Messenger RNA (mRNA) is transcribed from the coding DNA sequences by the RNA polymerase II. The nascently synthesised mRNA, called pre-mRNA, undergoes a series of maturation steps consisting of 5′ capping, removing non-coding sequences in the process of splicing and finally, 3′ end cleavage and polyadenylation. In mammals, 5′ cap structure includes template-independent terminal N7-methylguanosine (m7G), connected to mRNA via an unusual 5′ to 5′ triphosphate linkage and followed by the two first transcribed nucleotides, which are further modified with 2′-O-methyl (Nm) marks and, in the case of the first nucleotide being A, additionally N6-methyladenosine (m6A). The formation of the m7G cap is mediated by two enzymes: RNGTT and RNMT-RAM. Nm marks are added to the first and second nucleotide by the cap-specific RNA methyltransferases CMTR1 and CMTR2, forming cap1 and cap2 structures, respectively. Finally, if the first nucleotide is A, the PCIF1 enzyme deposits the m6A mark. While virtually all mRNA transcripts have cap1 modification, only approximately 50% have cap2. In addition to protecting mRNA from degradation and enhancing translation, these marks are essential components of the innate immune system, as uncapped transcripts are detected by the cytoplasmic sensors of foreign RNA, triggering interferon response and apoptosis.
The aim of my PhD was to further characterise the physiological and molecular functions of the mRNA cap1 and cap2 structure. Using a combination of mouse models and cell culture models, we show that both CMTR1 and CMT2 are vital for embryogenesis, with both mutant embryos dying around embryonic day E7.5, before the organogenesis stage. Sequencing of E6.5 and E7.5 mutant embryos showed that CMTR1 and CMTR2 regulate non- overlapping subset of genes indicating their separate functions. Interestingly, for both mutants, Cmtr1 and Cmtr2, this occurs without any activation of the innate immune response, suggesting that the functions of Nm extend beyond simply marking cellular RNAs as self. Conditional depletion of CMTR1 in male germ cells results in complete infertility, while only some females are infertile. Depletion of CMTR1 in fully developed organs, like the liver, leads to chronic activation of interferon expression. Interestingly, among commonly dysregulated genes in CMTR1 Knocked-out (KO) backgrounds are ribosomal genes overlapping with 5′ TOP transcripts and snoRNA host genes.
This investigation offers a comprehensive examination of the roles of CMTRs in development and in selected organs and illuminates their complex functions behind protecting from triggering autoimmune reactions.
