Capping mRNA

The activity of 4E is regulated in a second way, and this also involves phosphorylation. A recently discovered set of proteins bind to and inactivate 4E. These proteins include 4E-BP1 (BPl, also known as PHAS-1) and the closely related proteins 4E-BP2 and 4E-BP3. BPl binds with high affinity to 4E. The [4E] [BP1] association prevents 4E from binding to 4G (to form 4F). Since this interaction is essential for the binding of 4F to the ribosomal 40S subunit and for correctly positioning this on the capped mRNA, BP-1 effectively inhibits translation initiation. 

Figure 38-7. Activation of elF-4E by insulin and formation of the cap binding elF-4F complex. The 4F-cap mRNA complex is depicted as in Figure 38-6. The 4F complex consists of elF-4E (4E), elF-4A, and elF-4G. 4E is inactive when bound by one ofa family of binding proteins (4E-BPs). Insulin and mitogenic factors (eg, IGF-1, PDGF, interleukin-2, and angiotensin II) activate a serine protein kinase in the mTOR pathway, and this results in the phosphorylation of 4E-BP. Phosphorylated 4E-BP dissociates from 4E, and the latter is then able to form the 4F complex and bind to the mRNA cap. These growth peptides also phosphorylate 4E itself by activating a component of the MAP kinase pathway. Phosphorylated 4E binds much more avidly to the cap than does nonphosphorylated 4E.

Figure 38-10. Picornavimses disrupt the 4F complex. The 4E-4G complex (4F) directs the 40S ribosomal subunit to the typical capped mRNA (see text). 4G alone is sufficient for targeting the 40S subunit to the internal ribosomal entry site (IRES) of viral mRNAs. To gain selective advantage, certain viruses (eg, poliovirus) have a protease that cleaves the 4E binding site from the amino terminal end of 4G. This truncated 4G can direct the 40S ribosomal subunit to mRNAs that have an IRES but not to those that have a cap. The widths of the arrows indicate the rate of translation initiation from the AUG codon in each example.

Properties of ARCAs and ARCA-Capped mRNAs in Cell-Free... 

Properties of ARCA-Capped mRNAs in Mammalian Cells 221... 

Stability of ARCA-capped mRNAs in cultured mammalian cells 224... 

Synthetic capped mRNAs are useful tools to study all of the processes mentioned previously. To create capped mRNAs, DNA templates are transcribed with either a bacterial (Contreras etal., 1982) or bacteriophage (Konarska et al., 1984 Yisraeli and Melton, 1989) RNA polymerase in the presence of all four ribonucleoside triphosphates and a synthetic cap dinucleotide, m7Gp3G. The polymerase initiates transcription with a nucleophilic attack by the 3 -OH of the Guo moiety ofm7Gp3G on the a-phosphate... 

That this translational system can indeed detect a different (increased) translational activity when the extracts are programmed with capped mRNA can be seen from the results presented in Fig. 12.3C. On the other hand, a general reduction of the translational efficiency caused by... 

Our screen has the potential to identify inhibitors of cap-dependent initiation, IRES-mediated initiation, and translation elongation or termination. One assay to identify initiation inhibitors from hits obtained in the primary screen is to assess the ability of a given compound to prevent 48S and/or 80S initiation complex formation on a capped mRNA and on the HCV IBJ3S. Because initiation complexes are formed more efficiently in RRL than in... 

Borman, A. M., Michel, Y. M., and Kean, K. M. (2000). Biochemical characterization of cap-poly(A) synergy in rabbit reticulocyte lysates The eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5r-end. Nucleic Acids Res. 28, 4068-4075. 

This multisite inhibition of viral mRNA processing by RTP appears to be a result of biochemical differences between the viral and the natural cellular enzymes involved in capping mRNA. The viral enzymes are much more strongly inhibited, consequently, preventing viral protein synthesis or giving rise to short segments of viral protein that are nonfunctional. All capped viral mRNAs contain a purine exclusively in the 5f penultimate position (B1 in Fig. 9) and initiation of viral mRNA synthesis appears to be purine specific. In mammalian cell mRNA, B 1 may be a pyrimidine as well as a purine, and most often B1 is cytosine and B2 uracil. 

Hypermethylated guanosine-capped mRNA molecules are important in cellular transport and RNA splicing. The chemical synthesis of a 5 -terminal 2,2,7-trimethylguanosine-capped tri ribobonucleotide has been described by condensation of (185) with (186) in the presence of CDI in 40% yield. The synthesis includes a novel three step synthesis of 2N,2N-dimethylguanosine from guano-sine. The product was characterised by proton and phosphorus NMR. 

The second method for capping mRNA takes advantage of the activity of the vaccinia virus capping enzyme, also known as guanylyltransferase. This enzyme is commercially available. In the presence of GTP and S-adenosyl methionine (SAM), it can add a natural Cap structure (7-methylguanosine) to the 5 triphosphate of a RNA molecule. As it is an enzymatic reaction, it can bring a correct Cap to all mRNA molecules, and thus, it is optimal compared with in vitro transcription in the presence of standard Cap but similar theoretically to in vitro transcription in the presence of ARCA Cap. 

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. 

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