Part:BBa_K5082005

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.

              
         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.

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