Upregulation of RNA cap methyltransferase RNMT drives ribosome biogenesis during T cell activation

Abstract

The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.

CMTR1 is recruited to transcription start sites and promotes ribosomal protein and histone gene expression in embryonic stem cells

“…ChIP-PCR performed using a polyclonal anti-CMTR1 antibody was used to confirm that tagged and endogenous CMTR1 enriched equivalently at the TSS of individual genes, despite the HA-GFP-CMTR1 being expressed at a reduced level compared to the endogenous protein ( Supplementary Figure S7b ). Since the majority of RNAPII-transcribed RNA receives a 7-methylguanosine cap during transcription, RNMT may be recruited predominantly via the guanosine cap intermediate and/or its interaction with RNA ( 27 , 37 ). The limited binding of RNMT to the TSS in ESC is consistent with previous work performed in HeLa cells in which RNMT had to be overexpressed for RNMT binding to the TSS to be detected ( 38 ) (see Discussion).…”

“…For the ribosomal protein genes and histone genes, the impact of CMTR1 on transcript appeared direct; in isolated nuclei CMTR1 knockdown results in a reduction in their transcription and addition of recombinant CMTR1 increases their transcription. RNMT and its product the m7 G cap are linked to a transcriptional checkpoint involving co-transcriptional stability and protein:protein interactions ( 1 , 27 , 37 ). For CMTR1 and its product N1 2′-O-Me, it is likely that analogous influences on co-transcriptional stability and transcriptional mechanisms are governing CMTR1-dependent mRNA levels.…”

“…Unlike the other capping enzymes, RNMT does not have a CTD interaction domain and a direct interaction with RNAPII has not been demonstrated ( 22 , 23 , 44–46 ). Since the majority of nascent RNA has a m7 G cap, and a redundant N-7 cap guanosine methyltransferase is not observed in RNMT depleted extracts, RNMT must be recruited to nascent guanosine caps during transcription via a different mechanism ( 37 ). A previous study demonstrated that RNMT binds to RNA in correlation with the expression of that gene, without apparent preference for motifs or RNA regions ( 27 ).…”

Protein synthesis, degradation, and energy metabolism in T cell immunity

“…Indeed, the immune activation of T cells initiates large-scale remodeling of the transcriptome, with numerous mRNAs being created or destroyed. A modest 1.4-fold increase in total mRNA content was observed 6 h post immune activation in vitro [ 7 ], with a more substantial ~5–10-fold increase within 20–24 h post activation [ 7 , 32 ]. Despite this, mRNA quantity only moderately correlates with protein expression levels during T cell activation and in differentiated cytotoxic T lymphocytes (correlation coefficient ranging from 0.4 to 0.65) [ 6 , 7 , 33 ].…”

“…A plausible mechanism for a more substantial selective effect of mTORC1 on protein synthesis in T cells involves the pathways that couple mTORC1 to the translational control of 5' terminal oligopyrimidine tract (5'TOP) mRNAs, which include mRNAs encoding ribosomes and translation factors. While the exact regulatory mechanisms are still being determined, it is thought that 5'TOP-containing mRNA is bound by the repressive RBP Larp1, which releases the 5'TOP mRNA upon phosphorylation by mTORC1 [ 13 , 32 ]. mTORC1-mediated Larp1 phosphorylation has been detected within 2 h of T cell activation [ 6 ]; moreover, within 6–24 h of TCR activation, the expression levels of proteins encoded by 5'TOP mRNAs were found to be sensitive to mTOR inhibition [ 2 , 7 ].…”