Coding

Part:BBa_K5036018

Designed by: Emad hamdy Matter   Group: iGEM24_AFCM-Egypt   (2024-09-12)


NSP3A

Part Description

It is a non-structural large and multifunctional protein which has high affinity to the cap binding proteins such as eIF4GI.

Usage

We inserted this part to be attached to the cap of linear RNA.After cutting the poly A tail, the aptamer will be expressed to be attached to MCP. Once the injury occurs ,MMP9 increases and it will mediate the circularization of RNA through its nanobody.

this figure illustrates the structure of NSP3A in our TID switch.

Dry lab Characterization

We measured the effect of the nanobodies on the NSP3A-CAP binding stability, so we measured NSP3A-CAP before and after binding to nanobody1 and nanobody3

NSP3A-CAP binding stability

this figure illustrates The estimated binding stability (ΔG) between NSP3A and the Cap binding protein at the 5’ end of the mRNA equals -13.8 kcal mol-1 .


NSP3A-Cap-NB1 binding stability

this figure illustrates The estimated binding stability (ΔG) between NSP3A and the Cap binding protein, in the presence of nanobody1 , at the 5’ end of the mRNA equals -27.8 kcal mol-1. .

NSP3A-Cap-NB3 binding stability

this figure illustrates The estimated binding stability (ΔG) between NSP3A and the Cap binding protein, in the presence of nanobody 3 , at the 5’ end of the mRNA equals -34.9 kcal mol-1. .


Then we compared between the previous three states of the 5’ prime end and we conclude that Nanobodies presence stabilized the proteins at the 5’ end.

this figure shows that adding the NB1 to the NSP3A and the Cap increased their binding stability (ΔG) from -13.8 kcal mol-1 to -27.8 kcal mol-1. While adding the NB3 intensified their binding stability (ΔG) from -13.8 kcal mol-1 to -34.9 kcal mol-1 Therefore, we used the NB3 at the 5’ end to increase its stability, and NB1 at the 3’ prime end putting its high binding stability with MMP9 in our consideration .

Literature Characterization

To study the effect of a rotavirus protein (RF-NSP3A) on cellular translation, researchers chose MA104 cells. These cells are easily infected with rotavirus and better represent what happens during infection. Notably, the RF strain of the virus significantly reduces the translation of a specific type of cellular messenger RNA (poly(A) mRNA) . To create a comparison, they used the established cell line C20b. This line expresses a lower level of the protein of interest (NSP3A-RF) compared to MA104 cells infected with the strong RF strain (MOI of 10 for 3 hours, as measured by Western blot) (Figure A). Both C20b and MA104 cells were transfected with reporter messenger RNAs. Then, researchers purified RNA from these cells at two time points: immediately after transfection (time zero) and 6 hours later (time 6). They used a RT-qPCR to quantify the amount of reporter RNA present at each time point (Figure B). Finally, they measured the activity of a protein encoded by the reporter RNA at the 6-hour time point (Figures C to F).

The study found that even low levels of NSP3A significantly increased the production of proteins from messenger RNAs (mRNAs) with a specific ending sequence (GACC). Interestingly, this boost in protein production from these GACC-ending mRNAs wasn't simply due to NSP3A preserving the mRNA. Additionally, NSP3A itself seemed to promote the translation of both mRNAs with and without a poly(A) tail (poly(A) and nonpoly(A) mRNAs). This suggests that NSP3A might be able to take the place of a cellular complex (PABP-poly(A) complex) normally required to initiate translation.

Part (A) examined the levels of a viral protein called NSP3A within C20b cells. To achieve this, both C20b and MA104 cells were infected with a specific rotavirus strain (RF) at a set concentration (MOI of 10) for various time points. Western blotting was used to detect both the viral protein (NSP3A) and a cellular protein (GAPDH) acting as a reference. The intensity of the signals was measured to compare the amount of NSP3A relative to GAPDH. C20b cells served as the baseline for comparison (ratio set to 1). Parts (B) to (F) focused on reporter mRNAs, which are artificial RNAs used to track cellular processes. These reporter mRNAs (R-, N-, and pA-) were introduced into both MA104 and C20b cells using electroporation. RNA was then extracted from the cells at two time points: immediately after (T0) and 6 hours later (T6). RT-qPCR was employed to quantify the amount of reporter RNA present. Additionally, the activity of a protein produced from the reporter RNA was measured at the 6-hour time point (T6). To account for variations, the activity was normalized to the amount of reporter RNA present at either T0 or T6. Similar to part (A), mock-infected cells with reporter RNA served as the baseline (ratio set to 1).

Reference

Gratia, M., Sarot, E., Vende, P., Charpilienne, A., Baron, C. H., Duarte, M., ... & Poncet, D. (2015). Rotavirus NSP3 is a translational surrogate of the poly (A) binding protein-poly (A) complex. Journal of virology, 89(17), 8773-8782.‏



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 694
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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