Figure 5.

The MARF1 NYN displays endoribonuclease activity in vitro. (A) Upper panel: Schematic representation of the wild-type and mutant (MUT) MARF1 NYN proteins used for in vitro degradation assays. Mutant MARF1 protein contains tandem alanine substitutions for Asp426 and Asp427. Lower panel: Coomassie-stained SDS-PAGE gel of recombinant wild-type (WT) or mutant (MUT) MARF1 NYN proteins. (B) NYN^WT protein was incubated with a 5′-³²P-end-labelled U₃₀ oligonucleotide in the presence or absence of 3 mM Mn²⁺ or Mg²⁺. RNA was extracted at defined time points and resolved via denaturing PAGE. RNA signals were visualized by autoradiography. OH: hydrolysis ladder. (C) Time course analysis of NYN^WT and NYN^MUT proteins incubated with a 5′-³²P-end-labelled U₃₀ oligonucleotide in the presence of Mn²⁺. OH: hydrolysis ladder. (D) NYN^WT protein was incubated in the presence of Mn²⁺ with a 3′-³²P-end-labelled U₃₀ oligonucleotide. RNA was extracted at defined time points and resolved via denaturing PAGE. OH: hydrolysis ladder.