20

Noda Institute for Scientific Research GRANT
2014 Research Grant
Outline of Research Result

Construction of a highly efficient protein synthesis system byusing circular mRNAs and permuted/intronic tRNA processing
Akiko Soma
Graduate School of Horticulture, Chiba University

Research aims
Circular mRNAs are expected to be a powerful tool foruse in the production of recombinant proteins because theyshow high exonuclease resistance and efficient translation
initiation. Some reports have shown that circular mRNAs
can be used as a template via internal ribosome entry during bacterial translation.1), 2) On the other hand, the translational activity of circular mRNAs is generally inefficient,suggesting that the mRNA terminus is still required for efficient translation, although the details of the mechanism remain unclear. To facilitate the recombinant production ofdesired proteins and further understanding of the translation
mechanism in bacteria, the construction and analysis of thetranslation system using circular mRNAs are required.
Circularly permuted tRNAs found in the nuclear genomeof some unicellular algae (including Cyanidioschyzon
merolae 10D that inhabits an extreme environment) and in
the genome of some archaea are matured via a characteristic circular RNA intermediate during post-transcriptionalprocessing.3) In permuted tRNA genes, the sequences encoding the 5′-half and 3′-half of the specific tRNA are separated and inverted on the genome (Fig. 1). Biochemical
analyses have defined a processing pathway in which thetermini of tRNA precursors are ligated (probably dependingon the tRNA-intron splicing machinery) to form a circulartRNA, which is then cleaved to generate the typical clover-leaf structure. In the present study, we tried to constructmRNA circularization systems via either the tRNA-intron
splicing system or the self-splicing group I intron of bacterial tRNA. In addition, we assessed the translational activity of Escherichia coli and Bacillus subtilis mutant cells
that lacked tRNA and tRNA modification enzymes, to identify the mutants with increased protein production ability.
Methods
We prepared a marker gene that could produce a functional protein only when circularization of mRNA had occurred via either (1) the tRNA-intron splicing system or (2)the self-splicing group I intron. (1) The tRNA-intron splicing endonuclease from organisms harboring permutedtRNA genes is supposed to recognize substrate RNAs depending only on the specific motif, which is originallyfound in the exon-intron junction of tRNA. Plasmids encoding a marker gene flanked by this RNA motif (Fig. 2A)
and the C. merolae tRNA-intron splicing endonucleasegenes were introduced into E. coli. (2) A plasmid containing a marker gene with inverted group I intron splicing sites
derived from bacterial tRNA (Fig. 2B) was introduced intoE. coli cells and an E. coli cell-free system. The circularization of mRNA and expression of the marker protein wereassessed by RT-PCR and western blotting.
The β-galactosidase activity of E. coli and B. subtilis mutant strains defective in tRNA genes and anticodon modification enzyme genes was analyzed to identify the genesthat affect translational activity. Then, some strains with a
combination of the mutations for further increase of translational activity were constructed.
ResultsFig. 1. Processing model of a pre-tRNA of permuted tRNA genes via acircular RNA intermediate, probably depending on tRNA processing enzymes.

The active form of the C. merolae tRNA-intron splicing
endonuclease composed of multiple subunits could not be
produced in E. coli, and attempts to introduce expression ofthe enzyme caused severe growth defects. On the otherhand, a sufficient volume of marker protein was detectedfrom the circularization of mRNA derived from the marker
gene containing the inversed group I intron splicing sequence.
The β-galactosidase analysis using E. coli and B. subtilis
mutant strains identified genes for tRNA and tRNA-modi-		21Fig. 2. Plasmids containing marker genes that can be expressed by circularization of mRNA via either (A) the tRNA-intron splicing machinery and (B) theself-splicing group I intron. (C) A circularized form of mRNA.

fying enzymes that affected the translational activity. In B.
subtilis, the lack of tRNA or the enzyme modifying the anticodon corresponding to the four-codon boxes sometimes
increased the β-galactosidase activity. Some combinationsof these mutations further increased the translational activity, albeit only slightly. On the other hand, in E. coli, mostof the genes for tRNA and the tRNA-modifying enzymeappeared to be essential for the bacterium’s viability and efficient translation.
Conclusion
We attempted to construct a translation system usingmRNA circularized via either the tRNA-intron splicing machinery or the self-splicing group I intron. The active formof C. merolae tRNA-intron splicing endonuclease could not
be expressed in E. coli. This was probably due to a failureof appropriate protein folding, or to nonspecific cleavage ofintrinsic RNA molecules in E. coli cells. On the other hand,mRNA circularized via the group I intron produced a sufficient volume of marker protein, showing the utility of thissystem for further analysis. The introduction of cis-elements involved in ribosome binding may improve the efficiency of the internal translation initiation. In E. coli, mosttRNAs are essential for efficient translation, and the lack oftRNA genes generally decreases the translation of codons.
This tendency is observed in other bacteria.4) The results inthe present study showed the characteristic features ofcodon recognition in B. subtilis cells: the less the anticodon
repertoire exists, the more the translational activity increases. B. subtilis is a useful model for investigating thecodon recognition mechanism, and it could provide clues
for the construction of an efficient translation system using
bacterial ribosomes.
References
1)

Perriman R and Ares M Jr. (1998) Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo. RNA 4:1047–1054.
2) Abe N, Hiroshima M, Maruyama H, Nakashima Y, Nakano Y, Ma­
tsuda A, Sako Y, Ito Y, and Abe H (2013) Rolling circle amplification in a prokaryotic translation system using small circular RNA.
Angew. Chem. Int. Ed. 52: 7004–7008.
3) Soma A (2014) Circularly permuted tRNA genes: their expression
and implications for their physiological relevance and development.
Front. Genet. 5: 63. eCollection.
4) Näsvall JS, Chen P, and Björk GR (2007) The wobble hypothesis revisited: uridine-5-oxyacetic acid is critical for reading of G-endingcodons. RNA 13: 2151–2164.