Biology subjects

Bufton, J. C.

Publications and source records attributed to Bufton, J. C..

3 recordsLinked to original sources

bioRxiv2024

Dynamic RNA binding and unfolding by nonsense-mediated mRNA decay factor UPF2

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway involved in translational control and gene expression regulation. Core NMD factors UPF1, UPF2 and UPF3B are conserved from yeast to humans and essential to target mcRNAs with a premature stop codon for decay. UPF2 binding to UPF1 activates UPF1s ATPase and helicase activities, and UPF2 binding to UPF3B is important for its association with the exon-junction complex and efficient NMD. However, UPF2s association with RNA remains largely uncharacterized. Here, we analyze nucleic acid binding, identifying the first and third MIF4G domains of UPF2 as main RNA-/DNA-binding modules. We find that UPF2s MIF4G domain-3 has RNA annealing activity while full-length UPF2 unfolds our reporter hairpin-RNA structure. We show that UPF2 preferentially binds and stabilizes single-stranded RNA (ss-RNA) in a sequence-independent manner. Concomitant to ss-RNA binding, UPF2 undergoes a distinct conformational change in its otherwise highly dynamic structure. UPF2s RNA binding and unfolding activity may support UPF1s helicase and mRNP remodeling activity and, in combination with UPF3B, stabilize UPF1s association with nonsense mRNA.

biochemistry↗

bioRxiv2024

A BTB extension and ion-binding domain contribute to the pentameric structure and TFAP2A binding of KCTD1

KCTD family proteins typically assemble into Cullin-RING E3 ligases. KCTD1 is an atypical member that functions instead as a transcriptional repressor. Mutations in KCTD1 cause developmental abnormalities and kidney fibrosis in scalp-ear-nipple syndrome. Here, we present unexpected mechanistic insights from the structure of full-length KCTD1. Disease-causing mutation P20S maps to an unrecognized extension of the BTB domain that contributes both to its pentameric structure and TFAP2A binding. The C-terminal domain (CTD) shares its fold and pentameric assembly with the protein GFRP despite lacking discernible sequence similarity. Most surprisingly, the KCTD1 CTD establishes a central channel occupied by alternating sodium and iodide ions that restrict TFAP2A dissociation. The elucidation of the structure redefines the KCTD family BTB domain fold and identifies an unexpected ion pore for future study of KCTD1s function in the ectoderm, neural crest and kidney.

molecular biology↗

bioRxiv2022

Molecular basis of neurodevelopmental disorder-causing mutation in nonsense-mediated mRNA decay factor UPF3B

UPF3B is a key nonsense-mediated mRNA decay (NMD) factor required for surveillance of mRNA and regulation of eukaryotic gene expression. Mutations in UPF3B cause intellectual disability. The underlying molecular mechanisms remain unexplored as the mutations lie in an uncharacterized region of UPF3B. Here, we show that UPF3B shares structural and functional homology to the Drosophila Behavior/Human Splicing protein family comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended -helical domains essential for ribosome- and RNA-binding and RNA-induced oligomerization. A co-crystal structure of UPF3B with the third middle domain of eukaryotic initiation factor 4G (MIF4GIII) of UPF2 reveals an unexpectedly intimate binding interface. UPF3Bs disease-causing mutation Y160D located in the NOPS-L domain reduces the UPF2 binding affinity ~40-fold compared to wildtype UPF3B. UPF3Bs paralogue UPF3A, an NMD antagonist which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ~10-fold higher affinity than UPF3B, leading to impaired NMD activity and upregulation of mRNAs involved in neurodevelopment.

biochemistry↗