Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein-RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing

Abstract

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other's activity, indicating an essential role of protein-RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein-RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein-RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein-RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein-RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.

Keywords: CLIP; RNA polymerase II; RNA-binding protein; co-transcriptional RNA processing; tRIP.

Figure 1

Transcription and co-transcriptional RNA processing. RNA processing machinery is recruited to the transcribing RNAP II machinery depending on the phosphorylation status of C-terminal domain (CTD). Then, protein–RNA interactions are formed in the recruited machinery to process nascent RNA co-transcriptionally. Along with transcription termination and polyadenylation, pre-mRNA is released from the RNAP II.

Figure 2

Schematic representation of the CLIP workflow. RNA-binding protein (RBP)–RNA interactions are UV-crosslinked in living cells. Following the lysis of cells, RNA is partially digested with RNase, and the crosslinked complex is immunoprecipitated with an antibody against an RBP of interest. After stringent washes, the immunoprecipitants are separated on SDS-PAGE and transferred to nitrocellulose membrane, on which the RBP complexed with RNA migrate above the MW of the target RBP. Then, the complexes retaining the short segments (≈50 nt) are isolated from the membrane. Following the proteinase K-treatment and RNA purification, a cDNA library is constructed with RT-PCR.

Figure 3

Schematic representation of the tRIP analysis of protein–RNA interactions specifically assembled on RNAP II. Following UV-crosslinking, protein–RNA complexes were immunoprecipitated with an anti-RNAP II antibody (1st IP). RNA of the isolated RNAP II–RNA–RBP complexes was partially digested with RNase, and the released RBP–RNA complexes were further immunoprecipitated with antibody against an RBP of interests (2nd IP). Then, the immunoprecipitated RBP–RNA complexes are subjected to tRIP-seq analysis.

Figure 4

Schematic representation of TRIBE (targets of RNA-binding proteins identified by editing) and STAMP (surveying targets by APOBEC mediated profiling) strategies. A fusion protein of an RBP and an RNA editing enzyme edits the nucleotides around RBP-binding sites. In TRIBE, an RBP is fused to the catalytic domain of ADAR that catalyzes an adenosine-to-inosine conversion. In STAMP, it is fused to APOBEC1 that catalyzes a cytosine-to-uracil conversion. The edited sites can be detected using high-throughput RNA sequencing analysis.