eCRUIS captures RNA-protein interaction in vitro and in vivo

Available online 16 April 2024, 114051

Author links open overlay panel, , ABSTRACT

As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in various ways. From synthesis to degradation, RNA is associated with a range of RNA-binding proteins. Therefore, it is necessary to develop innovative methods to study the interactions between RNA and proteins. Previously, we developed an RNA-centric method, called CRISPR-based RNA-United Interacting System (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The current version allows us to label RNA-binding proteins on the target RNA within 30 minutes, potentially for in vivo use. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a broader range of in vitro and in vivo applications.

Section snippetsINTRODUCTION

The abundance, stability, and behavior of RNA are tightly regulated as it serves as the messenger that transmits gene information from DNA to proteins. As vital RNA regulators, RNA-binding proteins have attracted considerable attention 1, 2, 3, 4. Strategies for studying RNA protein interaction can be roughly divided into two categories: protein-centric methods and RNA-centric methods 5, 6.

The protein-centric methods widely used in the past decades mainly include the well-known RIP

The strategy of eCRUIS

To rapidly capture the RNA protein interactions of target RNA in living cells, our strategy is to replace the proximity labeling element of CRUIS with a system that can label the proximity proteins in a short time. A recent study reported a promiscuous engineered biotin ligase proximity labeling system, TurboID, which stems from the directed evolution of BioID [17]. TurboID exhibits extremely strong proximity labeling activity, which can efficiently label surrounding proteins within 30 minutes,

DISCUSSION

As an intermediary between DNA and protein, RNA regulates gene expression and function in many ways. From biogenesis to degradation, RNA interacts with a range of RNA-binding proteins. This also suggests that the study of RNA function and its regulatory mechanism must depend on the function and role of RNA-binding proteins throughout their lifecycle 26, 27.

Over the past decade, protein-centric methods for studying RNA-protein interactions have been widely developed, represented by RIP and CLIP.

Plasmid construction and transgenic fly generation

The dCas13d and TurboID were cloned in the downstream of TRE3GV promoter in a lentivirus transfer vector by ClonExpress One Step Cloning kit (Vazyme). Here, an HA-tag was added in the 3’ end of TurboID, and (GGGGS)×2 is used as a linker between dCas13d and TurboID. P2A-EGFP was added at the C-terminal as a selection marker. For the plasmids of Drosophila, the Drosophila codon-optimized dCas13d and TurboID sequences containing V5 epitope tag were synthesized. EcoRI and NotI were then used to

FUNDING

This work was funded by the Ministry of Science and Technology of the People’s Republic of China (Grant No. 2021YFA0804700), the National Natural Science Foundation of China (No. 32370744; 32350710195), the Shanghai Science and Technology Commission (20JC1410500), and the UK Medical Research Council (Grant No. MC_UU_12021/3 and MC_U137788471). This research was supported by Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University.

Declaration of Competing Interest

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

We thank the Molecular Imaging Core Facility (MICF) at School of Life Science and Technology, ShanghaiTech University for providing technical support.

REFERENCES (28)GerberA.P. RNA-Centric Approaches to Profile the RNA-Protein Interaction Landscape on Selected RNAs

Noncoding RNA

(2021)

KonigJ. et al.iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution

Nat Struct Mol Biol

(2010)

ZhangC. et al.Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data

Nature biotechnology

(2011)

RamanathanM. et al.RNA-protein interaction detection in living cells

Nature methods

(2018)