Fig. 4: RH24 is an ATP-dependent RNA helicase and accumulates under low temperatures.

a The molecular beacon helicase assays (MBHA). RH24 helicase unwinds a molecular beacon from a longer oligonucleotide. The fluorescent strand in the 3′-U₂₀ overhanging RNA substrate was modified with Cy5 at the 5′ end and BHQ-2 at the 3′ end. b Helicase activity of RH24 in MBHA. The decreasing intensity of the fluorescence indicates the activity of the helicase in the MBAH assays. Data are presented as mean values ± SD. c RH24 has ATP-dependent dsRNA unwinding activity. The unwinding activity was reflected by the double-stranded RNA template converting to a single-stranded product. The asterisk on the depiction of the short strand of the RNA duplex represents the biotin-labeled RNA. (ds: double strand, ss: single strand). d RH24^DAAD does not have the dsRNA unwinding activity. The assay was the same as in (c). The DEAD-box RNA helicase RH7 and RH24 were used as the positive controls. e ATPase activities of His-RH24 and His-RH24^DAAD. The ATPase activities of His-RH24 and His-RH24^DAAD were measured in the presence or absence of plant total RNA. Data are presented as mean values ± SD. f RH24 protein was accumulated under chilling stress. The 35S:RH24-ox Line 3 was grown on ½ MS at 22 °C for 12 days and then treated with 2 μM CHX at 4, 22, and 26 °C for 2 days, respectively. The protein levels of RH24 were examined by immunoblotting analysis. The expression levels of RH24 were examined by RT-qPCR. The band abundance was quantified by Image J. The relative protein level of RH24 was quantitatively analyzed with three repeats (lower panel). Different letters indicate the significant differences based on a two-factor ANOVA with Tukey’s HSD test (P ≤ 0.05, Data are means ± SD, n = 3 biological repeats). CHX: cycloheximide. The above experiments were repeated three times with similar results. Source data are provided as a Source Data file.