Difference between revisions of "Part:BBa K5082006"

Revision as of 08:38, 7 September 2024

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 EIF3B-MUT part was created based on the EIF3B-HSU part. We deleted and altered several nucleotides involved in the formation of the mRNA secondary structure through complementary base pairing. Subsequently, the HSU structure could no longer be formed. The predicted secondary structure for EIF3B-MUT is shown in Figure 1.

                
          Figure 1. EIF3B-MUT 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.

   
   Figure 2. Experimental outline. (A) GFP sensor system. (B) Luciferase sensor system.

In addition to the experimental group which used the EIF3B-HSU part, we also used the EIF3B-MUT parti, with a similar sequence but no HSU structure, to serve as a control group. This design ensured that the difference in experimental results was caused by HSU structures, verifying our hypothesis.

Characterization The EIF3B-MUT sequence was confirmed by Sanger sequencing, as shown in Figure 3.

                    
                     Figure 3. EIF3B-MUT validated by Sanger sequencing.

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=cdTwvRFDGx.

[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

Usage and Biology

Sequence and Features