Project
Circular mRNA -the world´s longest protein-
- Overall project summary
- Introduction
- Project Flow
- Theory and Methods
- Experiments
- Results&Data analysis
- Future Works
- Conclusions
- References
Overall project summary
In vivo, proteins are synthesized in transcription and translation.
Generally, mRNA is single-strand RNA, starting translation by binding ribosome on initiation codon, and ending 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, allowing ribosomes to have a semi-permanent translation mechanism by producing circular mRNA and causing a defect of termination codon.
To cyclize mRNA, we can use a splicing mechanism of T4 phage.
Splicing is a mechanism removing circular introns which don't code for proteins and joining in exons which code 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 introns to exons.
So we will introduce the plasmids which place the sequence of the end of introns 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 unbelievably 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 bond 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 is taken 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 bond to the downstream intron by an attack on an adenine of the upstream intron. The attack is taken 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)
- Pick out the intron and splice site in the exon.
- 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
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:
- RNase processing: to find the circular mRNA
- RT-PCR: to synthesize cDNA and to detect the cDNA synthesized from circular mRNA or endogenous RNA
- Electrophoresis: to detect the DNA synthesized from the cDNA
Protocol
Jump!
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!
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.
RFP -2- Western blotting
This experiment is now underway.
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.
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
-
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
- M. Puttaraju and Michael D. Been(1996)
“Circularizing ribozymes and decoy-competitors by autocatalytic splicing in vitro and in vivo”
SAAS Bull Biochem Biotechnol
- R. Perriman and M. Ares, Jr: (1998)
Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo.
- So Umekage et al. (2012)
“In Vivo Circular RNA Expression by the Permuted Intron-Exon Method”
Innovations in Biotechnology
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