Capping enzyme
The capping enzyme is a multifunctional enzyme complex essential for the co-transcriptional modification of eukaryotic messenger RNA (mRNA), catalyzing the addition of a 7-methylguanosine (m⁷G) cap structure to the 5′ end of nascent transcripts via a 5′–5′ triphosphate linkage.[1] This cap formation occurs shortly after transcription initiation by RNA polymerase II, typically when the first 25–30 nucleotides are synthesized, and involves three sequential enzymatic activities: RNA 5′ triphosphatase, which removes the γ-phosphate from the 5′ triphosphate end to generate a diphosphate; guanylyltransferase, which transfers guanosine monophosphate (GMP) from GTP to form an unusual 5′–5′ triphosphate bridge; and guanine-N7 methyltransferase, which adds a methyl group from S-adenosylmethionine (SAM) to the guanine base.[1] In organisms such as the yeast Saccharomyces cerevisiae, these activities are provided by three distinct proteins (Cet1, Ceg1, and Abd1), whereas in metazoans including humans, the triphosphatase and guanylyltransferase domains are fused into a single bifunctional protein known as RNA guanylyltransferase (RNGTT), with the methyltransferase often encoded separately.[2] Structurally, the guanylyltransferase domain features conserved motifs, including an OB-fold and key residues like an invariant lysine essential for forming a covalent enzyme-GMP intermediate during catalysis, with crystal structures from yeast and viral homologs revealing dynamic conformational changes that facilitate the reaction.[3] The m⁷G cap serves critical biological roles throughout the mRNA lifecycle, including protection against 5′–3′ exonucleolytic degradation, enhancement of mRNA stability, promotion of nuclear export, and recruitment of initiation factors like eIF4E for efficient ribosomal translation.[1] By distinguishing self mRNA from foreign RNAs (such as viral transcripts lacking caps), the cap structure also modulates innate immune responses, preventing aberrant activation of sensors like RIG-I.[1] Conservation of capping enzymes across eukaryotes, fungi, and even certain viruses underscores their evolutionary importance, with phylogenetic analyses identifying shared motifs that trace back to ancient origins in eukaryotic lineages.[3] In biotechnology, capping enzymes have enabled advances in synthetic mRNA production for vaccines and therapeutics, where precise cap addition is vital for immunogenicity and expression efficiency.[1]Overview
Definition and role
The capping enzyme, also known as RNA guanylyltransferase and classified under EC 2.7.7.50 with CAS number 56941-23-2, is a key enzyme that catalyzes the addition of a guanosine cap structure to the 5' end of nascent messenger RNA (mRNA) molecules during their synthesis in eukaryotic cells.[4] This process involves the transfer of a guanylyl moiety from guanosine triphosphate (GTP) to the diphosphorylated 5' end of the pre-mRNA, forming an unusual 5'-5' triphosphate linkage as an initial step toward the mature 7-methylguanosine (m⁷G) cap.[5] The enzyme is documented in major biochemical databases, including KEGG pathway hsa03020 for RNA polymerase and BRENDA entry EC 2.7.7.50, which detail its catalytic role in mRNA maturation. In its primary role, the capping enzyme facilitates a co-transcriptional modification that is specific to transcripts produced by RNA polymerase II, occurring shortly after the nascent RNA chain reaches a length of approximately 20-30 nucleotides.[6][7] This timing ensures the cap is added while the RNA is still associated with the transcription machinery, marking the pre-mRNA for subsequent processing events and distinguishing it from other RNA types.[8] As part of the essential triad of pre-mRNA processing—capping, splicing, and polyadenylation—the enzyme's activity coordinates with these steps to promote efficient gene expression in eukaryotes.[9] The capping enzyme functions as part of a multi-component complex that integrates RNA 5'-triphosphatase activity (to remove the γ-phosphate from the transcript's 5' triphosphate end), guanylyltransferase activity (EC 2.7.7.50 for GMP transfer), and subsequent methyltransferase activity to complete the m⁷G cap.[5] In humans, the core activities of triphosphatase and guanylyltransferase are encoded by the RNGTT gene (also known as HCE1 or MCE1), producing a bifunctional protein that is indispensable for mRNA capping.[5][10] This complex is recruited to the phosphorylated C-terminal domain of RNA polymerase II, underscoring its role in linking transcription directly to mRNA modification for stability and functionality.[7]Historical discovery
The discovery of the 5' cap structure on eukaryotic mRNA began in the early 1970s with investigations into viral transcripts. In 1975, Yasuhiro Furuichi and colleagues at the Roche Institute of Molecular Biology identified the unusual methylated, blocked 5' terminus, m⁷GpppNm, in reovirus mRNA through radiolabeling and enzymatic digestion experiments, revealing it as a distinctive feature of eukaryotic messenger RNA. This finding was rapidly extended to cellular mRNAs, as the same group demonstrated the presence of similar capped 5' ends in HeLa cell mRNA later that year using parallel biochemical analyses. These early observations highlighted the cap's role in distinguishing eukaryotic mRNAs from prokaryotic ones and set the stage for exploring its biosynthesis. Purification of capping activities marked key milestones in the mid-1970s. In 1975, Stewart A. Martin, Enzo Paoletti, and Bernard Moss isolated and purified guanylyltransferase and guanine-7-methyltransferase from vaccinia virus cores, showing these enzymes catalyze the transfer of GMP and a methyl group to form the cap structure on nascent viral RNA.40647-9) For eukaryotic cells, C.-M. Wei and B. Moss achieved partial purification of guanylyltransferase from HeLa cell nuclei in 1977, confirming the enzyme's ability to cap synthetic RNA substrates with a 5' diphosphate end and establishing its nuclear localization. By the early 1980s, studies demonstrated that capping occurs co-transcriptionally, tightly coupled to RNA polymerase II transcription; for instance, experiments with isolated HeLa cell nuclei showed cap addition to nascent heterogeneous nuclear RNA (hnRNA) within seconds of initiation, linking the process to Pol II activity.[11] Pioneering work by Aaron J. Shatkin, Amiya K. Banerjee, and their collaborators in the 1970s and 1980s defined the multi-enzyme nature of cap formation. Through fractionation and reconstitution assays on reovirus and vaccinia systems, they delineated the three-step pathway: RNA 5'-triphosphatase removes the γ-phosphate, guanylyltransferase adds GMP to form GpppN, and methyltransferase adds the N⁷-methyl group, with subsequent 2'-O-methylation in some cases.60258-3) Banerjee's group particularly advanced characterization of the viral triphosphatase and its distinction from guanylyltransferase activities.90379-5) Advances in the 1990s included gene cloning, providing genetic tools for functional studies. In 1992, Y. Shibagaki et al. cloned the Saccharomyces cerevisiae CEG1 gene encoding the guanylyltransferase subunit via immunological screening of a genomic library, showing its essentiality for viability.50122-3) The CET1 gene for the triphosphatase subunit was cloned in 1997, revealing a heterotetrameric complex conserved in yeast. For humans, the RNGTT gene (encoding RNA guanylyltransferase and 5'-phosphatase) was cloned in 1998 by K. Yamada-Okabe et al. from a cDNA library, demonstrating complementation of yeast mutants and conservation of the bifunctional enzyme architecture. These efforts underscored the evolutionary conservation of capping enzymes across eukaryotes, from yeast to mammals, with early yeast studies highlighting shared mechanisms for co-transcriptional recruitment via the Pol II C-terminal domain.00150-3)Mechanism of Cap Formation
Enzymatic steps
The formation of the m7G cap on eukaryotic mRNA occurs through three sequential enzymatic reactions catalyzed by the capping enzyme complex, which acts co-transcriptionally on nascent transcripts produced by RNA polymerase II (pol II).[12] The first step involves RNA 5'-triphosphatase activity, which hydrolyzes the γ-phosphate from the 5' triphosphate end (pppN) of the nascent pre-mRNA, generating a 5' diphosphate end (ppN) and inorganic phosphate (Pi). In yeast, this reaction is performed by Cet1, while in humans, it is catalyzed by the N-terminal domain of the bifunctional RNA guanylyltransferase and 5'-phosphatase (RNGTT). The reaction can be represented as:pppN-RNA + H₂O → ppN-RNA + Pᵢ.[13][1] In the second step, guanylyltransferase activity transfers a guanosine monophosphate (GMP) moiety from GTP to the 5' diphosphate end of the RNA, forming an unusual 5'-5' triphosphate linkage (GpppN). This process in yeast is mediated by Ceg1 and in humans by the C-terminal domain of RNGTT; it proceeds via a covalent enzyme-GMP intermediate where the guanine is linked to a lysine residue on the enzyme (E-Lys-GMP), followed by transfer to the RNA substrate and release of pyrophosphate (PPi). The reaction is reversible and can be depicted as:
ppN-RNA + GTP ⇌ GpppN-RNA + PPi.[14] The third step adds a methyl group to the N7 position of the guanine base using S-adenosylmethionine (SAM) as the methyl donor, yielding the mature cap 0 structure (m7GpppN) and S-adenosylhomocysteine (SAH) as a byproduct. This methylation is catalyzed by mRNA (guanine-N7)-methyltransferase, known as Abd1 in yeast and RNMT in humans. The reaction proceeds as:
GpppN-RNA + SAM → m7GpppN-RNA + SAH.[15] Collectively, these steps are highly specific to transcripts synthesized by pol II and depend on the phosphorylated C-terminal domain (CTD) of pol II for efficient recruitment and activation of the capping enzymes.[16]