Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)

Abstract

We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of a-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome.

The RNA‐binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs

“…With respect to canonical FL cDNA phage libraries, this approach has the advantage of generating a normalized library of protein domains (the Domainome) that are homogeneous in terms of peptide length and sequence coverage. It is noteworthy that despite the fact the library derives from only 3 human organs, almost all annotated RBPs and transcription factors are represented by at least 1 read (36). Therefore, the library can be considered universal and, as such, can be used as a tool for the initial identification of proteins interacting with any biomacromolecule of interest (protein, RNA, DNA, etc.…”

“…Sequences were processed with the NGS Transcriptome Profile Explorer (NGS-Trex) system ( https://www.ngs-trex.disit.unipmn.it/Trex/cms/ ) as previously described (36, 39). Briefly, sequences were mapped onto the human genome (U.S. National Center for Biotechnology Information Build 36) using genomic mapping (GMAP) software, and matching sequences were compared with annotated genes.…”

“…Screening of selected clones in ELISA-based assays, either in the phage format or as soluble GST fusion polypeptides, was performed according to protocols previously described in Patrucco et al . (36) with some modifications. Briefly, phage ELISA was performed with Microlon plates (Greiner Bio-One, Kremsmünster, Austria) coated overnight at 4°C with 10 μg/ml streptavidin.…”

“…The dynamics of SINEUP-ILF3 interactions were characterized by SPR using a Biacore T100 instrument (GE Healthcare) as previously described in Patrucco et al . (36). The biotinylated invSINEB2 RNA was immobilized on streptavidin-coated sensor chips (Series S Sensor Chip SA; GE Healthcare).…”

SINEUPs: a novel toolbox for RNA therapeutics

“…Recently, NMR 'fingerprints’ were used as sensitive probes to divide the full-length inverted SINEB2 sequence into minimal units that retain the original structure and function. One dynamic domain and two discrete structured domains (named C and M domains) were thus identified [ 75 ]. The 31–199 nts fragment, largely corresponding to the C domain, showed an identical fold and retained 80% of the SINEUP function of the full length inverted SINEB2 sequence.…”