGF receptor ligands. ADAM17 exists in complex with the rhomboid-like iRhom proteins, which act as cofactors that regulate ADAM17 substrate shedding. Here we report that the poorly characterised FERM domain-containing protein FRMD8 is a new component of iRhom2/ADAM17 sheddase complex. FRMD8 binds to the cytoplasmic N-terminus of iRhoms and is necessary to stabilise the iRhoms and ADAM17 at the cell surface. In the absence of FRMD8, iRhom2 and ADAM17 are degraded via the endolysosomal pathway, resulting in the reduction of ADAM17-mediated shedding. We have confirmed the pathophysiological significance of FRMD8 in iPSC-derived human macrophages and mouse tissues, thus demonstrating its role in the regulated release of multiple cytokine and growth factor signals.
All data generated or analysed during this study are included in the manuscript and supporting files.
- Matthew Freeman
- Ulrike Künzel
- Ulrike Künzel
- Boris Sieber
- Adam Graham Grieve
- Sally A Cowley
- Sally A Cowley
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Human iPSC lines were derived from dermal fibroblasts of donors that had given signed informed consent for the derivation of human iPSC lines from skin biopsies and SNP analysis (Ethics Committee: National Health Service, Health Research Authority, NRES Committee South Central, Berkshire, UK (REC 10/H0505/71)). All procedures on mice were conducted in accordance with the UK Scientific Procedures Act (1986) under a project license (PPL) authorized by the UK Home Office Animal Procedures Committee, project licenses 80/2584 and 30/2306, and approved by the Sir William Dunn School of Pathology Local Ethical Review Committee.
- Christopher G Burd, Yale School of Medicine, United States
© 2018, Künzel et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.
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