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. 2018 Aug 9;8:11904. doi: 10.1038/s41598-018-30383-z

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Validation of nucleomethylin (NML) as the human m¹A large subunit rRNA methyltransferase. (A) 3-D structure model of the human rRNA. 4UG0 pdb file together with UCSF-Chimera were used to create the rRNA surface model. The N1-methylated residue A1322 in the 28S rRNA is highlighted as a red sphere. For comparison, the corresponding region of the yeast 25S rRNA is shown in an inset. (B) siRNA-mediated depletion of NML is highly efficient at mRNA level. Total RNA extracted from HCT116 cells transfected with a siRNA specific for NML (siRNA #166 to #168) and incubated for 72 h, was tested by RT-qPCR. (C) NML is a nucleolar protein. Immunofluorescence with an antibody against NML was performed in HCT116 cells stably expressing a green fluorescent fibrillarin construct, which labels the dense fibrillar component of the nucleolus. The granular component protein PES1 was detected with a specific antibody (Bethyl Lab Inc). (D) Primer extension analysis of m¹A methylation after siRNA-mediated depletion of NML in HCT116 cells. ³²P-labeled primer complementary to nucleotides 1362 to 1381 of human 28S rRNA were used for methylation analysis of m¹A₁₃₂₂. (E) The reduction of stop intensity at the m¹A₁₃₂₂ in siRNA-mediated depleted cells was quantified using ImageJ software and estimated to be of ~60%.