600 - Direct circRNA Interactome Mapping: Amplification-Based Proteomics in Spastic Cerebral Palsy

Publication Number: 4588.600

Brigette M. Romero, University of Delaware, Newark, DE, United States; Karyn Robinson, Nemours Children's Hospital, Wilmington, DE, United States; Vijay Parashar, University of Delaware, Newark, DE, United States; Wade Shrader, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Robert Akins, Nemours Children's Health, Wilmington, DE, United States; Mona Batish, University of Delaware, Newark, DE, United States

Background: Spastic cerebral palsy (CP) is the most common motor disability disorder characterized by persistent muscle stiffness and contractures. These musculoskeletal deformities often worsen over time, highlighting the need for timely interventions and therapeutic targets. Unfortunately, the molecular and cellular mechanisms leading to motor dysfunction in spastic CP remain largely unknown. Our recently published work revealed that a specific circular RNA derived from the NFIX gene (circNFIX) regulates the expression of MEF2C, a muscle-specific transcription factor, by sponging microRNA miR373-3p. Given high-level expression of circNFIX, it is plausible that it has more functions than just miRNA sponging. Another functionally relevant role of circRNAs is to bind/sponge RNA-binding proteins (RBPs) or to serve as a scaffold for the formation of protein complexes, and both of these roles for circNFIX are yet to be characterized in muscle development.

Objective: We sought to identify RBPs that interact with circNFIX and to characterize their roles in satellite cell-derived myoblasts (SC-MBs) and myotubes (SC-MTs).

Design/Methods: SC-MBs were isolated from skeletal muscle biopsies of individuals with spastic CP and control subjects (CN), and validated by the expression of PAX7 and MYF5. These SC-MBs were cultured and differentiated into myotubes. To identify RBPs, we utilized CARPID (CRISPR-assisted RNA-protein interaction detection method) using two guide RNAs (one targeting the backsplice junction, the other linear NFIX). A complementary pull-down-free method was also employed to capture weaker or transient RNA-protein interactions and their respective binding sites. Identified RBP expression was validated by qRT-PCR, immunofluorescence (IF), and Western blot, while the interactions themselves were verified using IF and the circFISH technique.

Results: Differential RBP enrichment was found when comparing circNFIX to its linear counterpart (Fig 1). After comparing biological replicates and performing statistical analysis, 26 RBPs showed higher enrichment with circNFIX in MBs, and 9 RBPs were more enriched with circNFIX in MTs. MB-associated RBPs were primarily linked to mRNA 3'-end processing (BP) and choline transmembrane transporter activity (MF). MT-associated RBPs were linked to protein-containing complex assembly (BP) and tubulin binding (MF) (Fig 2).

Conclusion(s): As a highly expressed and stable circRNA, circNFIX function as an RBP sponge has a potential to impact multiple muscle development pathways affected in CP. identifying these pathways will pave the way for potential therapeutic target for spastic CP.

Figure 1. Overlap of RNA-binding proteins (RBPs) identified by CARPID.
Comparison between the set of RBPs specifically enriched with circNFIX, its linear counterpart (linNFIX), and the control (no guide RNA) in myoblasts (A) and myotubes (B).

Figure 2. Gene Ontology (GO) Biological Process enrichment of RNA-binding proteins (RBPs) differentially associated with circNFIX.
The top enriched terms are provided for the RBPs identified in myoblasts (A) and myotubes (B).