Capped mRNA with a Single Nucleotide Leader Is Optimally Translated in a Primitive Eukaryote, Giardia lamblia

Abstract

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.

Yeast-like mRNA Capping Apparatus in Giardia lamblia

“…We found, in agreement with the recent report from Li and Wang (25), that luciferase activity was detected after transfection of capped mRNA, but not after transfection of uncapped mRNA (Fig. 8).…”

“…This conclusion is consistent with a recent study (25) and our own data showing that a m 7 GpppN capped reporter mRNA, but not an uncapped reporter RNA, was translated in Giardia trophozoites in vivo when introduced into cells by electroporation. This result, and the fact that Giardia encodes two homologs of the cap-binding translation initiation factor eIF4E (data not shown), would argue that Giardia is not an exception to the general reliance on the cap structure for optimal gene expression in eukarya.…”

“…RNA was quantified by UV absorbance and RNA integrity was judged by denaturing agarose gel electrophoresis. For transfection experiments, 10 million trophozoites were resuspended in 800 l of cytomix with 50 units of RNAsin (Promega) and 10 g of yeast tRNA and then kept on ice (25). 20 g of luciferase transcripts were added and cells were electroporated at 800 V, 1260 microfarads, and 500 V in a 0.4-cm cuvette using a BTX ECM600 electroporator (Harvard Apparatus, Holliston, MA).…”

A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia

“…Additionally, Giardia mRNAs typically have short 59 and 39 untranslated regions (UTR) of <30 nucleotides (nt), which minimizes translational regulation. The mechanism of ribosome scanning, for instance, was found to be missing from Giardia mimicking that observed in Archaea (Li and Wang 2004). These data suggest that gene expression in Giardia is poorly controlled.…”

Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2)

“…Analysis of the Giardia genome is providing important insights to the early origins of RNA processing mechanisms that are regarded as uniquely eukaryotic (12). Although there had been some debate whether Giardia mRNAs even have a 5Ј-cap structure (13,14), recent studies show that Giardia does possess the enzymatic machinery for m 7 G cap synthesis (15), caps the 5Ј ends of its mRNAs, and exploits the m 7 G cap for enhanced translation of a reporter mRNA in vivo (15)(16)(17).The fact that Giardia encodes two homologs of the cap-binding translation initiation factor eIF4E (17) suggests that Giardia is not an exception to the general reliance on the cap structure for optimal gene expression in eukaryotes. However, characterization of the Giardia eIF4E proteins revealed that one paralog, eIF4E2, binds to m 7 G caps, whereas the other paralog, eIF4E1, binds to m 2,2,7 G caps (17).…”