Many eukaryotic and viral mRNAs are modified at their 5' ends by addition of 7-Methylguanosine (N⁷-methyl guanosine or m⁷G), known as "Cap". "Capping" of the mRNA structure plays a crucial role in a variety of cellular processes which include translation initiation^[1], splicing^[2], intracellular transport^[3] and turnover^[4].

Consistently, successful downstream application of in vitro transcribed mRNAs strongly depends on the 5' Cap structure. Capped mRNAs are generally more efficiently translated in wheat germ and reticulocyte in vitro translation systems^[5], and they are less susceptible to exonuclease degradation during microinjection experiments compared to uncapped mRNAs^[6].

In vitro synthesis of capped mRNAs can be performed by viral RNA polymerases (T7, SP6 or T3) that incorporate traditional cap analogs such as GpppG in its unmetylated, monomethylated or trimethylated form.

The ARCA cap analog yields transcripts which are more efficiently translated in vitro since the synthesized capped RNA is exclusively in the correct orientation^[7]. This is in contrast to traditional cap analogs where only 50 % of the cap analog is incorporated in the correct orientation.

The trimethylated cap analog is involved in RNA transport^[8] and RNA translation in nematodes^[9].

The unmethylated cap analog shows no significant difference in its translation properties compared to the monomethylated cap analog^[10].

References:

[1] Gingras et al. (1999) eIF4 initiation factors: Effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68:913.

[2] Izaurralde et al. (1994) A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell 78:657.

[3] Izaurralde et al. (1992) A cap binding protein that may mediate nuclear export of RNA polymerase II-transcribed RNAs. J. Cell Biol. 118:1287.

[4] Beelman et al. (1998) An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature 382:642.

[5] Paterson et al. (1979) Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure. Nature 279:692.

[6] Drummond et al. (1985) The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes. Nucl. Acids Res. 13:375.

[7] Grudzien et al. (2007) Synthesis of Anti-Reverse Cap Analogs (ARCAs) and their Applications in mRNA Translation and Stability. Methods Enzymol. 431:203.

[8] Fischer et al. (1991) Diversity in the signals required for nuclear accumulation of U snRNPs and variety in the pathways of nuclear transport. J. Cell Biology 113 (4):705.

[9] Maroney et al. (1995) Most mRNAs in the nematode Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency. RNA 1 (7):714.

[10] Krieg et al. (1984) In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155:397.

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