US-12618097-B2 - Method for determining long non-coding ribonucleic acid interaction proteins

US12618097B2US 12618097 B2US12618097 B2US 12618097B2US-12618097-B2

Abstract

The present invention provides a novel method for determining a long-chain non-coding ribonucleic acid interaction protein. The present invention provides a fusion protein formed by BASU and dCasRx, a mammalian expression vector for expressing said fusion protein. The method for determining the lncRNA interaction protein according to the present invention comprises: co-transfecting a mammalian expression vector that expresses the fusion protein and a gRNA that specifically targets the target lncRNA into target cells, thereby BASU specifically biotin-labeling effector proteins nearby; isolating the biotinylated proteins by using a streptavidin affinity coupled magnetic bead and then eluting, and digesting by trypsin and quantitatively analyzing by a label-free mass spectrometry. The present invention can highly credibly determine the proteins that interact with lncRNA.

Inventors

Liang Zhang
Jian Yan
Jingyu Li
Wenkai Yi
Ligang Fan

Assignees

CITY UNIVERSITY OF HONG KONG

Dates

Publication Date: 20260505
Application Date: 20210426
Priority Date: 20200430

Claims (5)

1 . A fusion protein formed by biotin ligase BASU and dCasRx, said fusion protein selected from the group consisting of B. subtilis biotin ligase with C-terminal mutation (BASU) as encoded by SEQ ID NO: 17-dCasRx as encoded by SEQ ID NO: 18 and dCasRx as encoded by SEQ ID NO: 18-BASU as encoded by SEQ ID NO: 17.
2 . A composition comprising: the fusion protein according to claim 1 ; and a pair of guide RNAs (gRNA) targeting a target long non-coding ribonucleic acid (lncRNA).
3 . A kit for determining lncRNA interaction proteins, comprising: the fusion protein according to claim 1 ; and a gRNA set targeting a target lncRNA.
4 . The composition according to claim 2 , wherein the target lncRNA is selected from the group consisting of X-inactivation specific transcript (XIST), Differentiation antagonizes non-protein-coding RNAs (DANCR), and Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1).
5 . The kit according to claim 3 , wherein the kit further comprises: a control reagent without gRNA.

Description

SEQUENCE LISTING The Sequence Listing file entitled “sequencelisting” having a size of 2,704 bytes and a creation date of Apr. 26, 2021, that was filed with the patent application is incorporated herein by reference in its entirety. TECHNICAL FIELD The present invention relates to a novel method for determining long non-coding ribonucleic acid interaction proteins. BACKGROUND ART Although only 2% sequences of the human genome code protein, more than 70% of genomic DNA can be transcribed into RNA at different stages of development. For decades, these huge numbers of non-coding RNAs (ncRNAs) have been considered as “dark matter” and their functions remained to be developed. These ncRNAs, especially long non-coding ribonucleic acids (lncRNA, defined as ncRNAs with more than 200 nucleotides in length) have been of interest recently, which were generally considered to be an important part participating in epigenetic regulation. For example, various lncRNAs were involved in cell cycle regulation and proliferation, the dysregulation of which is associated with progression and metastasis of various cancers. XIST (X-inactivation specific transcript) is one of the first widely studied lncRNAs, which directs X chromosome inactivation (XCI) in female mammals, thus balancing the dose of genes between XY males and XX females. Its ability to constrain chromatin binding proteins makes it possible to label allele and cis-regulate transcription. At present, XCI and XIST have become exemplary models for understanding the epigenetic regulation of lncRNA. The interaction between lncRNA and RNA binding proteins (RBP) determines the function and fate of RNA molecules. Up to 8.5 percent of the entire human proteome was predicted to have RNA binding properties, further demonstrating the multiple functions of lncRNA in various biological events. Mutations in lncRNA transcripts or changes in the abundance of lncRNA may alter their associated proteome, leading to health problems. The identification of lncRNA-related proteins will reveal the molecular mechanisms of the cell function in complex human diseases. Although there is a growing recognition of the functional importance of RBP, there are significant technical limitations in elucidating lncRNA-protein interactions in living cells at present. Current methods depend mainly on chemical or UV mediated cross-linking between RNA and protein molecules to achieve effective enrichment and separation for the complex. Such procedures may produce non-systematic biases in physiological situations and mask the interaction proteins. Recently, Ramanathan, M. et al. developed a RaPID method that integrates the promiscuous but efficient biotin ligase BASU with a AN peptide navigation system that recognizes the stem-loop of RNA BoxB. Compared with other biotin ligase variants, BASU remains inactive until it is rapidly activated by the high concentration of exogenous biotin in the culture medium, thereby labeling proteins nearby in a smaller labeling radius (˜10 nm) in a shorter reaction time. This feature greatly reduces non-specific background noise. However, the target RNA needs to be artificially fused with the BoxB stem-loop close to the RBP binding region, and it needs to be expressed ectopically in the cell. Therefore, RaPID must compromise on three important factors: firstly, the abundance of ectopically expressed target RNA greatly exceeds the endogenous level of the transcript, resulting in a non-physiological balance of the interaction between RNA and RBP; secondly, the incorporation of the hairpin structure BoxB into the target RNA may interfere with the natural structure of the transcript, thereby changing its binding protein; thirdly, BASU can only label the RBP adjacent to the stem loop of BoxB at the 5′ or 3′ end of the RNA, therefore, some important RBPs may be missed, especially for long transcripts such as XIST (˜19 kb). Briefly, the potential shortcomings, including the loss of cellular background, extensive molecular engineering and possible destruction of the natural structure of RNA, greatly limit the wide application of this method. SUMMARY OF INVENTION An object of the present invention is to provide a novel method for determining long non-coding ribonucleic acid (lncRNA) interaction proteins. The present invention provides a CRISPR-Assisted RNA-Protein Interaction Detection (CARPID) method, which integrates CRISPR/CasRx based RNA targeting and proximity markers to identify binding proteins of specific lncRNAs within cells at natural state. The inventive technology for detecting CRISPR-assisted RNA-protein interaction can be used as a novel and powerful method to find the interaction proteins of lncRNA in living cells. The method uses the highly specific CRISPR/CasRx system fused to the promiscuous but efficient biotin ligase BASU. The interaction with various proteins plays a central role in the regulatory activity of lncRNA. The present invention relates to a fusion protei