mRNA capping: biological functions and applications

Abstract

The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2'O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose.

Figure 1.

mRNA caps in eukaryotes.

Figure 2.

Enzymatic steps involved in RNA capping. The RNA triphosphatase activity (TPase) removes the γ-phosphate from 5′ triphosphate, generating a diphosphate 5′ end and inorganic phosphate (reaction [1]). The guanylyltransferase (GTase) activity consumes a GTP molecule and forms a covalent intermediate containing with a lysyl-Nζ-5′-phosphoguanosine (reaction [2.1]). In the presence of a 5′ diphosphate RNA, the GTase activity transfers the 5′-phosphoguanosine (GMP) to the 5′ diphosphate, forming a 5′-5′ triphosphate linkage between the first base of the RNA and the capping base (reaction [2.1]). In the presence of S-adenosylmethionine (SAM), the guanine-N7 methyltransferase (MTase) activity adds a methyl group to N7 amine of the guanosine cap to form the cap 0 structure (reaction [3]). Finally, the m7G cap-specific 2′O MTase modifies the 2′O of +1 ribose and generates the cap 1 structure (reaction [4]).

Figure 3.

Guanylyltransferase reaction. The GTase reaction consists of two steps: protein self-guanylylation and the transfer of the GMP group to the 5′ diphosphate RNA. The GTase reaction catalyzed by chlorella virus PBCV-1 capping enzyme is highly reversible. A detailed kinetics study of the forward and reverse reaction of both steps revealed that in the absence of RNA substrate, the k_cat/K_m value of the reverse reaction of the first step (the pyrophosphorolysis of the lysyl-Nζ-5′-phosphoguanosine intermediate into lysine and GTP) is 120 times higher than that of the forward reaction. The second step (the transfer of the GMP moiety from the lysyl-Nζ-5′-phosphoguanosine to diphosphate 5′ end) is largely forward-tending, with a 10-fold difference in the k_cat/K_m values (96).

Figure 4.

The opened and closed conformations of Chlorella virus PBCV-1 capping enzyme (PDB 1CKM). The PBCV-1 RNA capping enzyme adopts a bilobed structure where the N-terminal guanylyltransferase domain and C-terminal OB fold domain sit on either side of the protein with the active site situated at the interface of the two domains. The asymmetric unit of the crystal structure of Chlorella virus capping enzyme contains two protein units, each exhibiting a different conformation. In the opened conformer (blue), the cleft between the two domains is ∼15 Å at its widest, whereas in the closed conformer (orange), the cleft closes one end off to the solvent completely due to rigid movement of the OB-fold domain. Interestingly, Lys82, the nucleophile that forms the lysyl-Nζ-linkage with incoming GMP (red and violet), shows very limited movement within the active site (100).

Figure 5.

Multifunctional RNA capping enzymes. (A) Vaccinia capping enzyme structure co-crystallized with GTP and SAH (PDB 4CKB). The enzyme composes of two subunits D1 and D12. The functional domains are ordered as TPase (blue), GTase (orange) and guanine-N7 MTase (beige) from N- to C-terminus in D1. D12 subunit is colored in green. Note the β-barrel characteristic of triphosphate tunnel metaloenzymes (TTM). As indicated by the SAH molecule (magenta), the guanine-N7 MTase active site opens to the back, away from the GTase active site as indicated by the GTP molecule (red). (B) Bluetongue virus capping enzyme VP4 structure. The crystal form that contains two guanine molecules (red) and two SAH molecules (magenta) is shown (PDB 2JHP). The functional domains are arranged from N- to C-terminus in the order of kinase-like (KL) domain (pink), 2′O MTase domain (green), guanine-N7 MTase and the putative combined TPase/GTase domain (orange). The guanine-N7 MTase domain is composed of two discontinuous sequences which are colored in light brown and beige. Two stretches of polypeptide of 10 and 13 amino acid residues within the C-terminal TPase/GTase domain are missing in the structure. The SAH molecules (magenta) clearly identify the active sites of the guanine-N7 MTase and 2′O MTase in the structure. The putative TPase catalytic residue Cys518 is colored in bright green.