METTL3 alters capping enzyme expression and its activity on ribosomal proteins

Abstract

The 5' cap, catalyzed by RNA guanylyltransferase and 5'-phosphatase (RNGTT), is a vital mRNA modification for the functionality of mRNAs. mRNA capping occurs in the nucleus for the maturation of the functional mRNA and in the cytoplasm for fine-tuning gene expression. Given the fundamental importance of RNGTT in mRNA maturation and expression there is a need to further investigate the regulation of RNGTT. N6-methyladenosine (m⁶A) is one of the most abundant RNA modifications involved in the regulation of protein translation, mRNA stability, splicing, and export. We sought to investigate whether m⁶A could regulate the expression and activity of RNGTT. In this short report, we demonstrated that the 3'UTR of RNGTT mRNA is methylated with m⁶a by the m⁶A writer methyltransferase 3 (METTL3). Knockdown of METTL3 resulted in reduced protein expression of RNGTT. Sequencing of capped mRNAs identified an underrepresentation of ribosomal protein mRNA overlapping with 5' terminal oligopyrimidine (TOP) mRNAs, and genes are dysregulated when cytoplasmic capping is inhibited. Pathway analysis identified disruptions in the mTOR and p70S6K pathways. A reduction in RPS6 mRNA capping, protein expression, and phosphorylation was detected with METTL3 knockdown.

Keywords: Post‐transcriptional regulation; RNA capping; RNA methylation; RNGTT; m6A; mRNA decay.

Fig. 1

METTL3 regulates the expression and stability of RNGTT. (A) Genomic view of the 3’UTR of RNGTT. m⁶A pulldown peaks (REPIC) and METTL3 motif prediction (FIMO) are displayed as tracks in IgvR. (B) Representative western blot analysis of protein expression of METTL3, RNGTT, and loading control Vinculin done in triplicate. Original blots are presented in Supplementary Fig. 1. Band intensities were normalized to vinculin and sh- was set as 1. (C) mRNA levels of RNGTT analyzed by RT-qPCR normalized to GAPDH expression by ΔΔCT. Sh- was set to 1. Error bars represent S.E.M. in biological triplicates. * Represents student’s t-test p. value < 0.05. (D) m⁶A RNA immunoprecipitation on predicted site of m⁶A modification of RNGTT mRNA analyzed by RT-qPCR. Immunoprecipitation is represented as % input relative to recovery of m⁶A positive control. sh- was set to 100%. Error bars represent S.E.M. with biological replicates represented as a dot n ≥ 3. ** represents two-tailed student’s t-test p. value < 0.01. E) RNA stability assay in A549 sh-/shMETTL3 analyzed by RT-qPCR. RNA expression is represented as RNA levels normalized to GAPDH at the indicated timepoint relative to the RNA levels at timepoint 0 h in either sh- or shMETTL3. Error bars represent S.E.M. of biological replicates n = 4. * Represents two-tailed student’s t-test of sh- vs shMETTL3 p. value < 0.05 at each indicated timepoint, ** represents p. value < 0.01.

Fig. 2

Downregulation of METTL3 disrupts mRNA capping in select genes. (A) Schema of TeloPrime capped mRNA cDNA synthesis and DNAseq analysis. (B) Volcano plot on differentially expressed genes sh-/shMETTL3 A549. Grey points represent no significance (p. value > 0.01 and |Log2FC|< 0.58), green points represent |Log2FC|> 0.58, blue points represent p-value < 0.01, and red points represent significant genes (|Log2FC|> 0.58 and a p-value < 0.01). (C) Gene overlap between top mRNAs or cytoplasmic capped mRNAs and differentially expressed genes. Venn diagrams are scaled with list size. *** represents overlapping Fisher exact test p. value < 0.001. (D) Dot plot of the top 15 pathways identified by IPA for the downregulated capping genes. (E) Representative western blot analysis of protein expression of METTL3, RNGTT, P-RPS6, RPS6 and Ponceau staining as loading control done in triplicate. Original blots are presented in Supplementary Fig. 4. Band intensities were normalized to ponceau stain, and scr- was set to 1. (F) CAP-IP on selected genes in PC9 cells analyzed by RT-qPCR. Data were first normalized to input and then normalized to housekeeping control ACTIN and represented as fold change with scr enrichment set as 1. Error bars represent S.E.M. in N = 7. Ns represents non-significant two-tailed student t-test of scr vs siMETTL3. * Represents two-tailed student’s t-test of scr vs siMETTL3 p. value < 0.05, ** represents p. value < 0.01.