Abstract
SummaryThe 7-methylguanosine cap added to the 5′ end of mRNA is required for efficient gene expression in eukaryotes. In mammals, methylation of the guanosine cap is catalyzed by RNMT (RNA guanine-7 methyltransferase), an enzyme previously thought to function as a monomer. We have identified an obligate component of the mammalian cap methyltransferase, RAM (RNMT-Activating Mini protein)/Fam103a1, a previously uncharacterized protein. RAM consists of an N-terminal RNMT-activating domain and a C-terminal RNA-binding domain. As monomers RNMT and RAM have a relatively weak affinity for RNA; however, together their RNA affinity is significantly increased. RAM is required for efficient cap methylation in vitro and in vivo, and is indirectly required to maintain mRNA expression levels, for mRNA translation and for cell viability. Our findings demonstrate that RAM is an essential component of the core gene expression machinery.
Highlights
The methyl cap is the inverted 7-methylguanosine group linked to the first transcribed nucleotide of RNA polymerase II transcripts in eukaryotes (Cowling, 2009; Shatkin, 1976; Shuman, 2002)
The enzymes which catalyze methyl cap synthesis are essential from yeast to man; in mammals these are RNGTT (RNA guanylyltransferase and 50 triphosphatase) and RNMT (RNA guanine-7 methyltransferase) (Chu and Shatkin, 2008; Furuichi and Shatkin, 2000; Shuman, 2002)
Inhibition of RNMT expression results in loss of cap methylation and cell viability, and if there is another as yet undiscovered cap methyltransferase, it is not fully redundant with RNMT (Chu and Shatkin, 2008; Cowling, 2010; Shafer et al, 2005)
Summary
The methyl cap is the inverted 7-methylguanosine group linked to the first transcribed nucleotide of RNA polymerase (pol) II transcripts in eukaryotes (Cowling, 2009; Shatkin, 1976; Shuman, 2002). The 7-methylguanosine is linked by the 50 hydroxyl group through a triphosphate linkage to the transcript, to create a structure designated m7G(50)ppp(50)X (X is the first transcribed nucleotide). This is a unique molecular structure within the cell and is thought to target the 50 end of RNA pol II transcripts for several gene regulatory processes, including splicing, nuclear export of mRNA, and translation initiation (Bentley, 2005; Cowling, 2009; Moore and Proudfoot, 2009). Inhibition of RNMT expression results in loss of cap methylation and cell viability, and if there is another as yet undiscovered cap methyltransferase, it is not fully redundant with RNMT (Chu and Shatkin, 2008; Cowling, 2010; Shafer et al, 2005)
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