PLANS

Abstract

RNA and proteins interact in living cells at various stages, regulating processes such as transcription, splicing and translation. The alteration of RNA-protein interactions strongly affects cellular homeostasis and has been linked to human diseases[1]. We aim at developing a new methodology named PLANS (Proximity Ligation And Nanopore Sequencing) to i) characterize native RNA-protein interactions occurring in living cells, including transient interactions and interactions involving low abundant molecules; ii) identify RNA post-transcriptional modifications (PTMs) present in the interacting transcripts, expanding the role that PTMs play in the regulation of RNA-protein interactions; and iii) develop predictive tools that infer RNA-protein interactions and the effect of PTMs on RNA-protein binding. PLANS can be applied to any cellular RBP of interest. The selected protein is fused to a bacterial enzyme able to promote the promiscuous biotinylation of any RNA and protein in its close proximity. Biotinylated RNAs are then subjected to Nanopore direct RNA sequencing, hence analyzing native RNA molecules and their PTMs. (WP1). We will exploit PLANS to assess the RNA interactome of the RBPs involved in the epidermal growth factor receptor (EGFR) non-clathrin endocytosis (NCE) in response to EGF stimulation. These proteins, that bind RNA targets through canonical RNA binding domains and intrinsically disordered regions (IDRs), have been found as EGFR interactors in NCE[2]. The characterization of RNAs bound to NCE-associated proteins upon EGF stimulation will unveil new aspects that may be relevant for the EGFR physiological response and, if altered, contribute to aberrant EGFR signaling observed in cancer cells. (WP2). We will adopt PLANS to identify the human RNAs modified by pseudouridine synthase (PUS) enzymes. PUS enzymes transiently interact with their RNA targets and modify linear motifs included within specific consensus sequence[3, 4]. Through PLANS we aim at characterizing how PUS enzymes select their RNA targets, assess the transcript substrates specific to each enzyme and understand how other PTMs can influence the RNA target recognition. These data will bring relevant insights in the activity of Ministero dell’Università e della Ricerca MUR – BANDO 2022 PUS enzymes that have been found altered in human diseases[5]. (WP3) We will construct predictive mathematical models to identify RBP-binding regions involved in RNA-protein interactions. We will focus on IDRs, which are characterized by the lack of a fixed three dimensional structure and are known to bind RNA through multivalent promiscuous interactions. Through the data retrieved by PLANS in WP1 and WP2, we aim to define precise consensus sequences of IDRs required for the binding to RNA. As well as to identify the contribution that PTMs play in RNA-protein interactions. The predictions and the experimental data will be collected into a publicly available database that will be used as reference for RNA-protein interaction studies.

Progetto PLANS (PRIN 2022W93FTW) funded by the Italian Ministry of University and Research.