Poly(A) dependent translation in rabbit reticulocyte lysate.
Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)+ and poly(A)- mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease untreated rabbit reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs, stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.
Wakiyama
M
M
Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Tokyo, Japan.
Futami
T
T
Miura
K
K
D000818:Animals; D002474:Cell-Free System; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011061:Poly A / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D012333:RNA, Messenger; D011061:Poly A; D008156:Luciferases; D008836:Micrococcal Nuclease