Circular RNA-protein interactions: functions, mechanisms, and identification

Abstract

Circular RNAs (circRNAs) are covalently closed, endogenous RNAs with no 5' end caps or 3' poly(A) tails. These RNAs are expressed in tissue-specific, cell-specific, and developmental stage-specific patterns. The biogenesis of circRNAs is now known to be regulated by multiple specific factors; however, circRNAs were previously thought to be insignificant byproducts of splicing errors. Recent studies have demonstrated their activity as microRNA (miRNA) sponges as well as protein sponges, decoys, scaffolds, and recruiters, and some circRNAs even act as translation templates in multiple pathophysiological processes. CircRNAs bind and sequester specific proteins to appropriate subcellular positions, and they participate in modulating certain protein-protein and protein-RNA interactions. Conversely, several proteins play an indispensable role in the life cycle of circRNAs from biogenesis to degradation. However, the exact mechanisms of these interactions between proteins and circRNAs remain unknown. Here, we review the current knowledge regarding circRNA-protein interactions and the methods used to identify and characterize these interactions. We also summarize new insights into the potential mechanisms underlying these interactions.

Keywords: RNA binding proteins (RBPs); biogenesis; circular RNAs (circRNAs); degradation; translation.

Figure 1

Proteins play an indispensable role in the life cycle of circRNAs from biogenesis to biological function and degradation. (A-B) CircRNAs are formed by back-splicing into three major types of circRNA. (C-H) The functions of circRNAs in interacting with proteins. Several circRNAs have also been reported to encode proteins. (I) CircRNAs may be released from cells into the intracellular environment via exosomes or microvesicles. (J) The degradation of circRNAs mediated by RNase. It is still unclear whether the degradation of circRNAs by RNase happened mainly outside or within the cells. (K) Exogenous circRNAs can activate the RIG-1 cellular immune response pathway. (L) Linear mRNAs and linear non-coding RNAs (lncRNAs) can also be generated from spliceosome-mediated canonical splicing. IRES: internal ribosome entry site. RBPs: RNA-binding proteins. RIG-1: retinoic acid-inducible gene 1 protein. TET1: Tet methylcytosine dioxygenase 1. U1SnRNP: U1 small nuclear ribonucleoprotein.

Figure 2

Binding site-based vs. tertiary structure-based modes of circRNA-protein interactions. (A) Circ-Mbl contains several binding sites for the mannose-binding lectin (MBL) protein. Nuclear factor (NF) complexes 90/110 (NF90/NF110) promote circRNA production in the nucleus by associating with intronic RNA pairs and interacting with mature circRNAs in the cytoplasm through binding sites. (B) Circ-Foxo3 displays a variety of tertiary structures in various cell/tissue environments (see the text). (C) CircANRIL appears to form a stem-loop structure that mimics rRNA and binds to Pescadillo homolog 1 (PES1). (D) CircPOLR2A tends to form 16-26 bp of imperfect base-paired RNA duplexes to inhibit double-stranded RNA (dsRNA)-activated protein kinase (PKR). cdk2: cell division protein kinase 2. E2F1: E2F transcription factor 1. FAK: focal adhesion kinase. HIF-1α: hypoxia-inducible factor 1α. Id1: inhibitor of differentiation 1. Mdm2: Mouse double minute 2 protein. p21: cyclin-dependent kinase inhibitor 1.

Figure 3

The main approaches currently used to detect circRNA-protein interactions. (A) RNase protection assay (RPA). (B) RNA pull-down assay. (C) RNA immunoprecipitation (RIP) assay. (D) Electrophoretic mobility shift assay (EMSA).