Capped mRNA with a single nucleotide leader is optimally translated in a primitive eukaryote, Giardia lamblia.
The 5'-untranslated region (5'-UTR) of an mRNA plays an important role in translation initiation in eukaryotes. A minimal length of about 20 nucleotides is required to prevent leaky ribosome scanning. In one of the most primitive eukaryotes, Giardia lamblia, however, the mRNAs have 5'-UTRs mostly in the range of 0 to 14 nucleotides without a conserved sequence, which raises the question on how the ribosome could effectively scan such short 5'-UTRs for an accurate initiation of translation. In the present study, we expressed capped transcripts of luciferase gene in Giardia trophozoites via transfection and observed that when the 5'-UTR of the transcript was lengthened from 9 to 21 nucleotides, there was a corresponding decrease of translation efficiency. Conversely, shortening of the 5'-UTR from nine nucleotides down to a single nucleotide did not result in any reduced translation or leaky scanning. Translation appeared to initiate exclusively from the first initiation codon located downstream from the cap. Experimental evidence indicated also that a stem-loop structure immediately downstream from the initiation codon exerted significant inhibition on translation initiation when the 5'-UTR consisted of less than seven nucleotides. This inhibitory effect was abolished by increasing the distance between the stem-loop and the cap-G structure either upstream or downstream from the start codon, thus suggesting a spatial requirement for effective ribosome recruitment. Overall, our results suggest an absence of ribosome scanning for AUG in initiating translation in Giardia. A capped mRNA with a single nucleotide leader is apparently sufficient for recruiting ribosome and initiating translation.
Li
Lei
L
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280, USA.
Wang
Ching C
CC
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D003062:Codon; D018387:Codon, Initiator; D016829:Giardia lamblia / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry; D012315:RNA Caps / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D020121:5' Untranslated Regions; D003062:Codon; D018387:Codon, Initiator; D009097:Multienzyme Complexes; D012315:RNA Caps; D012333:RNA, Messenger; D012313:RNA; D008156:Luciferases; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases