Part:BBa_K5036019
MS2(x24)-HHR
Part Description
it is a non structural large and multifunctional protein which is encoded by coronavirus. NSP3 plays a crucial role in viral replication through its different activities as protease . Studying NSP3 enables the researchers to gain data about the mechanism of SARS-Cov2 infection and disease progression .
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 NSP3 in our switch .
literature characterization
To study the effect of a rotavirus protein (RF-NSP3) 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 (NSP3-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 technique called 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 NSP3 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 NSP3 preserving the mRNA. Additionally, NSP3 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 NSP3 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 NSP3 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 (NSP3) and a cellular protein (GAPDH) acting as a reference. The intensity of the signals was measured to compare the amount of NSP3 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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 7
Illegal XhoI site found at 1 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 20
Illegal AgeI site found at 238
Illegal AgeI site found at 456 - 1000COMPATIBLE WITH RFC[1000]
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