Capping of mRNA

This enzyme [EC 2.1.1.56] catalyzes the addition of the A -methyl group to the cap of mRNA thus, -adenosyl-L-methionine reacts with G(5 )pppR-RNA to produce... 

FIGURE 26-12 The 5 cap of mRNA. (a) 7-Methylguanosine is joined to the 5 end of almost all eukaryotic mRNAs in an unusual 5, 5 -triphosphate linkage. Methyl groups (pink) are often found at the 2 position of the first and second nucleotides. RNAs in yeast cells lack the 2 -methyl groups. The 2 -methyl group on the second nucleotide... 

The cap of mRNA binds, secondary structures unwind, and the ribosomal subunit moves along... 

RNA Polymerase II Initiates Transcription at DNA Sequences Corresponding to the 5 Cap of mRNAs... 

Because of the lack of a reliable quantitative assay to control for the presence of the Cap structure, aside from a functional assay of the mRNA expression (transfection of the mRNA in cells and detection of the translated protein), the enzymatic capping of mRNA is rarely used to produce mRNA for research or therapy. The utilization of the synthetic Cap (nonmodihed analog or ARCA) in the in vitro transcription reaction is the standard method to produce capped mRNA. 

The 43S complex recruite mRNA by interaction between specific elFs. Some elFs (e.g. eIF3) associate with the 43S complex and others (e.g eIF4) associate with the 5 cap of mRNA. eIF4 has helicase activity that unwinds secondary and tertiary structures in the mature mRNA. It also associates with proteins that are bound to the poly(A) tail. The interaction of mRNA with the 43S complex appears to be facilitated by looping that results from interactions between elF complexes... 

An initiator methionine tRNA forms a complex with the small ribosomal subunit, then together with a variety of initiation factors (enzymes and other proteins) binds to the initiator codon of mRNA, and finally to a large ribosomal subunit, to form the complete ribosome. The 5 cap of mRNA is important for this process, as it marks the position of the initiator codon. AUG is the only codon for methionine, and anywhere... 

In contrast to Millward and his colleagues, Detjen et al. (1982) find that protein synthesis in SC-1 cells remains cap dependent throughout infection and do not find a transition from cap-dependent to cap-independent translation in reovirus-infected L cells. These findings are consistent with their proposed mechanism of translation control by mRNA competition for a discriminatory factor. They suggest that a partial explanation for the difference of their results with those of Skup and Millward (1980u,Z>) may be the difference in experimental technique. They used m GTP inhibition to assess the extent of capping of mRNA species, which measures only mRNAs actively translated. Thus, their method would not detect the presence of uncapped mRNAs unless they were capable of translation. Thus, they cannot exclude the possibility that uncapped mRNAs are present but not translatable for some unclear reason. Millward and his coworkers also find that the translation of late reovirus mRNA is sensitive to m GTP but do not have an explanation for this observation (Zarbl and Millward, 1983). Clearly, further studies are necessary to explain the conflicts in the data reported by the laboratories of Mill-ward and Thach. 

Epstein DM, Chappell LL, Khalili H, Supkowski RM, Horrocks WD Jr, Morrow JR. Eu(III) macrocyclic complexes promote cleavage of and bind to models for the 5 -cap of mRNA. effect of pendent group and a second metal ion. Inorg Chem. 2000 39(10) 2130-2134. 

As mentioned above, mammahan mRNA molecules contain a 7-methylguanosine cap structure at their 5 terminal, and most have a poly(A) tail at the 3 terminal. The cap stmcmre is added to the 5 end of the newly transcribed mRNA precursor in the nucleus prior to transport of the mELNA molecule to the cytoplasm. The S cap of the RNA transcript is required both for efficient translation initiation and protection of the S end of mRNA from attack by S —> S exonucleases. The secondary methylations of mRNA molecules, those on the 2 -hydroxy and the N of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm. 

Figure 38-6. Diagrammatic representation of the initiation of protein synthesis on the mRNA template containing a 5 cap (G" TP-5 ) and 3 poiy(A) terminai [3 (A)J. This process proceeds in three steps (1) activation of mRNA ...

The advantage of mRNA over plasmid transfection is the ability of in vitro transcription to allow precise control over features contained within the mRNA (Humphreys et al., 2005 Pillai et al., 2005 Westman et al, 2005). For example, mRNA can be prepared either with or without the physiological m7G(5/)ppp(5/)G cap structure and S poly(A) tail, which are important mediators of canonical translation initiation (Gallie, 1991 Hentze et al., 2006 Iizuka et al, 1994 Kahvejian et al, 2005 Tarun and Sachs, 1995). 

Figure 6.2 Critical parameters of the miR/mRNA co-transfection method. (A) Titration of mRNA amount. HeLa cells were transfected with increasing amounts of cap tail R-luc-4 sites mRNA and a fixed amount of firefly (F-luc) mRNA. R-luc expression (luciferase activity) was measured 5 h after transfection. (B) Titration of miCXCR4 concentration. HeLa cells were transfected with cap tail R-luc-4 sites mRNA, F-luc mRNA, and varying concentrations of miCXCR4. Luciferase activity was measured 16 h after transfection and fold-repression by the miR was calculated as in Fig. 6.1D (C) Time-course of miR-mediated repression. HeLa cells were co-transfected with cap tail R-luc-4 sites and F-luc (control) mRNAs, either with or without miCXCR4, and harvested at different time points. Repression was calculated as detailed in Fig. 6.1 and plotted against time (mRNA transfection data series depicted by the circles). Analogous plasmid DNA transfections are shown for reference (pDNA, diamonds). Averaged results from several experiments are shown with standard deviation. Data were previously published (Humphreys etal., 2005). Copyright PNAS, reprinted with permission.

Iizuka, N., Najita, L., Franzusoff, A., and Sarnow, P. (1994). Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. Mol. Cell Biol. 14, 7322-7330. 

The nature of the cap structure can also influence mRNA stability. The stability of mRNA capped with different ARCAs is determined in MM3MG cells that are electroporated with luciferase mRNAs containing a 60-nt poly(A) tract and different cap analogs. The experiment is done... 

Grudzien, E., Kalek, M., Jemielity, J., Darzynkiewicz, E., and Rhoads, R. E. (2006). Differential inhibition of mRNA degradation pathways by novel cap analogs. J. Biol. Chem. 281, 1857-1867. 

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