RNA

Part:BBa_K5036039

Designed by: Emad hamdy Matter   Group: iGEM24_AFCM-Egypt   (2024-09-21)


HHR

Part Description

HHR is a type of self-catalytic RNA molecule that has been engineered to cleave specific RNA targets which is essential for various biological processes, such as gene regulation and viral replication.

Usage

CRISPR multiplexing employs a single RNA molecule that contains multiple guide RNAs targeting various locations so we have utilized our HHR to mediate self-cleavage so that we could separate these multiple guide RNAs into individual functional units.

this figure illustrates the structure and function of HHR in our CRISPER multiplexing system .


Characterization by Mathematical Modeling

The model provides the result of HHR action on our switch which is preventing the basal activity of our switch in absence of the MMP-9 , so once MMP-9 increases, its presence will initiate the switch circulation for translation of YAP-1. It is based on parametric values from literature. note we neglected the basal activity that may be due to cross talk between different MMPs

Graph(1). Illustrates the relation between circulation form activity of our switch (Yellow line) and their ability for YAP-1 production (Black line) upon their binding to MMP-9 .

Experimental Characterization

It is considered that manipulating the mRNA circularization process could be a promising approach to gain better control over translational initiation. Circularization and translation of this mRNA transcript would rely on a TID that imitates the natural PABP role by binding both the aptamer-based control region and any preinitiation complex member. TID consists of fusing the MCP to different eIF4F-binding proteins (eIFBPs). by sitting HHR-binding sites between aptamer region and the poly (A) region to remove the poly (A) tail through ectopic co-expression of HHR-binding sites, Due to high background activity of natural poly A tail located downstream of the synthetic aptamer region, which may attract endogenous factors (such as native PABP) to form a closed-loop configuration and activate translation even in the absence of TIDs, so SEAP expression from poly(A)-deficient mRNA showed markedly reduced background levels and increased TID-dependency.

(a) HEK-293 cells were co-transfected with a SEAP expression vector and the production of proteins from mRNA modified with a 3' UTR carrying a specific aptamer for RNA-binding proteins and a site for pre programmed poly (A) removal via HHR relies on the existence of TIDs made up of various RBPs linked to different eIFBPs as (PABP-MCP, pSL1315; eIF4G-MCP, pSL154; MCP-eIF4E, pSL1316; MCP-NSP3A, pSL95; MCP-VPg, pLYL47). Although this approach may improve control that relies on TID, it also carries possible drawbacks as Trans-removal has high background activity in ­ -shRNA on the other hand (b) cis-removal of poly A tail showed that MCP-NSP3A has the highest SEAP so it is the most preferred and MCP-Coh2 protein incapable of initiating translation (pSL674, negative control) .

Literature Characterization

To understand how various hammerhead ribozyme motifs affect gene activity, they incorporated eight different motifs into relevant mRNA sequences. To compare results across diverse genetic systems, they assessed reporter gene expression in human cells, baker's yeast (S. cerevisiae), and E. coli bacteria. Well-established plasmid-based gene expression constructs served as their reporter systems.

(A)The researchers placed the HHR motifs at the end of a reporter gene called Renilla luciferase (hRluc) within a vector named psi-CHECK2. The reporter gene is responsible for producing light. (B)They investigated how HHR motifs influence the production of a LacZ gene by inserting them into a specific region (3'-UTR) of a separate Gal4 gene on a plasmid. The LacZ gene, located on a chromosome, is controlled by a promoter that responds to Gal4. (C,D) It shows a comparison of how different ribozymes affect gene activity in living cells (in vivo analysis). Black bars represent a reporter gene controlled by a functional HHR motif, while gray bars show the same gene controlled by a non-functional HHR. The control group (Ctrl) lacks any ribozyme sequences. (C) of the figure displays results in human HeLa S3 cells after 18 hours of introducing the genetic material (transfection). (D) shows gene activity in baker's yeast (S. cerevisiae) grown for 18 hours in a special nutrient solution (synthetic complete medium) at room temperature (30°C).

Reference

Shao J, Li S, Qiu X, Jiang J, Zhang L, Wang P, Si Y, Wu Y, He M, Xiong Q, Zhao L, Li Y, Fan Y, Viviani M, Fu Y, Wu C, Gao T, Zhu L, Fussenegger M, Wang H, Xie M. Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells. Cell Res. 2024 Jan;34(1):31-46. doi: 10.1038/s41422-023-00896-y. Epub 2024 Jan 4. PMID: 38172533; PMCID: PMC10770082.


Wurmthaler, L. A., Klauser, B., & Hartig, J. S. (2018). Highly motif-and organism-dependent effects of naturally occurring hammerhead ribozyme sequences on gene expression. RNA biology, 15(2), 231-241.‏


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]


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