Identification of circRNA-Interacting Proteins by Affinity Pulldown

Abstract

Circular RNAs (circRNAs) comprise a vast class of covalently closed transcripts, generated primarily via backsplicing. Most circRNAs arise from full or partial exons, but they can also arise from introns, and from combinations of introns and exons. While high-throughput RNA-sequencing analysis has identified tens of thousands of circRNAs expressed in different tissues and growth conditions, the function of circRNAs has only been described for a handful of them. As most circRNAs appear not to encode peptides, their function is presumed to be linked to their interaction with a range of molecules, particularly other nucleic acids (notably microRNAs) and proteins. A major impediment to identifying circRNA-associated molecules is a lack of suitable methodologies capable of analyzing specifically circRNAs and not their linear RNA counterparts with which they share most of their sequence. Here, we describe a flexible and robust method for identifying the proteins that associate with a given circRNA. The affinity pulldown assay is based on the use of a biotinylated antisense oligomer that recognizes the circRNA-specific junction sequence. Following pulldown using streptavidin beads, the proteins are eluted from the circRNP (circribonucleoprotein) complex and identified by mass spectroscopy; validation by Western blot analysis and other methods would then confirm the identity of the circRNA-associated proteins. We present a detailed step-by-step protocol, tips to optimize the analysis, troubleshooting suggestions, and assistance in interpreting the results. In sum, this protocol enables the discovery of proteins present in circRNPs, a critical effort toward elucidating circRNA function.

Keywords: Antisense oligomer; Backsplice junction; Ribonucleoprotein complex; circRNA-Protein; circRNAs.

Fig. 1

Schematic of circular RNA-antisense oligo pulldown

Fig. 2

Validation of circRNA interacting proteins. (a) Biotinylated antisense oligomers (ASOs) complementary to the junction of circSamd4 and control (Ctrl) were incubated with C2C12 in GM as well as in DM. After affinity pulldown using streptavidin beads, the levels of circSamd4 enrichment in ASO pulldown samples were assessed by RT-qPCR analysis of circSamd4 (relative to the enrichment of GAPDH mRNA, a transcript that does not bind the ASOs and encodes a housekeeping protein) in the pulldown samples. Enrichment in Samd4 mRNA was monitored in parallel. (b) Top 5 proteins shared in the mass spec datasets from mouse and human myoblast pulldowns. (c) Validation in C2C12 of other proteins identified as being selectively enriched in circSamd4 and circSAMD4 ASO pulldowns. Following ASO pulldown, the presence of candidate proteins was assessed by Western blot analysis. Input, 5 μg. (Figure is reproduced from ref. 15)