53NISR Research GRANT
2010 Young Investigator Research Grant

Functional RNA production using the marine phototrophic
bacterium Rhodovulum sulfidophilum
So UMEKAGE
Department of Environmental and Life Sciences, Toyohashi University of Technology

Research aims
Functional RNAs, e.g., aptamers, antisense oligonucleotides, and double-stranded RNA (dsRNA), hold promise for use as RNA drugs in the near future. However, thelinear form of RNA without chemical modifications is rapidly degraded by endogenous nucleases. To address theproblems outlined above, our research group has considered
the possibility that functional RNAs can be expressed invivo as circular RNAs for inexpensive RNA drug production because circular RNA molecules are resistant to exoribonucleases without any chemical modifications under cellular conditions1,2).
Circular RNA can be produced by two methods. The first
method makes use of ligase to ligate both ends of the linear
form of RNA transcripts, while the second method uses aspontaneous group I intron self-splicing system, designatedas the permuted intron-exon (PIE) method. The latter technique is the only methodology available for in vivo circularRNA production because it has no requirement for proteinaceous components, such as ligases. Therefore, the PIE
method is a promising economical method for producing
circular RNA drugs.
To increase the circular RNA expression in marine phototrophic bacteria Rhodovulum sulfidofilum, we developed
the TOP (tandem one-way transcription of PIE) technique,which is a simple methodology for increasing the copy
number of the PIE sequence in a single plasmid.
In this study, we constructed plasmids for circular RNA
expression by the TOP method, evaluated the expression ofthe circular RNA from these TOP plasmids in the modelbacterium Escherichia coli, and finally attempted to produce extracellular circular RNA using the marine phototrophic bacterium R. sulfidophilum as the host.
Methods
First, we amplified the transcriptional unit, which consists of the lpp promoter, the PIE sequence and the rrnC
terminator in pM3-3E5, with both the 5 flanking sequence
containing KpnI–XhoI sites and the 3 flanking sequence
containing a SalI site. Next, we digested the amplified sequence with KpnI and SalI, and the resulting fragment wasinserted into the M3 plasmid double-digested with KpnIand XhoI. The digested XhoI site on the M3 plasmid andthe SalI site on the amplified fragment can hybridize withFig. 1. Construction of the pTOP vectors, and the application of theTOP method for generating circular RNA in JM101Tr. (A) Outline of theTOP method. (B) Illustration of sequential insertion of the PIE sequence
into the same plasmid. (C) Nucleotide sequence of one unit of the TOPsystem. Arrows represent splicing positions of this PIE sequence: yellow,the PIE sequence; blue box, lpp promoter sequence; italicized sequence inthe blue box, 35 and 10 regions of the lpp promoter; red upper caseletters, aptamer sequence and rrnC terminator sequence; lower case lettersin the yellow region, intron sequence of the td gene; bold lower case letters, exon sequence of the td gene; bold, circularized sequence; boxed sequence, ligated sites. (D) Northern blotting analysis of the circular RNA
expression by each member of the pTOP plasmid series. Arrows indicate
the migration positions of the circular RNA (circular), nicked RNA(nicked) and 5S rRNA. Lane “M”, the circular RNA control marker prepared by in vitro transcription (Umekage & Kikuchi, 2009a); lane “”,
total RNA from JM101Tr; lane “pM3”, negative control of the TOP system lacking the PIE sequence. Roman numerals I, II, III and IV represent
the total RNA from JM101Tr harbouring pTOP(I), pTOP(II), pTOP(III)and pTOP(IV), respectively.

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mutual 3 protruding ends of the palindoromic TCGA sequence, and the resulting ligated fragment forms the sequence GTCGAG, which can be digested with neither XhoInor SalI (Fig. 1B). Therefore, the inserted sequence is asfollows: 5-KpnI-XhoI-lpp promoter-PIE sequence-rrnC
terminator sequence-GTCGAG site-3 (Fig. 1C). Thus, thesubsequent transcriptional unit can be inserted at theKpnI–XhoI site. We constructed a series of four pTOP vectors from M3 [i.e., pTOP(I), pTOP(II), pTOP(III) andpTOP(IV), which were designated according to the numberof inserted transcriptional units they contains]. These plasmids were transformed into the E. coli JM101Tr and thetransformants obtained were cultivated for 18 hour at 30°Cin the 2YT broth. After the cultivation, total RNA waspurified by the AGPC method and the circular RNAs in thetotal RNA fraction were detected by Northern analysis (Fig.
1D).
Next, the TOP plasmid was introduced into the Rhodovulum sulfidophilum DMS 1374T with a heat shock procedure.
The transformants were cultivated under light-anaerobicconditions. Total RNAs were recovered with the AGPC
method and the expression of circular RNA was monitoredby Northern analysis.

the amount of circular RNA produced in 1 L of culture
medium was estimated to be approximately 0.19 mg, whichis the highest yield of circular RNA in E. coli reported todate. In contrast, the expression level of the circular RNA
was dramatically low with the E. coli host carryingpTOP(IV); the reason for this low expression is not yetclear. To address this problem, we recovered plasmid DNA
from the E. coli strain JM101Tr carrying pTOP(IV) after 18
h of cultivation. The plasmid DNA was then digested withHindIII, which has a single cleavage site in the plasmid, andsubjected to 1% agarose gel electrophoresis. Several fragments of pTOP(IV) with different size were observed (datanot shown). These unexpected results suggested a marked
instability of the pTOP(IV) in the E. coli host cells during
cultivation.
Next we introduced the pTOP(II) plasmid into R. sulfidophilum with the heat pulse method and circular RNA expression was monitored by Northern analysis; however theexpression of the circular RNA was not detected (data notshown). We currently investigate the possibility of using
other promoters, such as the rrnA promoter, for the effective circular RNA expression by R. sulfidophilum.
ConclusionResults
We found that the circular RNA was expressed fromall pTOP plasmids (pTOP(I), pTOP(II), pTOP(III) andpTOP(IV)) (Fig. 1D). As shown in the Fig. 1D, the expression level of circular RNA from the plasmid with two tandem insertions of the PIE [pTOP(II)] is the highest among
the four plasmids constructed. These results indicated that
the TOP system is a simple and potentially useful methodology for increasing the expression of circular RNAs in E.
coli. The level of circular RNA expression with pTOP(II)was estimated to be about 9.71.0 ng per 1 m g of total
RNA after 18 h of cultivation and this yield was approximately 2.7-fold higher than that obtained with the expression procedure using the pM3-3E5 system2). In addition,

The TOP system was effective when it was used with E.
coli. However the system did not work well with R. sulfidophilum. Further optimization of the TOP system will berequired to achieve effective expression of circular RNAs
by R. sulfidophilum.
References1)

Umekage, S. & Kikuchi, Y., (2009). In vitro and in vivo production
and purification of circular RNA aptamer. J. Biotechnol. 139: 265–272.
2) Umekage, S. & Kikuchi, Y., (2009). In vivo circular RNA production
using a constitutive promoter for high-level expression. J. Biosci.
Bioeng. 108: 354–356.