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Does capping of in vitro transcribed mRNAs increase their translation in rabbit
reticulocyte lysates?
Eukaryotic mRNAs contain a modified structure at their 5´-end that consists of a
7-methylguanylate residue linked by a 5´ to 5´ pyrophosphate attachment to the first
transcribed base of the mRNA. This complex methylated structure is known simply as the
5´-cap structure. This cap structure plays an important role in translation in
eukaryotes, being recognized by the translation initiation factor eIF-4E. The binding of
eIF-4E to the mRNA cap structure is an early step in the initiation of translation (1).
Early studies with cell-free translation systems showed that decapping mRNAs with
pyrophosphatase or competing with cap analog reduced their translation efficiency. The
generally accepted view, therefore, is that a 5´ cap structure is required for efficient
translation in eukaryotes (2-6). Recently, Promega introduced the Ribo m7G Cap
Analog (Cat.# P1711),
which is used in in vitro transcription reactions with either SP6, T7 or T3 RNA Polymerase
(Cat.# P1081, P2075 and P2083) to generate RNA
with a cap structure at their 5´-end.
The early cell-free translation studies were performed in wheat germ extracts. A second
type of commonly used in vitro translation system includes the Rabbit Reticulocyte Lysate
Systems, Nuclease Treated(a) (Cat.# L4960) and the
Flexi® Rabbit Reticulocyte Lysate System(a) (Cat.# L4540).
Cap-dependent stimulation of translation has been seen in rabbit reticulocyte lysate for
several mRNAs (7,8). However, translation in rabbit reticulocyte lysates is not dependent
on a cap structure and can occur quite efficiently in its absence (9). Translation in
rabbit reticulocyte lysates can be made more cap-dependent by the addition of general RNA
binding proteins such as La and hnRNP A1 (9). Apparently, the low level of general RNA
binding proteins such as these in rabbit reticulocyte lysates makes it possible for these
lysates to translate uncapped mRNAs efficiently. Thus, the ability of a 5´ cap structure
to stimulate translation in rabbit reticulocyte lysate may depend, to a certain extent, on
the mRNA being translated. However, the presence of a 5´ cap structure is not an absolute
requirement for translation to occur in this system, and would not decrease translation
efficiency.
References
- Rhoads, R.E. (1991) In: Translation in Eukaryotes, Trachsel, H., ed., CRC Press,
Boca Raton, FL.
- Both, G.W., Banerjee, A.K. and Shatkin, A.J. (1975) Methylation-dependent translation of
viral messenger RNAs in vitro. Proc. Natl. Acad. Sci. USA 72, 1189.
- Muthukrishnan, S. et al. (1975) 5´-Terminal 7-methylguanosine in eukaryotic
mRNA is required for translation. Nature 255, 33.
- Zan-Kowalczewska, M. et al. (1977) Removal of 5´-terminal m7G from eukaryotic
mRNAs by potato nucleotide pyrophosphatase and its effect on translation. Nucl. Acids
Res. 4, 3065.
- Shimotohno, K. et al. (1977) Importance of 5´-terminal blocking structure to
stabilize mRNA in eukaryotic protein synthesis. Proc. Natl. Acad. Sci. USA 74,
2734.
- Hickey, E.D., Weber, L.A. and Baglioni, C. (1976) Inhibition of initiation of protein
synthesis by 7-methylguanosine-5´-monophosphate. Proc. Natl. Acad. Sci. USA 73,
19.
- Song, H.J., Gallie, D.R. and Duncan, R.F. (1995) m7GpppG cap dependence for efficient
translation of Drosophila 70-kDa heat-shock-protein (Hsp70) mRNA. Eur. J.
Biochem. 232, 778.
- Ohlmann, T. et al. (1995) Proteolytic cleavage of initiation factor eIF-4 gamma
in the reticulocyte lysate inhibits translation of capped mRNAs but enhances that of
uncapped mRNAs. Nucl. Acids Res. 23, 334.
- Svitkin, Y.V. et al. (1996) General RNA binding proteins render translation cap
dependent. EMBO J. 15, 7147.
(a)U.S. Pat. No. 5,283,179 and other patents.
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Flexi is a trademark of Promega Corporation and is registered with the
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