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. 2012 Oct 12;151(2):278-88.
doi: 10.1016/j.cell.2012.08.041.

Human RNA methyltransferase BCDIN3D regulates microRNA processing

Blerta Xhemalce  1 , Samuel C Robson, Tony Kouzarides
Affiliations

Human RNA methyltransferase BCDIN3D regulates microRNA processing

Blerta Xhemalce et al. Cell. .

Abstract

MicroRNAs (miRNAs) regulate key biological processes and their aberrant expression may lead to cancer. The primary transcript of canonical miRNAs is sequentially cleaved by the RNase III enzymes, Drosha and Dicer, which generate 5' monophosphate ends that are important for subsequent miRNA functions. In particular, the recognition of the 5' monophosphate of pre-miRNAs by Dicer is important for precise and effective biogenesis of miRNAs. Here, we identify a RNA-methyltransferase, BCDIN3D, that O-methylates this 5' monophosphate and negatively regulates miRNA maturation. Specifically, we show that BCDIN3D phospho-dimethylates pre-miR-145 both in vitro and in vivo and that phospho-dimethylated pre-miR-145 displays reduced processing by Dicer in vitro. Consistently, BCDIN3D depletion leads to lower pre-miR-145 and concomitantly increased mature miR-145 levels in breast cancer cells, which suppresses their tumorigenic phenotypes. Together, our results uncover a miRNA methylation pathway potentially involved in cancer that antagonizes the Dicer-dependent processing of miR-145 as well as other miRNAs.

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Figures

Figure 1
Figure 1. BCDIN3D is a methyltransferase that targets the 5′ mono-phosphate of nucleic acids
(A) Alignment of the S-Adenosyl Methionine (SAM) Binding domain of the Bin3 family members from S. pombe, A. thaliana, C. elegans, D. melanogaster, M. musculus and H. sapiens, generated with the CLUSTAL W algorithm. The asterisks indicate the residues that, when mutated to Alanine, abolish the methyltransferase activity of human BCDIN3D (Figure 1F). The arrow indicates human BCDIN3D. (B) Phylogenetic tree based on the alignment shown in (A), generated with the Geneious software. The dashed ellipsoid underlines the BCDIN3D cluster and the arrow indicates human BCDIN3D. (C) In vitro methyltransferase assay with recombinant human BCDIN3 and BCDIN3D, 3H-radioactive SAM as methyl group donor and either histones or nucleosomes as substrate. The reactions were loaded on a 15% SDS-PAGE gel, fixed and subjected to autoradiography. The radioactive band observed in the BCDIN3D/nucleosome lane co-migrates with histone H3. (D) In vitro methyltransferase assay with recombinant BCDIN3D, 3H-SAM and nucleosomes, in which histone H3 is either full length (H3), or is truncated of the 31 first amino-acids (H3ΔNter). The truncation makes H3ΔNter migrate faster (Coomassie staining, right panel), but does not modify the migration of the radioactive band (autoradiography, left panel). (E) 1%g of histones and nucleosomes were loaded on a 15% SDS-PAGE gel and stained with Ethidium Bromide. The band observed in the nucleosome lane corresponds to the 601-DNA used to assemble the nucleosomes. (F) In vitro methyltransferase assay using recombinant human BCDIN3D-WT and BCDIN3D-D72G74A catalytic mutant, 3H-radioactive SAM as methyl group donor and purified 601-DNA as substrate. The 601-DNA is generated by cleavage from a plasmid harbouring multiple tandem repeats of the sequence flanked by EcoRV restriction sites. Prior to the methyltransferase assay with BCDIN3D, the 601-DNA was either CpG methylated using the Ssp1 bacterial methyltransferase and/or treated with Alkaline Phosphatase (AP) that removes the 5′-alpha-phosphate left from the EcoRV digestion. The DNA from the methyltransferase assays with BCDIN3D was purified, loaded on a native 6% PAGE gel, stained with Ethidium Bromide (lower panel), fixed, and subjected to autoradiography (upper panel). (G) Schematic representing the inferred methyltransferase activity of BCDIN3D on a 5′ mono-phosphorylated ribonucleotide. The dashed ellipsoid underlines the 5′ alpha phosphate group. See also Figure S1.
Figure 2
Figure 2. BCDIN3D depletion suppresses tumorigenic phenotypes in vitro
(A) Stable depletion of BCDIN3D in the MDA-MB-231 breast cancer cells abolishes anchorage independent growth. Microscopic image of MDA-MB-231 [shBCDIN3D] and MDA-MB-231 [shNC] cells growing in soft agar after 4 weeks incubation (see Extended Experimental Procedures for further detail). (B-D) Stable BCDIN3D depletion substantially impairs invasiveness, but not growth and migration, of MDA-MB-231 cells. MDA-MB-231 [shBCDIN3D] and MDA-MB-231 [shNC] cells were analyzed with the xCELLigence system, which allows to assess cellular growth (B, Growth assay), migration through a 8 %M pore size membrane (C, Migration assay) or invasion through a 8 %M pore size membrane coated with Matrigel (D, Invasion assay) in parallel and in Real-Time. The cells were trypsinized and loaded in quadruplets onto an E-plate (B, Growth assay), CIM-plate (C, Migration assay) or CIM-Plate coated with 1/40 dilution of Matrigel (D, Invasion assay). The cell index was measured every 15 min for 48 h. The results of the assay are represented as normalized cell index. Error bars represent Standard Error of the Mean (SEM) values. (E) MDA-MB-231 [shBCDIN3D+BCDIN3DshR(esistant)-GFP] express BCDIN3DshR-GFP at levels similar to endogenous BCDIN3D protein. MDA-MB-231 [shBCDIN3D+BCDIN3DshR-GFP], MDA-MB-231 [shBCDIN3D+GFP] and MDA-MB-231 [shNC+GFP] cell lines were generated as indicated in the Extended Experimental Procedures. Whole cell extracts were analyzed by Western Blot with anti-BCDIN3D antibody and the loading control alpha-tubulin. The two panels showing the endogenous and BCDIN3DshR-GFP are from the same exposure of the same blot. (F) Expression of BCDIN3DshR-GFP in MDA-MB-231 [shBCDIN3D] cell lines suppresses their invasion defect. The MDA-MB-231 [shBCDIN3D+BCDIN3DshR-GFP], MDA-MB-231 [shBCDIN3D+GFP] and MDA-MB-231 [shNC+GFP] cell lines were analyzed as in (B-D) and the invasion assays are shown. Error bars represent SEM values. See also Figure S2.
Figure 3
Figure 3. siRNA mediated depletion of BCDIN3D changes the levels of precursor and mature forms of miR-145
MCF-7 cells were transfected with a siRNA targeted against the first exon of BCDIN3D (siBCDIN3D) or with non targeting siRNAs as a negative control (siNC). (A) The levels of the indicated miRNAs were analyzed by quantitative Reverse Transcription and PCR (qRT-PCR) analysis. The data are normalized to the U6 RNA and to siNC cells. Error bars represent SEM values. (B) Transcript levels of the pri-miR-145 were not significantly altered upon BCDIN3D depletion. The pri-miRNA levels were analyzed by qRT-PCR and normalized to the ALAS1 and B2M mRNAs and to siNC cells. Error bars represent SEM values. (C) The levels of pre-miR-145 are reduced upon BCDIN3D depletion. The pre-miRNA levels were analyzed by qRT-PCR and normalized to the 5S and U6 RNAs and to siNC cells. Error bars represent SEM values. (D) Schematic view of the IRS-1 mRNA and the sequences targeted by miR-145. (E) In a similar manner to miR-145 over-expression, BCDIN3D depletion affects IRS-1 expression at the protein level. These effects are not due to p53 stabilization. MCF-7 cells were co-transfected with miR-145 mimic, siBCDIN3D and siNC as indicated and analyzed by Western Blot with antibodies against IRS-1, p53 and the loading control GAPDH. (F) BCDIN3D depletion significantly reduces the expression of the luciferase reporter gene fused to the 3′UTR of the IRS-1 mRNA. siBCDIN3D and siNC transfected MCF-7 cells were transfected with pMirTarget (Origene) containing or not the 3′-UTR of IRS1 fused to the Luciferase Reporter gene and after 24h, the cells were lysed and the luciferase activity was measured in a luminometer. Shown are results from a typical experiment in quadruplets. Error bars represent SEM values. The transfection efficiency of the plasmids, as assessed by the expression of RFP gene contained in the pMirTarget plasmid, was identical in all samples. (G) miR-145 inhibition rescues the invasion defect of MDA-MB-231 cells transfected with siBCDIN3D. MDA-MB-231 were transfected according to the procedure schematized on the left with siRNAs against BCDIN3D and Negative Control, followed by anti miR-145 inhibitors and negative control. 24 h after the transfection of the miRNA inhibitors, the cells were trypsinized and analyzed as in Figure 2, (B-D) and the invasion assays are shown. Error bars represent SEM values. See also Figure S3.
Figure 4
Figure 4. BCDIN3D dimethylates the 5′Phosphate group of (pre-)miR-145 in vitro
(A) BCDIN3D preferentially methylates pre-miR-145. In vitro methyltransferase assay using recombinant GST-BCDIN3D (1-8) or GST (9-10), 3H-radioactive SAM as methyl group donor and the following synthetic DNA and RNA molecules as substrate (1) deoxy-pre-miR-145 [5′-OH]; (2) deoxy-pre-miR-145 [5′-P]; (3&9) pre-miR-145 [5′-OH]; (4&10) pre-miR-145 [5′-P]; (5) duplex miR-145 [5′-P]; (6) miR-145 [5′P]; (7) miR-145* [5′P]; (8) mock. The nucleic acids were purified and analyzed by autoradiography (left) and liquid scintillation (right). Error bars represent Standard Deviation (SD) values. Full recovery of nucleic acids after purification was verified with a spectrophotometer. (B-D) The product of BCDIN3D methylation is a 5′-Phospho-dimethyl. (B) In vitro methyltransferase assay as in (A) using as substrate synthetic pre-miR-145 molecules that have [5′-OH], [5′-P], [5′-Pme1] or [5′-Pme2] ends (see Figure 1G). The lower panel shows the Staining with ethidium bromide of the gel used for the autoradiography shown in the upper panel. (C) Synthetic pre-miR-145 molecules that have [5′-OH], [5′-P], [5′-Pme1] or [5′-Pme2] ends were ligated with the T4 RNA Ligase 1. The circular RNA molecules migrate faster than the linear molecules. pre-miR [5′-Pme2] is resistant to ligation. (D) Synthetic pre-miR-145 [5′-P] was in vitro methylated as in (A) prior to ligation with T4 RNA Ligase 1. The left panel shows the ethidium bromide staining of the gel used for the autoradiography shown in the right panel. The pre-miR-145 molecules that were methylated by BCDIN3D were also resistant to ligation.
Figure 5
Figure 5. Pre-miR-145 is also modified in a BCDIN3D dependent manner in vivo
(A) Synthetic pre-miR-145 molecules that have [5′-OH], [5′-P], [5′-Pme1] or [5′-Pme2] 5′ ends were treated with Terminator 5′-P Dependent exonuclease that is a processive 5′-3′ exonuclease that digests RNAs having a 5′ mono-phosphate. pre-miR [5′-Pme2] is resistant to this treatment. (B) RNA from MCF-7 cells treated or not with Terminator was migrated on an Agilent Total Eukaryotic RNA pico Chip. The treatment was specific as the Terminator fully digested the 18S and 28S rRNA which have 5′mono-phosphates, but did not digest the 5S RNA which has a 5′ tri-phosphate. (C) RNA treated as in (B) was analyzed by Reverse Transcription and PCR with primers specific for pre-miR-21, pre-miR-145, miR-21, miR-145 and 5S. The 5S RNA is known to be 5′ triphosphate and is used to control for equal RNA recovery. The indicated RNAs were analyzed by quantitative RT-PCR and normalized to the 5S RNA and to the [-Terminator] sample (graph on the right). Error bars represent SEM values. To facilitate the visualization of the assay, the PCRs were also analyzed by semi-quantitative PCR (panels on the left). For each primer, the semi-quantitative PCRs were performed in parallel with the quantitative PCRs and were stopped at the same cycle as the quantitative PCR reached the threshold of the less abundant sample. The treatment with Terminator fully digests miR-145 but not pre-miR-145. The asterisk indicates a non-specific band. See also Figures S4, S5 & S6.
Figure 6
Figure 6. BCDIN3D interacts with Dicer and its depletion affects miR-145 association with Dicer
(A) BCDIN3D specifically interacts with Dicer. Flag eluates from isogenic HELA-S3-Flp-In (-) and HELA-S3-Flp-In-BCDIN3D-Flag (BCDIN3D-Flag) stable cell lines co-immunoprecipitated with an anti Flag Antibody were analyzed by Western Blotting with the indicated antibodies (see Extended Experimental Procedures for further detail). (B) The interaction between BCDIN3D and Dicer is sensitive to treatment with RNase A. Flag co-Ips were performed as in (A) in the presence of mock or 1 or 5 %l of RNAse A (see Extended Experimental Procedures for further detail). The co-IPs were analyzed by Coomassie Staining to confirm equal recovery of BCDIN3D (lower panel) and by Western Blot with the Dicer antibody (upper panel). (C) Upon BCDIN3D depletion, the association of Dicer with miR-145, the product of pre-miR-145 processing, is significantly increased. MCF-7 cells transfected with siBCDIN3D or siNC were subjected to RNA-immunoprecipitation with the indicated antibodies (see Extended Experimental Procedures for further detail). RNA from the immunoprecipitates was purified and the levels of miR-21 and miR-145 were analyzed by qRT-PCR. The data are normalized to the 5S and U6 RNAs and to siNC cells. Error bars represent SEM values. (D-E) Synthetic pre-miR-145 molecules that have [5′-OH], [5′-P], [5′-Pme1] or [5′-Pme2] 5′ ends were incubated with human Dicer or Mock at 1mM of MgCl2 for 30, 60 or 120 min as indicated (see Extended Experimental Procedures for further detail). The Dicer processing reactions were loaded on a 15% Urea-PAGE gel and stained with Ethidium Bromide. The gel was photographed with the Chemidoc XRS+ system from Biorad (D, lower panel) and analyzed by Northern Blot with an anti-miR-145 probe (D, upper panel). The miR-145 product was quantified using the Image Lab Software (E). See also Figure S7.
Figure 7
Figure 7
Model for the mode of action of BCDIN3D.
See this image and copyright information in PMC

Comment in

  • Small RNAs: controlling maturity.
    David R. David R. Nat Rev Mol Cell Biol. 2012 Dec;13(12):753. doi: 10.1038/nrm3475. Epub 2012 Nov 15. Nat Rev Mol Cell Biol. 2012. PMID: 23151665 No abstract available.

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