Difference between revisions of "Part:BBa K4140022"

Latest revision as of 17:34, 7 October 2022

Peg10

This is a paternally expressed imprinted gene that binds its own mRNA and self-assembles into virion-like extracellular vesicles that encapsulate their own mRNA and are released from cells, enabling intercellular transfer of PEG10 mRNA. It is thought to have originated from the Ty3/Gypsy family of retrotransposons

Usage

PEG10 mRNA can be transferred between cells thanks to a paternally expressed imprinted gene that binds its own mRNA and self-assembles into extracellular vesicles that resemble virion capsules. Making VLPs is one of the SEND platform's components for mRNA delivery. Therefore, we took advantage of it by attaching the 5' and 3'UTR of PEG10 to the mRNA of PAH in order to make the PEG10 protein encapsulate the PAH mRNA as shown in figure 1.

Figure(1) SEND delivery to hepatocytes

Characterization of Mutational Landscape

After creating a multiple sequence alignment of the protein sequence and predicting mutational landscapes, the effect of these mutations on the evolutionary fitness of the protein is tested. The prediction of the mutational landscape by saturation mutagenesis of the Peg10 protein. The (P631D) mutation, as depicted in the chart, had the greatest score when compared to other mutations. On the other hand, it's clear that the (Q632R) had the least evolutionary fitness for Peg10 protein. As displayed in Figure(2)

Figure 2. shows the mutational landscape of the Peg10 protein.

Literature Characterization

The illustration below shows:-
(A) Detecting nucleic acids secreted from CA domain-containing proteins in the VLP fraction. (B) CRISPR activation of endogenous MmPeg10 leads to distinct RNA abundances and significance in the VLP fraction of N2a cells. NT, nontargeting gRNA. (C) Sequencing coverage of MmPeg10 mRNA shown by alignment of sequencing reads from (B). After heterologous transfection of MmPeg10 into N2a cells, differential RNA abundance and significance are shown in (D). n = 3 replicates. CMV, cytomegalovirus. (E) Four domains of MmPEG10 are translated into two isoforms. These are self-processed by the PEG10 protease into separate domains, of which the NC and RT bind RNA. (F) Fold enrichment of MmPeg10 mRNA compared with GFP in the VLP fraction from N2a cells transfected with wild-type MmPeg10 or deletions of the predicted nucleocapsid (∆NC) and reverse transcriptase (∆RT) domains. (G) Log2 fold change and significance of bound RNAs from eCLIP data comparing HA-GFP with wild-type MmPEG10-HA. (H) Representative sequencing alignment histogram of the MmDdit4 locus generated from eCLIP of N2a cells transfected with wild-type or mutant MmPeg10. (I) Representative sequencing alignment histogram of the MmPeg10 locus generated from eCLIP data of n = 3 HA-PEG10 and n = 3 untagged animals.

References

Segel, M., Lash, B., Song, J., Ladha, A., Liu, C. C., Jin, X., ... & Zhang, F. (2021). Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery. Science, 373(6557), 882-889.‏ Sequence and Features