Please also check out related works on dual labeling RBPs: TRIBE-STAMP genesdev.cshlp.org/content/36/1... from Meyer lab PIE-seq www.nature.com/articles/s41... from Zhang lab and preprint from Yeo lab doi.org/10.1101/2023...

We imagine REMORA can be used together with many other RNA modification-based information encoding strategies. Our rABE plasmid is available from addgene www.addgene.org/191383/ and data analysis scripts are available in my github github.com/linyz/remora (7/7)

By combining rABE with the C-to-U deaminase APOBEC1, we measured binding by two RNA-binding proteins on single messenger RNAs. Orthogonal labeling of Pumilio proteins PUM1 and PUM2 shows that they co-bind mRNA molecules containing certain microRNA target sites.(6/7)

By combining REMORA with long-read RNA sequencing, we identified isoform-specific RNA:protein interactions driven by isoform changes distal to the binding site. For example, alternative intron retention in upstream exons of DDX5 lead to reduced PUM1 binding in the 3’UTR. (5/7)

In REMORA, RBP-rABE fusion protein introduce A-to-I edits adjacent to RBP binding sites. Adenosine deamination serves as a molecular record of RNA-protein interactions that are identified by A-to-G mutations in sequencing. (4/7)

However, currently available RNA editors such as TRIBE, DART or STAMP have limitations due to RNA structure and sequence dependence. We engineered an RNA A-to-I deaminase (named rABE) with directed protein evolution that has high activity, low bias and low background. (3/7)

Using RNA editor as molecular recorders for RNA binding protein (RBP) footprinting has its benefits over CLIP-like methods that it is less labor intensive, does not rely on RBP specific antibody and does not require RNase digestion. (2/7)