CMTR1-Catalyzed 2'-O-Ribose Methylation Controls Neuronal Development by Regulating Camk2α Expression Independent of RIG-I Signaling

Abstract

Eukaryotic mRNAs are 5' end capped with a 7-methylguanosine, which is important for processing and translation of mRNAs. Cap methyltransferase 1 (CMTR1) catalyzes 2'-O-ribose methylation of the first transcribed nucleotide (N1 2'-O-Me) to mask mRNAs from innate immune surveillance by retinoic-acid-inducible gene-I (RIG-I). Nevertheless, whether this modification regulates gene expression for neuronal functions remains unexplored. Here, we find that knockdown of CMTR1 impairs dendrite development independent of secretory cytokines and RIG-I signaling. Using transcriptomic analyses, we identify altered gene expression related to dendrite morphogenesis instead of RIG-I-activated interferon signaling, such as decreased calcium/calmodulin-dependent protein kinase 2α (Camk2α). In line with these molecular changes, dendritic complexity in CMTR1-insufficient neurons is rescued by ectopic expression of CaMK2α but not by inactivation of RIG-I signaling. We further generate brain-specific CMTR1-knockout mice to validate these findings in vivo. Our study reveals the indispensable role of CMTR1-catalyzed N1 2'-O-Me in gene regulation for brain development.

Keywords: 2′-O-ribose methylation; CMTR; CaMK2; RIG-I; cap1 modification; dendrite development; innate immunity.

Graphical abstract

Figure 1

Nuclear CMTR1 Is Required for Dendritic Development (A) The chemical structure of capped RNAs. RNMT (mRNA cap guanine-N⁷ methyltransferase), CMTR1, and CMTR2, which transfer the methyl group from S-adenosylmethionine (SAM) to the corresponding positions, are indicated. SAH, S-adenosylhomocysteine. (B) Developmental expression of CMTR1 in neurons at the denoted days in vitro (DIV). The level of CMTR1 was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as a relative ratio to DIV2. Data are mean ± SEM from three cultures. (C–E) CMTR1 domain structure (C). The K239A and ΔNLS mutations render catalytic dead and cytoplasm-localized CMTR1, respectively. DIV2 rat neurons were infected with lentivirus expressing siCTL or siCM1 ± RFP or RFP-tagged human CMTR1 WT or mutants. The infected neurons at DIV7 were used for immunoblotting or immunostaining of RFP and MAP2 (D and E). Scales, 50 μm. (F) Sholl analysis to count the dendritic intersections in every 10-μm segment away from the soma. (G) Total dendritic number and length are expressed as mean ± SEM (n = 60 neurons from three cultures). ^∗∗∗p < 0.001, two-way analysis of variance (ANOVA). See also Figures S1 and S2.

Figure 2

CMTR1-KD Impairs Dendritic Development Independent of Secretory Factors and RIG-I/MAVS-Activated Signaling (A) Schematic diagram of the co-culture experiment. Neurons on coverslips were co-cultured with siCTL or siCM1 neurons from DIV3 to DIV7. The neurons on dishes were harvested for western blotting. (B) The neurons on coverslips were fixed for MAP2 immunostaining and analyzed for the number and total length of dendrites. (C) PCR genotyping of E17.5 embryos from RIG-I heterozygous mating. (D) RIG-I-KO neurons infected with the designated lentivirus were immunostained for MAP2. MAVS-KO neurons were processed similarly. (E and F) The dendritic intersections, number, and length were determined. (B, E, and F) Data are mean ± SEM (n = 60 neurons from three cultures). ns, not significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, two-way ANOVA. Scales, 50 μm. See also Figure S2F.

Figure 3

Knockdown of CMTR1 Decreases CaMK2α Expression to Affect Dendrite Development (A) Gene Ontology (GO) analysis of enrichment clusters from duplicate microarrays. The statistical significance of each cluster is expressed as −log₁₀ P. (B) The list of genes from the dendrite GO cluster with most transcriptomic changes in siCM1 neurons. Ltbp1, latent transforming growth factor β binding protein 1; Cdkn1a, cyclin-dependent kinase inhibitor 1; Pdyn, prodynorphin; Sort1, sortilin1; Atp7a, copper-transporting P-type ATPase; Camk2α, calcium/calmodulin-dependent protein kinase 2α; Ptk2b, protein tyrosine kinase 2b; Twf1, twinfilin-1; Pafah1b1, platelet-activating factor acetylhydrolase 1b subunit 1; Prkcg, protein kinase C gamma type; Kif5a, kinesin family member 5a. (C) The normalized RNA levels relative to Gapdh were determined by qRT-PCR. (D) The CaMK2α level relative to GAPDH in siCTL and siCM1 neurons ± ectopic expression of denoted mutant. (E) DIV2 neurons infected with the denoted lentivirus were immunostained with MAP2. Scales, 50 μm. (F) Sholl analysis. (G) Total dendritic number and length per neuron (n = 60). Data are mean ± SEM collected from ≥3 independent experiments. ns, not significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; Student’s t test in (C) and (D) and two-way ANOVA in (F) and (G). See also Figure S3.

Figure 4

Defective Dendritic Arborization of Cortical Pyramidal Neurons in CMTR1-cKO^Emx1 Mice without Evident Inflammation (A) The fCmtr1 mice were used to generate cKO^Emx1 or cKO^Nes mice. (B) The dorsal view of postnatal day 22 brains with medial sagittal sections stained with hematoxylin and eosin. The outlined cortical areas are denoted. (C) The poly(A) RNAs isolated from cortices were used to detect N1 2′-O-Me (pAm and pCm, circled by dotted line) by thin-layer chromatography. (D) CaMK2α level in P7 cortices (n = 3 mice) were determined as mean ± SEM. (E) 4-Thiouridine (4sU)-labeled transcripts were biotinylated, examined by dot blotting, and isolated by streptavidin beads for qRT-PCR. The nascent mRNA level of Camk2α relative to that of β-actin is expressed as mean ± SEM (n = 3 experiments). (F) Representative z stack images of YFP-expressing cortical layer 5 neurons. Scales, 100 μm. (G) Total dendritic intersections, number, and length from both apical and basal dendrites are mean ± SEM from 20 cWT neurons and 16 cKO neurons (three mice per group). (H) qRT-PCR. The levels of denoted mRNAs relative to Gapdh mRNA in cortices are mean ± SEM (four mice per group). Total RNAs isolated from enterovirus-71-infected spinal cord (SC) and Japanese-encephalitis-infected cortex (brain) were positive controls of innate immunity. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; Student’s t test in (D), (E), and (H) and two-way ANOVA in (G). See also Figures S4–S6.