Abstract
The gamma-monomethylphosphate cap structure is found in several eukaryotic small RNAs including nuclear U6, U6atac, 7SK, plant nucleolar U3, and rodent cytoplasmic B2 RNAs. In the case of human U6 snRNA, the 5' end sequence corresponding to nucleotides 1-25 serves as the capping signal and directs the formation of methylphosphate cap structure. In this study, we show that the U6 RNA capping signal, when introduced at the 5' end of RNAs, can efficiently direct the methylphosphate cap formation in RNAs of up to 2.7 kb long, as well as in different mRNAs. These data show that the methylphosphate capping signal functions in mRNAs having different primary sequences and different lengths. Presence of the methylphosphate cap structure on the 5' end of a luciferase mRNA with EMCV 5' noncoding region, which is translated in an IRES-dependent pathway, resulted in a 6- to 100-fold inhibition of translation compared to the same mRNA with a 5' triphosphate when microinjected into frog oocytes or expressed in mouse cells in tissue culture. Thus, conversion of the pppG structure to a methyl-pppG structure on the 5' end of an mRNA, which is translated in an IRES-dependent pathway, results in severe inhibition of translation. These data show that the 5' end motif of mRNAs plays an important role even in the IRES-mediated mRNA translation.