Fig. 3

NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2, nsun5-1, nop2b-1 and nop2c-1. Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b, nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product