Difference between revisions of "Part:BBa K5082005"
 
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__NOTOC__
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<partinfo>BBa_K5082005 short</partinfo>
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===Usage and Biology===
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RNA is a common type of biological molecule. In cells, RNAs exist in many forms including messenger RNA (mRNA), ribosomal RNA (rRNA), translational RNA (tRNA), etc. Although RNA molecules are single-stranded, unlike DNA, they still form secondary structures through intramolecular complementary base-pairing to minimize free-energy (−ΔG/nt) and become more thermodynamically stable. mRNA secondary structures are dependent on factors including base sequence, protein binding, and guanine-cytosine content. mRNA secondary structures with −ΔG/nt≥0.3 are defined to be highly structured while those with −ΔG/nt ≤0.2 are defined to be poorly structured [1]. mRNA secondary structures are often found in non-coding regions, especially in the 3’UTR [2]. Highly structured 3’UTR are abbreviated as HSU while poorly structured 3’ UTR are abbreviated as PSU. Although HSU regions are non-coding, they serve vital roles in the regulation of gene expression [1]. Previously, the EIF3B gene has been reported to contain an HSU structure [3]. Therefore, this sequence can be used to regulate gene expression once fused downstream to a gene. The secondary structure of an RNA sequence can be predicted by computer software such as mFold or ViennaRNA [4]. The predicted secondary structure for EIF3B-HSU is shown in Figure 1.
 
RNA is a common type of biological molecule. In cells, RNAs exist in many forms including messenger RNA (mRNA), ribosomal RNA (rRNA), translational RNA (tRNA), etc. Although RNA molecules are single-stranded, unlike DNA, they still form secondary structures through intramolecular complementary base-pairing to minimize free-energy (−ΔG/nt) and become more thermodynamically stable. mRNA secondary structures are dependent on factors including base sequence, protein binding, and guanine-cytosine content. mRNA secondary structures with −ΔG/nt≥0.3 are defined to be highly structured while those with −ΔG/nt ≤0.2 are defined to be poorly structured [1]. mRNA secondary structures are often found in non-coding regions, especially in the 3’UTR [2]. Highly structured 3’UTR are abbreviated as HSU while poorly structured 3’ UTR are abbreviated as PSU. Although HSU regions are non-coding, they serve vital roles in the regulation of gene expression [1]. Previously, the EIF3B gene has been reported to contain an HSU structure [3]. Therefore, this sequence can be used to regulate gene expression once fused downstream to a gene. The secondary structure of an RNA sequence can be predicted by computer software such as mFold or ViennaRNA [4]. The predicted secondary structure for EIF3B-HSU is shown in Figure 1.
  
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              https://static.igem.wiki/teams/5407/eif3b-hsu.png
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                  https://static.igem.wiki/teams/5407/eif3b-hsu.png
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          Figure 1. EIF3B-HSU secondary structure predicted by RNAfold [5].
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            Figure 1. EIF3B-HSU secondary structure predicted by RNAfold [5].
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===Design===
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In our project, we found that the G3BP1 protein was overexpressed in gastric cancer (GC) cells [6]. Meanwhile, G3BP1 could bind with HSU structures and lead to mRNA degradation [7]. Therefore, we fused the EIF3B-HSU sequence downstream to reporter genes: GFP and luciferase, to monitor G3BP1 levels and hence diagnose GC. The experimental outline is shown in Figure 2.
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                    https://static.igem.wiki/teams/5407/eif3b-hsu-2.png
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      Figure 2.Experimental outline. (A) GFP sensor system. (B) Luciferase sensor system.
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===Reference ===
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References
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[1] Fischer, Joseph W et al. “Structure-Mediated RNA Decay by UPF1 and G3BP1.” Molecular cell vol. 78,1 (2020): 70-84.e6. doi:10.1016/j.molcel.2020.01.021
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[2] Ermolenko, Dmitri N, and David H Mathews. “Making ends meet: New functions of mRNA secondary structure.” Wiley interdisciplinary reviews. RNA vol. 12,2 (2021): e1611. doi:10.1002/wrna.1611
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[3] Mestre-Fos, Santi et al. “eIF3 engages with 3'-UTR termini of highly translated mRNAs in neural progenitor cells.” bioRxiv : the preprint server for biology 2023.11.11.566681. 11 Nov. 2023, doi:10.1101/2023.11.11.566681. Preprint.
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[4] Gaspar, Paulo et al. “mRNA secondary structure optimization using a correlated stem-loop prediction.” Nucleic acids research vol. 41,6 (2013): e73. doi:10.1093/nar/gks1473
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[5] “RNAfold Web Server.” Univie.ac.at, 2024, rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3&ID=aRk6YHn0WG.
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[6] Ge, Yidong et al. “The roles of G3BP1 in human diseases (review).” Gene vol. 821 (2022): 146294. doi:10.1016/j.gene.2022.146294
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[7] Xiong, Rui et al. “G3BP1 activates the TGF-β/Smad signaling pathway to promote gastric cancer.” OncoTargets and therapy vol. 12 7149-7156. 2 Sep. 2019, doi:10.2147/OTT.S213728
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5082005 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K5082005 parameters</partinfo>
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Latest revision as of 06:42, 15 September 2024

EIF3B-HSU

Usage and Biology

RNA is a common type of biological molecule. In cells, RNAs exist in many forms including messenger RNA (mRNA), ribosomal RNA (rRNA), translational RNA (tRNA), etc. Although RNA molecules are single-stranded, unlike DNA, they still form secondary structures through intramolecular complementary base-pairing to minimize free-energy (−ΔG/nt) and become more thermodynamically stable. mRNA secondary structures are dependent on factors including base sequence, protein binding, and guanine-cytosine content. mRNA secondary structures with −ΔG/nt≥0.3 are defined to be highly structured while those with −ΔG/nt ≤0.2 are defined to be poorly structured [1]. mRNA secondary structures are often found in non-coding regions, especially in the 3’UTR [2]. Highly structured 3’UTR are abbreviated as HSU while poorly structured 3’ UTR are abbreviated as PSU. Although HSU regions are non-coding, they serve vital roles in the regulation of gene expression [1]. Previously, the EIF3B gene has been reported to contain an HSU structure [3]. Therefore, this sequence can be used to regulate gene expression once fused downstream to a gene. The secondary structure of an RNA sequence can be predicted by computer software such as mFold or ViennaRNA [4]. The predicted secondary structure for EIF3B-HSU is shown in Figure 1. 

                  eif3b-hsu.png
           Figure 1. EIF3B-HSU secondary structure predicted by RNAfold [5].

Design

In our project, we found that the G3BP1 protein was overexpressed in gastric cancer (GC) cells [6]. Meanwhile, G3BP1 could bind with HSU structures and lead to mRNA degradation [7]. Therefore, we fused the EIF3B-HSU sequence downstream to reporter genes: GFP and luciferase, to monitor G3BP1 levels and hence diagnose GC. The experimental outline is shown in Figure 2. 

                    eif3b-hsu-2.png
      Figure 2.Experimental outline. (A) GFP sensor system. (B) Luciferase sensor system.

Reference

References

[1] Fischer, Joseph W et al. “Structure-Mediated RNA Decay by UPF1 and G3BP1.” Molecular cell vol. 78,1 (2020): 70-84.e6. doi:10.1016/j.molcel.2020.01.021

[2] Ermolenko, Dmitri N, and David H Mathews. “Making ends meet: New functions of mRNA secondary structure.” Wiley interdisciplinary reviews. RNA vol. 12,2 (2021): e1611. doi:10.1002/wrna.1611

[3] Mestre-Fos, Santi et al. “eIF3 engages with 3'-UTR termini of highly translated mRNAs in neural progenitor cells.” bioRxiv : the preprint server for biology 2023.11.11.566681. 11 Nov. 2023, doi:10.1101/2023.11.11.566681. Preprint.

[4] Gaspar, Paulo et al. “mRNA secondary structure optimization using a correlated stem-loop prediction.” Nucleic acids research vol. 41,6 (2013): e73. doi:10.1093/nar/gks1473


[5] “RNAfold Web Server.” Univie.ac.at, 2024, rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3&ID=aRk6YHn0WG.

[6] Ge, Yidong et al. “The roles of G3BP1 in human diseases (review).” Gene vol. 821 (2022): 146294. doi:10.1016/j.gene.2022.146294

[7] Xiong, Rui et al. “G3BP1 activates the TGF-β/Smad signaling pathway to promote gastric cancer.” OncoTargets and therapy vol. 12 7149-7156. 2 Sep. 2019, doi:10.2147/OTT.S213728


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]