CN-121991936-A - Fusion protein, RNA editing level detection method and application thereof

Abstract

The invention discloses a fusion protein, a detection method of RNA editing level and application thereof, and relates to the technical field of nucleic acid editing, wherein the fusion protein comprises a first protein and a second protein, and the first protein comprises any one of a translation initiation factor and a translation termination factor; the second protein comprises deaminase, and the fusion protein is used for editing RNA at the beginning or the end of translation. Wherein the translation initiation factor and the translation termination factor are respectively combined with an RNA chain at the time of translation initiation and translation termination, so that the fusion protein is contacted with the RNA and more accurately positions all RNAs which begin translation and translation termination, and deaminase is subjected to deamination editing on nucleotides near a contact point when the fusion protein is contacted with the RNA, thereby leaving a mark for RNA editing, and the RNA translation level can be detected by using the mark.

Inventors

• CHEN WEI
• FANG LIANG
• SUN ZHIYUAN
• WEN XIAOZHEN

Assignees

• 南方科技大学

Dates

Publication Date

20260508

Application Date

20241101

Claims (10)

1. 1. A fusion protein comprising a first protein and a second protein, The first protein comprises any one of a translation initiation factor and a translation termination factor; The second protein comprises a deaminase; The fusion protein is used for editing RNA at the beginning or end of translation.
2. 2. The fusion protein of claim 1, wherein the deaminase comprises any of a ADAR, APOBEC, tadA, ADAR variant, an apodec variant, and a TadA variant.
3. 3. The fusion protein of claim 1, wherein the translation initiation factor comprises any one of EIF1AX, EIF3G, EIF D, and EIF 4E.
4. 4. The fusion protein of claim 1, wherein the translation termination factor comprises GSPT a.
5. 5. The fusion protein of claim 1, wherein the fusion protein is obtained by gene cloning.
6. 6. A method for detecting RNA editing level, comprising the steps of: S10, obtaining a cell sample to be tested; S20, expressing a fusion protein in the cell sample to be tested, and screening to obtain a cell sample to be tested enriched with the fusion protein, wherein the fusion protein is the fusion protein of any one of claims 1 to 5; S30, extracting RNA of the cell sample to be detected enriched in the fusion protein; S40, calculating the editing level of the RNA according to the RNA of the cell sample to be detected enriched with the fusion protein.
7. 7. The method for detecting an RNA editing level according to claim 6, wherein S40 comprises: S401, sequencing the RNA to obtain the read quantity (R) of the RNA covered to a trusted editing site and the read quantity (E) of the RNA subjected to trusted editing in the cell sample to be tested of the enriched fusion protein; S402, calculating the RNA editing level of each gene in the cell sample to be tested of the enrichment fusion protein according to a formula Z=E/R, wherein Z is the RNA editing level of each gene in the cell sample to be tested of the enrichment fusion protein, E is the read number of the RNA which is subjected to trusted editing in the cell sample to be tested of the enrichment fusion protein, and R is the read number of the RNA which covers the trusted editing site of each gene in the cell sample to be tested of the enrichment fusion protein.
8. 8. The method of claim 6, wherein the means for expressing the fusion protein in the sample of cells to be tested in step S20 comprises transient transfection or stable transfection.
9. 9. The method of claim 7, wherein in step S401, the method of sequencing the RNA comprises high throughput sequencing and Sanger sequencing.
10. 10. Use of a method for detecting the level of RNA editing according to any one of claims 6 to 9 for detecting the level of translation of RNA.

Description

Fusion protein, RNA editing level detection method and application thereof Technical Field The invention relates to the technical field of nucleic acid editing, in particular to a fusion protein, a detection method of RNA editing level and application thereof. Background According to the central rule, genetic information is stored in deoxyribonucleic acid (DNA), which is transcribed into ribonucleic acid (RNA), which is subsequently translated into protein, ultimately exhibiting a specific biological function. Regulation of translation levels plays an important role in many physiological and pathological processes, such as organ development, cancer, and the occurrence and development of neurodegenerative diseases. Therefore, the systematic detection of RNA translation levels is critical for the deep exploration of the molecular mechanisms of gene expression regulation, development and disease. Although RNA translation levels can be detected using existing techniques such as ribosomal imprinting, there are challenges in that 1) a large amount of biological sample is required, 2) high-throughput RNA sequencing must be additionally performed on each sample to estimate translation levels, and 3) the experimental procedure is complex and the reagents are expensive. These factors make RNA translation level identification applicable only to a small number of biological samples. Disclosure of Invention The invention mainly aims to provide a fusion protein, a detection method of RNA editing level and application thereof, and aims to provide a method for calculating RNA translation level, which has the advantages of less biological sample consumption, less sequencing times and simpler experimental flow. To achieve the above object, the present invention provides a fusion protein comprising a first protein and a second protein, The first protein comprises any one of a translation initiation factor and a translation termination factor; The second protein comprises a deaminase; The fusion protein is used for editing RNA at the beginning or end of translation. In one embodiment, the deaminase comprises any of the deaminase comprising ADAR, APOBEC, tadA, ADAR variant, apodec variant, and TadA variant. In one embodiment, the translation initiation factor comprises any one of EIF1AX, EIF3G, EIF D, and EIF 4E. In one embodiment, the translation termination factor comprises GSPT a 1. In one embodiment, the fusion protein is obtained by gene cloning. The invention also provides a method for detecting the RNA editing level, which comprises the following steps: S10, obtaining a cell sample to be tested; S20, expressing a fusion protein in the cell sample to be tested, and screening to obtain a cell sample to be tested enriched with the fusion protein, wherein the fusion protein is the fusion protein; S30, extracting RNA of the cell sample to be detected enriched in the fusion protein; S40, calculating the editing level of the RNA according to the RNA of the cell sample to be detected enriched with the fusion protein. In one embodiment, S40 includes: S401, sequencing the RNA to obtain the read quantity (R) of the RNA covered to a trusted editing site and the read quantity (E) of the RNA subjected to trusted editing in the cell sample to be tested of the enriched fusion protein; S402, calculating the RNA editing level of each gene in the cell sample to be tested of the enrichment fusion protein according to a formula Z=E/R, wherein Z is the RNA editing level of each gene in the cell sample to be tested of the enrichment fusion protein, E is the read number of the RNA which is subjected to trusted editing in the cell sample to be tested of the enrichment fusion protein, and R is the read number of the RNA which covers the trusted editing site of each gene in the cell sample to be tested of the enrichment fusion protein. In one embodiment, in step S20, the means for expressing the fusion protein in the test cell sample comprises transient transfection or stable transfection. In one embodiment, in step S401, the method of sequencing the RNA comprises high throughput sequencing and Sanger sequencing. The invention also provides an application of the method for detecting the RNA editing level in detecting the translation level of RNA. In the technical scheme of the invention, the fusion protein comprises deaminase and translation initiation factors/translation termination factors, wherein the translation initiation factors and the translation termination factors are respectively combined with RNA chains at the time of translation initiation and translation termination, so that the fusion protein is contacted with RNA and more accurately positioned to all RNAs which begin translation and translation termination, and the deaminase performs deamination editing on nucleotides near a junction when the fusion protein is contacted with RNA, thereby leaving marks for RNA editing. Drawings In order to more clearly illustrate the embodiments of