Arabidopsis thaliana NudiXes have RNA-decapping activity

Abstract

Recent discoveries of various noncanonical RNA caps, such as dinucleoside polyphosphates (Np _n N), coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD) in all domains of life have led to a revision of views on RNA cap function and metabolism. Enzymes from the NudiX family capable of hydrolyzing a polyphosphate backbone attached to a nucleoside are the strongest candidates for degradation of noncanonically capped RNA. The model plant organism Arabidopsis thaliana encodes as many as 28 NudiX enzymes. For most of them, only in vitro substrates in the form of small molecules are known. In our study, we focused on four A. thaliana NudiX enzymes (AtNUDT6, AtNUDT7, AtNUDT19 and AtNUDT27), and we studied whether these enzymes can cleave RNA capped with Np _n Ns (Ap_2-5A, Gp_3-4G, Ap_3-5G, m⁷Gp₃G, m⁷Gp₃A), CoA, ADP-ribose, or NAD(H). While AtNUDT19 preferred NADH-RNA over other types of capped RNA, AtNUDT6 and AtNUDT7 preferentially cleaved Ap₄A-RNA. The most powerful decapping enzyme was AtNUDT27, which cleaved almost all types of capped RNA at a tenfold lower concentration than the other enzymes. We also compared cleavage efficiency of each enzyme on free small molecules with RNA capped with corresponding molecules. We found that AtNUDT6 prefers free Ap₄A, while AtNUDT7 preferentially cleaved Ap₄A-RNA. These findings show that NudiX enzymes may act as RNA-decapping enzymes in A. thaliana and that other noncanonical RNA caps such as Ap₄A and NADH should be searched for in plant RNA.

Fig. 1. Screening of the RNA-decapping activity of AtNUDT6, 7, 19, and 27. (A) Scheme of the experimental setup. ³²P labelled RNA was prepared by in vitro transcription with T7 RNA polymerase in the presence of the small molecules Np_nNs, NAD(H), CoA, or ADP-ribose. The side product, uncapped triphosphate RNA, was degraded by treatment with 5′ polyphosphatase and Terminator™ exonuclease. (B) A representative example of PAGE analysis of the RNA-decapping activity of AtNUDT6, 7, 19, and 27 on 5′-capped Ap₄A-RNA and NADH-RNA.

Fig. 2. (A) Kinetic parameters of AtNUDT6, AtNUDT7 and AtNUDT27 cleaving Ap₄A expressed as mean ± standard deviation. (B) Cleavage efficiency of free Ap₄A (2.5 μM) and Ap₄A-RNA (2.5 μM) by AtNUDT6, AtNUDT7, and AtNUDT27 (50 nM) at 15- and 60 min time points. (C) Inhibition study of Ap₄A-RNA (1 μM) cleavage by AtNUDT6 (500 nM), AtNUDT7 (500 nM) and AtNUDT27 (50 nM) with addition of 1, 2 or 4 μM Ap₄A.