Project

Circular mRNA -the world´s longest protein-

1. Overall project summary
2. Introduction
3. Project Flow
4. Theory and Methods
5. Experiments
6. Results&Data analysis
7. Future Works
8. Conclusions
9. References

Overall project summary

In vivo, proteins are synthesized in transcription and translation. Generally, mRNA is single-strand RNA and starts translation by binding ribosome on start codon. And then the translation ends by separating ribosome from mRNA. In this study, we aim to build the method of synthesizing long-chain, massive proteins and to improve translation efficiency. That is, circular mRNA allows ribosomes to have a semi-permanent translation mechanism and improves a defect of stop codon. To cyclize mRNA, we can use a splicing mechanism of T4 phage. Splicing is a mechanism removing circular intron which doesn't code for proteins and joining in exon which codes for ones. It occurs after transcription. Splicing is catalyzed by several base sequences of the ends of introns as a ribozyme, being subjected to nucleophilic attack from intron to exon. So we will introduce the plasmid which places the sequence of the end of intron as a splicing ribozyme on the end of a gene coding for proteins into E. coli, and cyclize mRNA for synthesis of long-chain, massive proteins.

Introduction

We want to create an unbelievablely long protein. In vivo, the process of protein synthesis is transcription and translation. Generally, mRNA is linear. Translation is started by ribosome binding on a start codon and then it is stopped by dissociation of ribosome from mRNA. The size of synthesized protein is constant. So we planned the construction of circular mRNA without stop codon. It enables infinite translation, so it enables mass production of the protein. This is just like a protein factory!

Project Flow

We can use the group I intron self-splicing mechanism in td gene of T4 phage to circularize mRNA. The group I intron self-splicing is a mechanism that circularizes an intron and connects exons. It occurs after transcription. The self-splicing is catalyzed by several base sequences of the ends of introns as a ribozyme. We permuted exons and introns with the mechanism and attempted an exon circularization. So we constructed mRNA circularization devices. We induced a protein coding sequence and them into E. coli. We created circular mRNA and synthesized massive long-chain protein with it.

Theory & Methods

The circularization mechanism of group I intron

The group I intron is capable of self-splicing. The mRNA circularization device is based on the mechanism. We explain the circularization mechanism of group I intron with td gene of T4 phage as an example. Td gene consists of an upstream exon, an upstream intron, an ORF, a downstream intron and a downstream exon. As the first step, a nucleophilic attack by a guanosine separates the upstream exon from the upstream intron and then the guanosine bonds to the 5’ end of the upstream intron. As the second step, the downstream exon is separated from the downstream intron by a nucleophilic attack. The nucleophilic attack takes place by a hydroxy group at the 3’ end of the upstream exon. (Figure 1)


Figure 1. Self-splicing in T4 phage: the first and second step (Blue: intron, Orange: exon)

As the third step, the upstream intron bonds to the downstream intron by an attack on an adenine of the upstream intron. The attack takes place by a hydroxyl group of an end of the downstream intron. And then a circular intron is formed.(Figure 2)

Figure 2. Self-splicing in T4 phage: the third step (Blue: intron, Orange: exon)

The permuted intron-exon method: PIE method

Two exons are connected with each other in the circularization system; furthermore an exon can theoretically be circularized by the system. (Figure 3)



Figure 3. An idea of mRNA circularization (Blue: intron, Orange: exon)

The method that puts the theory into practice is the PIE method. The PIE method stands for the Permuted Intron-Exon method. A circular mRNA is made by the method.

The protocol of PIE method (Figure 4)

1. Pick out the intron and splice site in the exon.
2. Sandwich the sequence that you want to circularize between preceding fragments.

Figure 4. PIE method

Experiments

Parts construction

Parts assembly

We picked out the two fragments (5’ side and 3’ side) for self-splicing from td gene of T4 phage. The fragment consists of an intron and the fragment of the exon (splicing site). We integrated a promoter, the fragment of self-splicing (3’ side) and RBS(binding-site for ribosome) into a plasmid. (→ mRNA circularization device (5´ side)) We integrated the fragment of self-splicing (3’ side) and DT (double terminator) into a plasmid. (→ mRNA circularization device (3´ side))(Figure 5)

Figure 5. Parts assembly

The mRNA which becomes a loop

Detecting circular mRNA

Summary of the experiment

The existence of circular mRNA is confirmed by RNase processing. RNA is decomposed by RNaseA (endoribonuclease). Endogenous RNA (linear RNA)(GAPDH) is decomposed by RNaseR (exoribonuclease), but circular RNA is not decomposed. Double-stranded DNA from undecomposed RNA can be gained with RT-PCR. So the existence of circular mRNA is confirmed by the observation of the DNA with electrophoresis.

Flow of the experiment

Purpose: proving the existence of circular mRNA
Goal: finding the RNA that is decomposed by endoribonuclease but is not decomposed by exoribonuclease.
Protocol:

1. RNase processing: to find the circular mRNA
2. RT-PCR: to synthesize cDNA and to detect the cDNA synthesized from circular mRNA or endogenous RNA
3. Electrophoresis: to detect the DNA synthesized from the cDNA

Protocol

Jump!

Elucidation of the cyclization mechanism

Summary of the experiment

I read the sequence of the binding site of the circular mRNA to confirm how an intronic ribozyme acted

Synthesizing long-chain RFP

The ability of coloration

Summary of the experiment

We compared RFPs derived from RNAs in various states to assay the coloration of a long-chain RFP.

The existence of long-chain protein -1- SDS-PAGE

Summary of the experiment

We confirmed the existence of the long-chain RFP derived from the circular mRNA by SDS-PAGE.

The existence of long-chain protein -2- Western blotting

Summary of the experiment

This experiment is now underway.

The determination of long-chain RFP

Summary of the experiment

Synthesizing long-chain SmtA (Metallothionein)

Summary of the experiment

We cultured E. coli that the SmtA semi-permanent generator is integrated into in the presence of zinc to examine the activity of a long-chain SmtA.

Results&Data analysis

Detecting circular mRNA

Positive: 3,5,6
Negative: 1,2,4,7,8

1. To detect the sequence of the circular mRNA in the cDNA derived from the RNA after RNaseA processing
2. To detect the sequence of the linear mRNA in the cDNA derived from the RNA after RNaseA processing
3. To detect the sequence of the circular mRNA in the cDNA derived from the RNA after RNaseR processing
4. To detect the sequence of the linear mRNA in the cDNA derived from the RNA after RNaseR processing
M. Marker
5. To detect the sequence of the circular mRNA in the cDNA derived from the non-treated RNA
6. To detect the sequence of the linear mRNA in the cDNA derived from the non-treated RNA
7. To detect the sequence of the circular mRNA in the non-treated RNA
8. To detect the sequence of the linear mRNA in the non-treated RNA

See the lane 7,8. → RNA is not detected by the electrophoresis, namely, the matter detected is cDNA.
See the lane 5,6,7,8. → The factor involved in the existence of cDNA is the ribonuclease processing.
See the lane 1,2,5,6. → The endoribonuclease decomposes the all RNA.
See the lane 3,4,5,6. → There is the RNA decomposed by the exoribonuclease.
Therefore, the RNA that is decomposed by the endoribonuclease but is not decomposed by the exoribonuclease exists. We think this RNA is the circular mRNA!

Elucidation of the cyclization mechanism

After having reverse-transcripted it, I amplified a joining part between the intron by PCR and read sequence. I show below the sequence for the junction of 3'intron parts and 5'intron parts.

As a result of having read Sequence, I understood that U of 5'intron was combined with C of 3'intron and mRNA became a loop.

The existence of a long-chain protein

RFP -1- SDS-PAGE

1. RFP from linear RNA (with stop codon)
2. RFP from circular RNA (with stop codon)
3. RFP from circular RNA (without stop codon)
4. RFP from circular RNA (with the stop codon of mRNA circular device)
S. supernatant
P. precipitation
M. marker

There is a long-chain protein near a band that indicates 250 kDa. The molecular weight of a monomeric RFP is 25423.7(→ BBa_E1010), so we guess that the protein is not less than decameric RFP. In addition, long chain protein looked like a ladder. This reason is because it dissociates while ribosome continues translating it. There will be three reasons that ribosome leaves.

1. It is caused by a distortion to occur to the cyclic mRNA.
2. When other ribosome was binded, the ribosome which turned around was pushed outside of the circular mRNA.
3. Originally there may be little tRNA like a rare codon.

RFP -2- Western blotting

We separated “long-chain RFP” from cell bodies by 12%, 10%, 7% of SDS-PAGE and transferred PVDF membrane. After that we performed Western blot by using peroxidase-labeled RFP antibody (rabbit) and color coupler (DAB). We showed below the result.

As a result of having dyed gel of SDS-PAGE in CBB after the membrane transfer.

As a result of dyed gel after the membrane transcription. The protein more than 100KDa was not transferred and stayed in gel of 12%. The protein more than 250KDa was not transferred and stayed in gel of 10% Most of the long chain protein more than 250KDa was transferred on PVDF membrane.

The result of having performed Western blot of membrane.

A band was detected and was able to confirm that an antibody was connected, and it was developed a pigment by DAB. A band of RFP (27KDa) was detected clearly when we watched the membrane which transferred from 12% and 10% gel after color development. However, if gel density is high, it is hard to elute the long chain protein. So it is hard for us to show that long chain protein was detected from this date.

The determination of long-chain RFP

We aimed to find how much long-chain RFP would be synthesized per cell body of E. coli. RFP combines with Histag. So it ought to have gained monomer RFP and long-chain RFP by conducting affinity chromatography with Ni-NTA. However, the long-chain RFP was insolubilized and produced precipitation because it is polymer compound. And it could not combine with the Ni-NTA column, flowing out in flow-through. So it was difficult to refine the long-chain RFP, which has different size. On the other hand, the monomer RFP combined with the Ni-NTA column, being able to refine them using imidazole. By using these monomer RFP, we tried to calculate protein mass of the long-chain RFP.

Protocol

1. E. coli, which can synthesize long-chain protein, was cultivated for 3 hours, after that, IPTG, which is an inducing substance, was added, and it was cultivated for further 10 hours. 5mL of culture solution that reached the stationary phase was taken, it was centrifuged, the supernatant was removed, and 300µL of 1×PBS was added. The cell body was crushed, it was centrifuged, and supernatant was separated from precipitation. The supernatant was refined with Ni-NTA, creating almost pure monomer RFP solution. The solution was refined in 1, 2, 4, 8, 16, and 32 times, measuring Abs (280nm) of each solution by using an absorbance meter. And protein concentration of each solution from the molecular absorbance coefficient and the molecular weight of RBS was calculated.
2. 1mL of culture solution that reached the stationary phase was taken, it was centrifuged, the supernatant was removed, and 1mL of 1×PBS was added. Using this solution as a sample, an undiluted solution and a 10 times diluted solution are prepared.
3. 4µL of each sample solution and each monomer RFP solution of different dilution rate were applied on the gel. Each of them was separated by the 10% SDS-PAGE, dyeing by CBB. After that, using the image processing software (ImageJ), analytical curve based on “the total sum of its brightness and area of dyeing” and “the already known concentrate of monomer solution” was made. Adapting the former, the concentration of long-chain RFP was calculated.
4. On the other hand, to calculate the number of cultivated E. coli easily, OD¬600 of culture solution was measured. Using the conversion formula of E. coli (the number of cell bodies), the number of cell bodies from the value of OD600 was calculated.
5. From the number of cell bodies and the concentration of long-chain RFP, the mass of long-chain RFP per cell body was calculated.
6. And also, if silk fabrics are made of 900g raw silk by this E. coli, the number of cell bodies that is needed for it was estimated. We assumed that the fibers of same length from the same weight of raw silk and the long-chain RFP.

Result

The following table is the concentration of each monomer RFP solution determined from the absorbance.

Table 1. The concentration of each monomer RFP solution corresponding to the absorbance on 280nm

We calculated the sum of the stained area with the chromaticity from the picture. We made a calibration curve from “the sum of the stained area with the chromaticity of the gel” and “known concentration of the monomer solution”. The result is shown in the following table.

Table 2. The concentration of monomer RFP and polymer RFP

Figure 6. calibration curve

Figure 7. The result of 10% SDS-PAGE

Following table shows the number of bacteria which synthesizes long-chain protein calculated by OD600. And we calculate the amount of the proteins which one bacterial cell (E. coli) synthesized from concentration of the protein (the above).

Table 3.

examination

It showed that Circular mRNA / liner mRNA=0.026 in Modeling. It can not be said that a high ratio. However, the ratio of polymer RFP / monomer RFP=0.71, we found that long-chain protein is synthesized in relatively large amounts. This shows Circular RNA is better in the ability to synthesize protein than linear RNA.

There is a problem that the way to synthesize of long-chain proteins using circular RNA. It can not adapt to all kind of proteins, Efficiency of RNA cyclization is low, and so on. However, if you improve that problems or devise a way to use, the method of　synthesizing protein using circular mRNA will be useful enough basis.

Activation of a long-chain protein

The ability of coloration

1.RFP from linear RNA (with stop codon)
2.RFP from circular RNA (with stop codon)
3.RFP from circular RNA (without stop codon)：using this device
4.RFP from circular RNA (with the stop codon of mRNA circular device)

The RFP (+histidine tag) polymer didn’t show the fluorescence.
Possible factor
1.The RFP polymer is too huge, so it becomes an inclusion body.
2.The repetitive amino acid sequences are too near, so the conformation of the RFP polymer is in disorder.

On the other hand, we understand that long chain protein was detected than 150KDa on the membrane which transferred from 7% gel. Because we used RFP antibody derived from a rabbit, this date shows that detected long chain protein is protein derived from RFP.

Future Works

Our future work is the improvement of a functionality of a long-chain protein. For example, SmtA (Metallothionein) can catch heavy metal ions such as Zn²⁺. We think that a protein sheet made of long-chain SmtA (Metallothionein) prevents heavy metal from leak from factories.

If the improvement of a long-chain protein is achieved, we can gain practical achievements in many directions.

Conclusions

The existence of circular mRNA

Yes! ← RNase processing

The existence of a long-chain protein

Yes! ← SDS-PAGE

Yes! ← Western blotting

Activation of a long-chain protein

RFP

No… ← Comparing RFPs derived from RNAs in various states

SmtA

The experiment is now underway.

References

1. Rederick K. chu, Gladys F. Maley, and Frank Maley(1998) “RNA splicing in the T-even bacteriophage” Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201, USA
2. M. Puttaraju and Michael D. Been(1996) “Circularizing ribozymes and decoy-competitors by autocatalytic splicing in vitro and in vivo” SAAS Bull Biochem Biotechnol
3. R. Perriman and M. Ares, Jr: (1998) Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo.
4. So Umekage et al. (2012) “In Vivo Circular RNA Expression by the Permuted Intron-Exon Method” Innovations in Biotechnology