Circular RNA: metabolism, functions and interactions with proteins — TEI-tagged view

Zhou Wei-yipersName, Zerong CaipersName, Jia LiupersName, De‐Shen WangpersName, Huai‐Qiang JupersName, Rui‐Hua XupersName (2020date). “Circular RNA: metabolism, functions and interactions with proteins.” Molecular Cancertitle·j.

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Abstract. Circular RNAs (CircRNAsterm·dom) are single-stranded, covalently closed RNA molecules that are ubiquitous across species ranging from viruses to mammals. Important advances have been made in the biogenesis, regulation, localization, degradation and modification of circRNAsterm·dom. CircRNAsterm·dom exert biological functionsterm·dom by acting as transcriptional regulators, microRNA (miR) sponges and protein templates. Moreover, emerging evidence has revealed that a group of circRNAsterm·dom can serve as protein decoys, scaffolds and recruiters. However, the existing research on circRNA-protein interactionsterm·dom is quite limited. Hence, in this reviewterm·mth, we briefly summarize recent progress in the metabolismterm·dom and functionsterm·dom of circRNAsterm·dom and elaborately discuss the patterns of circRNA-protein interactionsterm·dom, including altering interactions between proteins, tethering or sequestering proteins, recruiting proteins to chromatin, forming circRNA-protein-mRNA ternary complexes and translocating or redistributing proteins. Many discoveries have revealed that circRNAsterm·dom have unique expression signatures and play crucial roles in a variety of diseases, enabling them to potentially act as diagnostic biomarkers and therapeutic targets. This reviewterm·mth systematically evaluates the roles and mechanismsterm·dom of circRNAsterm·dom, with the hope of advancing translational medicine involving circRNAsterm·dom. Introduction Single-stranded, covalently closed circRNAsterm·dom were first reported as viroids, which are pathogens of certain plants, in 1976 [ 1 ] and were first detected in human HeLa cells by electron microscopy in 1979 [ 2 ]. Later, more studies found or synthesized circular forms of RNAs in various species, including viruses [ 3 ], prokaryotes [ 4 ], unicellular eukaryotes [ 4 , 5 ] and mammals [ 6 ]. With the development of high-throughput RNA-sequencing and bioinformatic tools, scientists have found that circRNAterm·dom is a general feature of the human transcriptome and is ubiquitous in many other metazoans [ 7 – 9 ]. More recently, an increasing number of investigations have identified multiple functionsterm·dom of circRNAsterm·dom, including serving as protein scaffolds or miR sponges and being translated into polypeptides [ 7 , 8 , 10 ]. The unique structure of circRNAsterm·dom provides them with a longer half-life and more resistance to RNase R than linear RNAs [ 11 ], which makes them potential candidates for diagnostic biomarkers and therapeutic targets. Plenty of studies have uncovered their distinct expression signatures and crucial biological roles in a variety of diseases, such as cancer [ 12 – 14 ], cardiovascular disease [ 15 ], neurological disorder [ 16 ] and autoimmune disease [ 17 ]. However, the mechanismsterm·dom underlying the abnormal landscape of circRNAsterm·dom and how circRNAsterm·dom exert physiological or pathological roles in diseases remain poorly understood. Moreover, the majority of functional studies have shown that circRNAsterm·dom act as miR sponges, which are monotonous and stereotypical. Antisense transcript of cerebellar degeneration-related protein 1 (CDR1as) represents the earliest functionally studied circRNAsterm·dom and expands the competing endogenous RNA (ceRNA) crosstalk network [ 18 – 20 ]. Interestingly, a recent study reported that CDR1as interacts with IGF2BP3 and compromises its pro-metastatic functionsterm·dom [ 21 ]. Another one found that CDR1as interacts with p53 and blocks it from MDM2 [ 22 ]. This inspires us that circRNAsterm·dom can be versatile and previously-studied circRNAsterm·dom may also possess other abilities. Actually, one circRNAterm·dom can simultaneously functionterm·dom as both a miR sponge and a protein template [ 23 ] (or interactor [ 24 , 25 ]). Additionally, proteins are direct effectors of almost all vital activities. CircRNA-protein interactionsterm·dom have remarkably rejuvenated the field of circRNAsterm·dom and enlightened our insights into their biological significance. However, studies on circRNA-protein interactionterm·dom are still lacking, and its mechanismsterm·dom fascinate scientists. Therefore, in this article, we reviewed the important progress in the metabolismterm·dom and functionsterm·dom of circRNAsterm·dom and highlighted the modes of circRNA-protein interactionsterm·dom. Hopefully, this reviewterm·mth will help to reveal intricate circRNA-related issues and to develop circRNA-targeted translational research and clinical applications. Metabolismterm·dom of circRNAterm·dom In this module, we briefly introduce the cutting-edge investigations into circRNAterm·dom metabolismterm·dom and discuss how its modification influences metabolic programs (Fig. 1 ). To date, the upstream regulatory machinery of circRNAterm·dom remains intriguing. Fig. 1 Metabolismterm·dom of circRNAterm·dom. Regulation of circRNAterm·dom biogenesis. RBP can modulate circRNAterm·dom biogenesis by dimerization, ICS stabilization or ICS impairment. ICS in flanking introns can facilitate exon circularization. Biogenesis of circRNAterm·dom . a In the lariat model, back-spliced exons are skipped and extruded to form an intronic lariat that undergoes further back-splicing, while the remaining exons directly link with each other and form a mature mRNA. b In the direct model, back-splicing occurs first to form a circRNAterm·dom, leaving an immature linear RNA containing introns. Localization of circRNAterm·dom c| Long (> 800 nt) or short circRNAsterm·dom can be translocated to the cytoplasm with the assistance of UAP56 or URH49, respectively. d CircRNAsterm·dom can be translocated to the cytoplasm in m6A-dependent manner mediated by YTHDC1. e CircRNAsterm·dom can be excreted to the extracellular space by exosomes. Degradation of circRNAterm·dom . f Upon viral infection, RNase L activated by 2′-5′-oligoadenosine(2′-5′A) causes the global degradation of circRNAsterm·dom, which relieves the suppression of PKR. g M6A-containing circRNAsterm·dom can be recognized by YTHDF2, which interacts with the RNase P/MRP complex bridged by HRSP12, and then the complex endoribonucleolytically cleaves circRNAsterm·dom. h UPF1 and G3BP1 can bind to imperfect base-paired regions of circRNAsterm·dom and induce their degradation Biogenesis Generally, pre-mRNA transcribed by RNA polymerase II (Pol II) contains introns and exons, followed by a 7-methylguanosine cap and poly-adenosine tail adding to its 5′- and 3′-ends, respectively. Then with the assistance of spliceosomes, pre-mRNA undergoes splicing at canonical splice sites (5′-GU and 3′-AG at introns) to become mature and translatable. CircRNAterm·dom is generated by a special alternative splicing manner termed back-splicing, in which the 3′-end of an exon ligates to the 5′-end of its own or an upstream exon through a 3′,5′-phosphodiester bond, forming a closed structure with a back-splicing junction site [ 7 , 8 , 26 ]. Initially, circRNAsterm·dom were regarded as splicing errors containing so-called “scrambled exons” [ 27 ]. According to the order of splicing events and different intermediates, two models of biogenesis were proposed [ 28 ] and validated [ 29 ]: the lariat model and the direct back-splicing model [ 26 ]. Recently, an excellent study assembled the spliceosome E complex on pre-mRNA and carried out structural and biochemical analyses, proposing an integral model for intron definition, exon definition, remodeling and the back-splicing-mediated circRNAterm·dom biogenesis [ 30 ]. Regulation CircRNAterm·dom biogenesis relies on canonical splicing machinery, including splice signal sites and spliceosomes [ 31 ]. However, inhibiting the pre-mRNA processing machinery shifts the output of genes to circRNAsterm·dom [ 32 ], which implies that there is competition between circRNAsterm·dom and their linear counterparts. Cis-elements (intronic complementary sequences, ICSs) and trans-factors (RNA binding proteins, RBPs) can regulate circRNAterm·dom production [ 33 ]. Inverted repeated Alu pairs in flanking introns facilitate exon circularization, while pairs in the same intron promote the extrusion of themselves and canonical splicing [ 34 ]. Mammalian-wide interspersed repeat family elements also contribute to the circularization of circRNAsterm·dom [ 35 ]. Additionally, a group of RBPs modulate circRNAterm·dom biogenesis by binding to flanking introns. Some directly draw introns into proximity and facilitate circularization [ 33 , 36 ], and others stabilize [ 37 ] or impair [ 38 ] Alu pairs to promote or prevent back-splicing, respectively. A recent study found that N6-methyladenosine (m6A) controls circRNAterm·dom biogenesis. Methyltransferase-like 3 (METTL3) or YTH domain-containing 1 (YTHDC1) depletion both regulate approximately 20% of a subset of circRNAsterm·dom, with no significant changes in the linear isoforms [ 39 ]. Another study found that alkB homolog 5 (ALKBH5) inhibition increases the production of translatable circRNAsterm·dom through m6A enrichment at junction sites. Meanwhile, the m6A-modified start codon is recognized by YTHDF3 and mediates translation initiation [ 40 ]. However, whether there are any other regulatory factors and how m6A deposition affects the choice between back and canonical splicing remain unclear. Localization CircRNAsterm·dom formed from exons are generally localized to the cytoplasm [ 41 ]. The mechanismsterm·dom of their nuclear export remained elusive until a recent study found that UAP56 or URH49 depletion causes long or short circRNAsterm·dom, respectively, to be enriched in the nucleus, which suggests that their transportation is partially length-dependent [ 42 ]. Another study showed that the nuclear export of circNSUN2 is mediated by YTHDC1 recruitment, which provides the first evidence that m6A controls circRNAterm·dom translocation [ 43 ]. Additionally, scientists have identified certain intron-containing circRNAsterm·dom that are retained in the nucleus and regulate their parental gene expression [ 44 , 45 ]. Some exonic circRNAsterm·dom are predominantly distributed in the nucleus as well and increase the nuclear retention of proteins [ 46 ] or recruit proteins to chromatin [ 47 ]. Moreover, circRNAsterm·dom can be delivered by extracellular vesicle (EV) and detected in the circulation and urine [ 48 ]. The sorting of these exosomal circRNAsterm·dom seems to be regulated by associated miR levels in producer cells, while the specific biological activities transferred to recipient cells are largely unknown in diverse settings [ 48 , 49 ]. The packaging, delivery and absorption of them also remain elusive so far. Recently, a handful of studies identified mitochondria-located circRNAsterm·dom and examined their functionsterm·dom, which broaden our knowledge of circRNAterm·dom derivation and mitochondrial transcriptome [ 50 – 52 ]. Nevertheless, circRNAsterm·dom located in other organelles or subcellular compartments deserve further investigations. Degradation CircRNAsterm·dom are stable and accumulate in many cell types, especially in neural tissues [ 53 ]. In contrast, another study reported a global reduction in circRNAterm·dom abundance in highly proliferative tissues, possibly due to dilution by proliferation [ 54 ]. Regarding how circRNAsterm·dom maintain a dynamic balance, recent research has shed light on the mechanismsterm·dom of circRNAterm·dom degradation. One study revealed that circRNAsterm·dom can be globally degraded by RNase L. Endogenous circRNAsterm·dom tend to form imperfect duplexes and inhibit PKR (dsRNA-activated protein kinase), while their reduction leads to aberrant PKR activation and autoimmunity [ 55 ]. Another study found that m6A-containing circRNAsterm·dom are recognized by YTHDF2 that interacts with RNase P/MRP (mitochondrial RNA processing) bridged by heat-responsive protein 12 (HRSP12). Then, these circRNAsterm·dom are endoribonucleolytically cleaved [ 56 ]. A more recent study proposed a structure-dependent mechanismterm·dom mediated by UPF1 RNA helicase and ATPase (UPF1) and G3BP stress granule assembly factor 1 (G3BP1), which bind to highly structured base-paired regions and direct both mRNA and circRNAterm·dom decay [ 57 ]. Additionally, miR-671 directs the cleavage of CDR1as in an Argonaute2 (Ago2)-dependent manner [ 58 ]. GW182 (a key component of P-body and RNAi machinery) is also involved in circRNAterm·dom degradation [ 59 ]. Apart from intracellular pathways, circRNAterm·dom excretion may contribute to their clearance [ 60 ]. Further studies are needed to fully understand circRNAterm·dom decay mechanismsterm·dom and interpret their homeostasis and differential distribution across cell types. Modification M6A is the most common RNA modification and is regulated by readers, writers and erasers [ 61 ]. Emerging studies have revealed a transcriptome-wide and cell-type-specific map of m6A-containing circRNAsterm·dom [ 62 ] and m6A involvement in circRNAterm·dom metabolismterm·dom [ 63 ]. In addition to abovementioned biogenesis [ 39 , 40 ], nuclear export [ 43 ] and degradation [ 56 ], m6A also modulates circRNAterm·dom translation. One study reported that m6A drives the translation initiation of circRNAsterm·dom mediated by the interaction between eIF4G2 and YTHDF3, which is enhanced by METTL3/14 and suppressed by FTO [ 64 ]. Additionally, the YTHDF2 binding of m6A-markedself-circRNAs abrogates innate circRNAterm·dom immunity [ 65 ]. Other types of modifications in circRNAsterm·dom await further identifications. Functionsterm·dom of circRNAterm·dom In this module, we discuss how circRNAsterm·dom functionterm·dom at the molecular level and underlying mechanismsterm·dom mainly involve interactions with other molecules. CircRNAsterm·dom have long been considered “non-coding” RNAs (ncRNAs) with regulatory potency [ 20 , 66 ]. Later, scientists identified translatable circRNAsterm·dom [ 67 , 68 ], and more recent studies presented evidence for their prevalence [ 69 , 70 ] (Fig. 2 ). Briefly, downstream pathways of circRNAterm·dom are mostly related to miR sponges and other brilliant talents that circRNAterm·dom yields require brand new pursuits. We will systematically reviewterm·mth the current progress in circRNA-protein interactionsterm·dom and discuss this topic in detail in the next module. Fig. 2 Functionsterm·dom of circRNAterm·dom. a CircRNAsterm·dom can bind to the host genes at their synthesis locus and cause transcriptional pausing or termination through the formation of RNA-DNA hybrid (R-loop structure), upregulating the exon-skipped or truncated transcripts. b EIciRNAs can combine with U1 snRNP and then interact with Pol II to enhance parental gene expression. c CircRNAsterm·dom can act as miR sponges and upregulate the miR target mRNAs. d CircRNAsterm·dom can interact with proteins. e IRES-containing circRNAsterm·dom can directly recruit ribosomes and be translated. f M6A-containing circRNAsterm·dom can be recognized by YTHDF3, which recruits eIF4G2, thus triggering translation. This process can be enhanced by METTL3/14 and suppressed by fat mass and obesity-associated protein (FTO) Transcriptional regulation CircSEP3 originating from exon 6 of SEPALLATA3 increases the abundance of cognate exon 6-skipped variant by binding to the host DNA locus and forming an RNA-DNA hybrid or R-loop, resulting in transcriptional pausing and splicing factor recruitment [ 71 ]. Similarly, circSMARCA5 causes transcriptional termination at exon 15 of SMARCA5 through R-loop formation, upregulating the truncated nonfunctional isoform [ 72 ]. CircRNAsterm·dom with introns retained between exons (exon-intron circRNAsterm·dom, EIciRNAs) can combine with U1 small nuclear ribonucleoprotein through RNA-RNA interactions between snRNA and EIciRNAs and then interact with Pol II at parental gene promoters, enhancing their expression [ 44 ]. Likewise, circular intronic RNAs (ciRNAs) formed from lariats that escape debranching can accumulate at their synthesis sites and increase parental gene expression by modulating elongating Pol II activity [ 45 ]. MicroRNA sponging Plenty of studies have confirmed that circRNAsterm·dom exert biological functionsterm·dom by acting as ceRNAs or miR sponges [ 73 , 74 ]. CDR1as contains 63 conserved miR-7 binding sites [ 20 ] and increases the levels of miR-7 target mRNAs, which are mostly associated with tumor progression [ 75 , 76 ]. By sponging miR-7, CDR1as regulates the development of zebrafish midbrain [ 20 ] and the processes of many other diseases [ 77 ]. Apart from miRs, circPan3 binds to and stabilizes the mRNA that encodes interleukin-13 (IL-13) receptor subunit IL-13Rα1, eventually leading to the maintenance of intestinal stem cells [ 78 ]. However, the stoichiometric relationship between circRNAsterm·dom and other molecules (miRs and proteins) has received more attention [ 7 , 13 ]. In view of the generally low abundance of circRNAsterm·dom and the limited number of binding sites, exactly how circRNAsterm·dom exert sufficient/detectable effects remains unclear. Translation into proteins The translation is performed by ribosomes and involves initiation, elongation, termination and ribosome recycling [ 79 ]. The Initiation on eukaryotic mRNAs involves scanning by 43S preinitiation complexes from the 5′ cap-proximal point of attachment to the initiation codon, followed by ribosomal subunit joining and factor displacement [ 80 ]. Lacking the 5′-cap and 3′-tail, circRNAterm·dom can only adopt cap-independent manners. In addition to previously described m6A-mediated translation [ 39 , 64 , 81 ], artificial [ 67 ] and endogenous circRNAsterm·dom containing an internal ribosome entry site (IRES) that directly recruits ribosomes [ 82 ], can also be translated [ 63 ]. These two approaches may be coupled with each other. For example, m6A improves the efficiency of IRES-mediated translation of circZNF609 [ 39 , 83 ]. Additionally, circRNAterm·dom with an infinite ORF undergoes rolling circle amplification in an IRES-independent manner, leading to a hundred-fold higher productivity than linear transcript [ 84 ]. Peptides encoded by circRNAsterm·dom are generally truncated and their functionsterm·dom are mostly analogous to the full-length protein counterparts (circFBXW7-185aa [ 23 , 85 ]). However, some proteins originating from circRNAsterm·dom exert functionsterm·dom independent of or even opposed to those of their host gene products (circFNDC3B-218aa [ 86 ]). These results broaden the range of human proteome. However, the regulatory mechanismsterm·dom of circRNAterm·dom translation and the processes of elongation and termination are still not completely understood. CircRNA-protein interactionsterm·dom A handful of studies have reported that circRNAsterm·dom functionterm·dom by interacting with proteins [ 87 – 89 ]. In addition to the abovementioned RBPs that participate in circRNAterm·dom metabolismterm·dom [ 33 , 36 ], and circRNAsterm·dom that interact with Pol II [ 44 , 45 ], and miR-sponging circRNAsterm·dom associated with Ago2 [ 58 ], a group of circRNAsterm·dom serve as protein decoys, scaffolds and recruiters in diverse physiological and pathological contexts. One circRNAterm·dom may exclusively bind to a single protein or interact with multiple proteins under different circumstances. One RBP can also combine with a subgroup of circRNAsterm·dom and form circRNA-protein complex (circRNP) families (IGF2BP3) [ 90 ]. However, bioinformatic analyses indicated that circRNAsterm·dom possess a lower RBP binding density than linear RNAs [ 91 ], which implies that many circRNAsterm·dom are incapable of interacting with proteins. Thus, urgent solutions are needed to more efficiently carry out studies on circRNA-protein interactionsterm·dom. In this module, we classify circRNA-protein interactionsterm·dom into five sections (Fig. 3 ). Proteins involved in circRNAterm·dom upstream processes (metabolismterm·dom), and some special species (e.g., Ago2 and Pol II) will not be included (all relevant studies are listed in Table 1 ). Fig. 3 CircRNA-protein interactionsterm·dom. A (a) CircRNAterm·dom binds to both proteins and strengthens their interaction. (b) CircRNAterm·dom binds to protein A and reinforces its interaction with protein B, which does not directly bind to circRNAterm·dom. (c) CircRNAterm·dom binds to both proteins that originally combine with each other and then disrupts their interaction. B CircRNAterm·dom blocks proteins from interacting with DNA, RNA or other proteins, thus compromising their original functionsterm·dom. C CircRNAterm·dom recruits transcription factors, chromatin remodelers and DNA or histone modifying enzymes to the promoters and alters transcription (including activating and inhibiting). D CircRNAterm·dom helps RBPs to combine with mRNA and stabilizes mRNA (indirectly promoting translation) or directly regulates translation (including promoting and inhibiting). E ( a ) Nuclear circRNAterm·dom causes the nuclear retention of proteins. ( b ) Cytoplasmic or shuttling circRNAterm·dom facilitates the nuclear import of proteins. ( c ) Cytoplasmic circRNAterm·dom causes the cytoplasmic retention of proteins. ( d ) Nuclear or shuttling circRNAterm·dom facilitates the nuclear export of proteins. ( e-g ) Furthermore, circRNAsterm·dom can transport proteins to the nucleolus, mitochondria and membrane, respectively Table 1 Known circRNA-protein interactionsterm·dom classified by manners of action Manner CircRNAterm·dom Protein Direct Effect Biological Functionterm·dom Correlation a Refs Altering interactions between proteins circFoxo3 p53, MDM2 induce mutant p53 ubiquitination pro-apoptosis P [ 92 ] circFoxo3 p21, CDK2 dampen the activity and accessibility of CDK2 cell cycle arrest in G1 phase P [ 93 ] circNfix YBX1, Nedd4l induce YBX1 ubiquitination and inhibit its nucleartranslocation cell cycle arrest, anti-angiogenesis,anti-regeneration U [ 25 ] circADD3 CDK1, EZH2 phosphorylate EZH2 and induce its ubiquitination anti-metastasis P [ 94 ] circAmotl1 PDK1, AKT phosphorylate AKT and promote pAKT nucleartranslocation anti-apoptosis, cardio-protection P [ 95 ] circGLI1 p70S6K2, GSK3β phosphorylate GSK3β pro-metastasis P [ 96 ] circCTNNB1 DDX3, YY1 transactivate YY1 tumor progression P [ 97 ] circCUX1 EWSR1, MAZ transactivate MAZ pro-Warburg effect, tumor progression P [ 98 ] circACC1 β and γ subunits of AMPK stabilize and activate AMPK metabolic reprogramming N [ 99 ] circCcnb1 H2AX, (wild-type p53) free Bclaf1 from p53 cell survival P [ 100 ] circCcnb1 H2AX, (Bclaf1) wrap Bclaf1 by H2AX cell death N [ 100 ] circCcnb1 Ccnb1, CDK1 deactivate Ccnb1 and retain it in the cytoplasm cell cycle arrest in G2 phase N [ 101 ] Blocking proteins from DNA circHuR CNBP sequester CNBP from the HuR promoter tumor suppression N [ 102 ] circSCMH1 MeCP2 tether MeCP2 and relieve its repression upon thetarget gene transcription neuroprotection post stroke P [ 103 ] circSamd4 PURA, PURB tether PURA/B and relieve their repression uponMHC transcription pro-myogenesis U [ 104 ] ACR DNMT3B tether DNMT3B and decrease the methylation of thePink1 promoter anti-autophagy U [ 105 ] cia-cGAS cGAS block cGAS from self-DNA and inhibit its enzymatic activity maintaining HSCs quiescent U [ 106 ] Blocking proteins from RNA circSMARCA5 SRSF1 tether SRSF1 and suppress its splicing activity (SRSF3, PTBP1) anti-migration N [ 107 ] circSMARCA5 SRSF1 tether SRSF1 and suppress its splicing activity (VEGFA) anti-angiogenesis N [ 108 ] circPABPN1 HuR sequester HuR and destabilize PABPN1 mRNA anti-proliferation N [ 109 ] circPABPN1 HuR sequester HuR and destabilize Atg16L1 mRNA anti-autophagy U [ 110 ] circZKSCAN1 FMRP sequester FMRP from combing with CCAR1 mRNA anti-stemness, tumor quiescence U [ 111 ] circMTO1 TRAF4 deactivate Eg5 translation chemosensitization U [ 112 ] circMMP9 AUF1 relieve the inhibition of MMP9 mRNA pro-metastasis P [ 113 ] circANRIL PES1 prevent pre-rRNA maturation and impair ribosome biogenesis anti-atherosclerosis N [ 114 ] circPPM1F HuR sequester HuR and destabilize PPM1F mRNA M1 macrophage activation N [ 115 ] Blocking proteins from proteins circGSK3β GSK3β block GSK3β from β-catenin pro-metastasis N [ 116 ] CDR1as p53 block p53 from MDM2 tumor suppression U [ 22 ] circ102171 CTNNBIP1 block CTNNBIP1 from β-catenin tumor progression U [ 117 ] circH19 PTBP1 tether PTBP1 and inhibit its ability to cleave and activate SREBP1 adipogenesis U [ 118 ] circECE1 c-myc block c-myc from SPOP pro-Warburg effect P [ 119 ] SCAR ATP5B block mPTP from CypD anti-metaflammation U [ 50 ] Recruiting transcription factors to chromatin circRHOT1 TIP60 recruit TIP60 to the NR2F6 promoter and initiate transcription tumor progression P [ 47 ] circAnks1a YBX1 recruit YBX1 to the VEGFB promoter and activate transcription central sensitization, painbehavioral hypersensitivity U [ 120 ] circ0005276 FUS recruit FUS to the XIAP promoter tumor progression P [ 121 ] circPOK ILF2/3 complex potentiate the affinity of ILF2/3 to the IL-6 promoter pro-angiogenesis N [ 122 ] Recruiting modifying enzymes to chromatin circFECR1 TET1 induce demethylation and activate FLI1 transcription pro-tumorigenesis P [ 123 ] circMRPS35 KAT7 induce the acetylation of H4K5 and activate FOXO1/3a transcription tumor suppression U [ 124 ] circAGFG1 EZH2 induce H3K27me3 of the p53 promoter tumor progression U [ 125 ] circLRP6 LSD1, EZH2 induce H3K27me3 and H3K4me2 of the KLF2 and APC promoter tumor progression P [ 126 ] Recruiting chromatin remodelers circDONSON NURF complex recruit the NURF complex to the SOX4 promoter and activate itstranscription tumor progression U [ 127 ] circKcnt2 NuRD complex recruit the NuRD complex to the Batf promoter and inhibit itstranscription anti-inflammation U [ 128 ] Ternary complexes regulating RNA stability circNSUN2 IGF2BP2, (HMGA2 mRNA) stabilize HMGA2 mRNA pro-metastasis P [ 43 ] circPOK ILF2/3, (mRNA of IL-6 andVEGF) stabilize the mRNA of IL-6 and VEGF tumor progression, pro-angiogenesis N [ 122 ] circFNDC3B IGF2BP3, (CD44 mRNA) stabilize CD44 mRNA tumor progression P [ 129 ] Ternary complexes regulating translation circMALAT1 Ribosome, (PAX5 mRNA) retard PAX5 translation self-renewal of HCC stem cells U [ 130 ] circYap eIF4G, PABP, (Yap mRNA) interrupt the assembly of Yap translation initiation machinery tumor suppression N [ 131 ] circMYBL2 PTBP1, (FLT3 mRNA) promote FLT3 translation AML progression P [ 132 ] Translocating proteins to the nucleus circAmotl1 c-myc retain c-myc in the nucleus and increase its affinity to targets pro-tumorigenesis P [ 46 ] circAmotl1 STAT3 facilitate the nuclear translocation of STAT3 pro-wound repair P [ 133 ] circDNMT1 p53, AUF1 facilitate the nuclear translocation of p53 and AUF1, elevatingLC3B level and stabilizing DNMT1 mRNA, respectively pro-autophagy, anti-senescence,tumor progression P [ 134 ] circABCC1 β-catenin redistribute β-catenin tumor progression U [ 135 ] circSOX4 β-catenin translocate β-catenin to the nucleus tumor progression P [ 136 ] Translocating proteins to the cytoplasm circFoxo3 ID-1, E2F1, FAK, HIF1α retain these proteins in the cytoplasm and arrest their functionsterm·dom pro-senescence P [ 137 ] circFOXP1 PTBP1 translocate PTBP1 to the cytoplasm and stabilize PKLR mRNA pro-Warburg effect, tumor progression N [ 138 ] circSTAG1 ALKBH5 retain ALKBH5 in the cytoplasm anti-depression U [ 139 ] circBACH1 HuR translocate HuR to the cytoplasm cell cycle progression P [ 140 ] circZFP609 HIF1α retain HIF1α in the cytoplasm anti-angiogenesis U [ 141 ] circCCAC1 EZH2 retain EZH2 in the cytoplasm pro-metastasis P [ 24 ] Other regions circERBB2 PA2G4 translocate PA2G4 to the nucleolus and promote rDNA transcription tumor progression P [ 142 ] circSKA3 Tks5, Integrin β1 recruit Tks5 to the membrane and co-localize with integrin β1;induce the formation of invadopodia pro-invasion, pro-metastasis P [ 143 ] mecciND1, mecciND5 RPA32, hnRNPA1, TOM40 interact with TOM40 and facilitate the mitochondrial importationof RPA32 and hnRNPA1, respectively metabolismterm·dom of mtDNA and mtRNA;not clear U [ 143 ] mecciRNAs PNPASE control the mitochondrial importation of mecciRNAs not clear U [ 52 ] Unknown or unconfirmed manners circ0011460 PGT just verify the interaction by RIP; increase PGT level positive correlation with pre-eclampsia P [ 144 ] circ0075932 PUM2 just verify the interaction by RNA pull-down; increase PUM2 level pro-inflammation, pro-apoptosis U [ 145 ] circFndc3b FUS just verify the interaction by RIP; decrease FUS level pro-angiogenesis, anti-apoptosis P [ 146 ] circZNF292 LDHA just verify the interaction by RIP; increase LDHA level pro-glycolysis N [ 147 ] circAmotl1 pAKT just verify the interaction by RIP; activate AKT pathway chemoresistance P [ 148 ] circ0075804 (circE2F3) HNRNPK stabilize E2F3 mRNA; the formation of ternary complex is notconfirmed pro-proliferation, anti-apoptosis P [ 149 ] circPTK2 vimentin just verify the interaction by RNA pull-down tumor progression U [ 150 ] circ406961 ILF2 just verify the interaction by RNA pull-down; decrease ILF2 leveland suppress the STAT3/JNK pathway anti-inflammation (induced by PM 2.5 ) U [ 151 ] circBbs9 Ccnd2 just verify the interaction by RIP; increase Ccnd2 level pro-proliferation U [ 152 ] circHECTD1 ZC3H12A reduce ZC3H12A ubiquitination by attenuatinginteraction between ZC3H12A and HECTD1; this study only showscircHECTD1 negatively correlates with HECTD1 deactivation and pro-apoptosis ofalveolar macrophage activated bysilica N [ 153 ] circFOXK2 YBX1, hnRNPK enhance the interaction of YBX1 and hnRNPK with NUF2 andPDXK; lack a detailed mechanismterm·dom tumor progression N [ 154 ] circMUC16 ATG13 just verify the interaction by RNA pull-down; increase ATG13 level pro-autophagy P [ 155 ] circUBR5 QKI, NOVA1 (U1 snRNA) probably participate in RNA splicing; lack a detailed mechanismterm·dom non-functional phenotype U [ 156 ] circNF1–419 Dynamin-1, Adaptor protein2 B1 (AP2B1) just verify the interaction by RNA pull-down and RIP; lack a detailed mechanismterm·dom pro-autophagy, senile dementiadelay U [ 157 ] circNOL10 SCML1 just verify the interaction by RNA pull-down; increase SCML1 level tumor suppression U [ 158 ] circHipk3 Notch1 intracellular domain(N1ICD) verify the interaction by RNA pull-down and RIP; increase N1ICDexpression, stability, acetylation; lack a detailed mechanismterm·dom cardiac regeneration U [ 159 ] cIARS ALKBH5 inhibit the ALKBH5-mediated interaction between Beclin1 andBcl-2; lack a detailed mechanismterm·dom pro-autophagy, pro-ferroptosis U [ 160 ] a approximate functional correlation between circRNAsterm·dom and their parental genes. P positive, N negative, U unknown or unrelated Altering interactions between proteins In this section, we summarize three modes of circRNA-protein interactionsterm·dom: (1) one circRNAterm·dom binds to both proteins and cements their interaction; (2) one circRNAterm·dom binds to protein A and cements or dissociates its interaction with protein B that does not directly bind to circRNAterm·dom; and (3) one circRNAterm·dom binds to both proteins, which originally combine with each other, dissociating their interaction. The circRNA-protein A/B ternary (or more) complex arises in all three modes, but the effects are different. Cementing interactions between proteins In the first mode, circRNAterm·dom mainly mediates posttranslational modifications (ubiquitination and phosphorylation) of protein A catalyzed by protein B or the transactivation of protein A by protein B, followed by a subsequent downstream cascade. Silencing circFoxo3 reinforces cell viability, while its overexpression enhances tumor sensitivity to chemotherapy by potentiating apoptosis. Mechanistically, circFoxo3 interacts with p53 and mouse double minute 2 (MDM2), enhancing p53 ubiquitination. This also occupies MDM2 and relieves Foxo3 ubiquitination [ 92 ]. CircFoxo3 also increases Foxo3 translation by sponging miRs [ 161 ]. In addition, circFoxo3 is associated with cell cycle. The circFoxo3-p21-CDK2 ternary complex reinforces the interaction of CDK2 with p21 (CDK inhibitor 1A) and dampens CDK2 phosphorylation activity. This impedes the formation of CDK2/cyclin E and CDK2/cyclin A complexes, and thus blocks G1/S transition and S progression, respectively, ultimately leading to cell cycle arrest in G1 phase [ 93 ]. This study addressed that a circRNAterm·dom modulates protein-protein interactions for the first time. Similarly, the loss of circNfix promotes cardiomyocyte proliferation and angiogenesis, and inhibits apoptosis post myocardial infarction. Mechanistically, circNfix reinforces the interaction between Y-box binding protein 1 (YBX1) and NEDD4-like E3 ubiquitin ligase (Nedd4l), inducing YBX1 ubiquitination. CircNfix also inhibits the nuclear translocation of YBX1, which binds to the promoters of cyclin A2 and cyclin B1 and activates transcription. In addition, circNfix sponges miR-214 and increases glycogen synthase kinase 3 beta (GSK3β) expression, subsequently repressing VEGF secretion and β-catenin activity [ 25 ]. Other examples of this mode, such as circADD3 [ 94 ], circAmotl1 [ 95 ], and circGLI1 [ 96 ] are listed in Table 1 . Regarding transactivation, circCTNNB1 promotes cancer progression by enhancing the transactivation of Yin Yang 1 (YY1) by DDX3 and thus upregulating target genes involved with β-catenin activation [ 97 ]. This mode is also illustrated by circCUX1, which promotes aerobic glycolysis and neuroblastoma progression by strengthening the transactivation of MYC-associated zinc finger protein (MAZ) by EWS RNA binding protein 1 (EWSR1) [ 98 ]. An outstanding study revealed that in metabolic adaptation to serum deprivation, the production of acetyl-CoA carboxylase 1 (ACC1) RNA switches from the linear to the circular form circACC1, which facilitates the assembly of the AMP-activated protein kinase (AMPK) holoenzyme by combining with the regulatory β and γ subunits and enhancing their interaction. This also stabilizes and activates AMPK, which inhibits anabolism and boosts catabolism, thus promoting β-oxidation and glycolysis. A tetramer is formed by AMPK and circACC1; however, circACC1 does not directly combine with the α subunit or enhance its interaction with another two subunits. The mechanismterm·dom through which ACC1 pre-mRNA shifts to back-splicing under metabolic stress remains unexplored [ 99 ]. The second mode can be exemplified by circCcnb1. In p53 wild-type cells, circCcnb1 precipitates p53 bridged by H2A.X variant histone (H2AX), while in p53 mutant cells, it precipitates Bclaf1 also bridged by H2AX. However, circCcnb1 cannot directly access p53 or Bclaf1. The wild-type p53 has a greater affinity to H2AX than Bclaf1 and circCcnb1 augments this interaction, and thus the former ternary complex allows Bclaf1 to bind to Bcl2 and results in cell survival. While the mutant p53 is unable to bind to H2AX, and thus the latter complex wraps Bclaf1 by H2AX and results in cell death [ 100 ]. Dissociating interactions between proteins In addition to H2AX, circCcnb1 can simultaneously interact with Ccnb1 and CDK1 and dissociates their interaction by forming a large ternary complex. This also decreases their nuclear translocation, thus arresting Ccnb1 functionterm·dom and decelerating cell cycle [ 101 ]. The situation in which a circRNAterm·dom binds to protein A and dissociates its interaction with protein B that does not directly bind to circRNAterm·dom is sorted into the third type (“Blocking proteins from other proteins”) of the next section. Actually, this disruption of interactions is the same as blocking protein A from B. Therefore, we see the diversity of relationships between circRNAterm·dom and two proteins and foresee the complications that arise when three or more proteins are involved. However, apart from mutual binding sites or sequences, little is known about exactly how circRNAterm·dom alters protein-protein interactions. We speculate that circRNAsterm·dom probably change the spatial distance of proteins, expose or cover their activity sites, and allosterically transform their conformation fit for interactions. More investigations and novel technologies are warranted to validate these hypotheses. Tethering or sequestering proteins In this section, circRNAterm·dom combines with only one protein and compromises original functionterm·dom or creates new effects. We classify relevant studies into three types based on the downstream consequences: circRNAterm·dom blocks proteins from interacting with DNA, RNA or other proteins. Blocking proteins from DNA In the first type, DNA binding proteins (e.g., transcription factors, TFs) represent the majority, and thus circRNAterm·dom adversely alters transcription. CircHuR inhibits gastric cancer (GC) proliferation, invasion and metastasis by sequestering CCHC-type zinc finger nucleic acid binding protein (CNBP) from the human antigen R (HuR) promoter, thus downregulating HuR and repressing tumor [ 102 ]. Similarly, circSCMH1 delivered by EVs improves neuronal plasticity, and inhibits glial activation and immune cell infiltration post-stroke by sequestering methyl-CpG binding protein 2 (MeCP2) and compromising its role in transcriptional repression [ 103 ]. CircSamd4 [ 104 ] and autophagy-related circRNAterm·dom (ACR) [ 105 ] also conform to this type. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a DNA sensor that catalyzes cGAMP synthesis when it binds to DNA. Then, cGAMP activates the STING pathway and turn on type I interferon expression. This evolves as a defense mechanismterm·dom against microbial infections, while cGAS activated by self-DNA triggers autoimmunity [ 162 ]. Under homeostatic conditions, circRNAterm·dom antagonist for cGAS (cia-cGAS) occupies cGAS and avoids its binding with self-DNA. Meanwhile, this interaction inactivates synthase activity, thereby abrogating the cGAS-mediated production of type I interferon and protecting long-term hematopoietic stem cells (LT-HSCs) from exhaustion [ 106 ]. Blocking proteins from RNA The second type introduces a group of RBPs that control mRNA splicing, stability and translation. Therefore, circRNAterm·dom indirectly intervenes in posttranscriptional processes. CircSMARCA5 inhibits glioblastoma multiforme (GBM) cell migration by sequestering serine and arginine rich splicing factor 1 (SRSF1) that enhances exon 4 skipping in SRSF3 pre-mRNA. Both SRSF1 and SRSF3 (the isoform without exon 4) upregulate PTBP1, which contributes to glioma cell migration and adhesion [ 107 , 163 ]. VEGFA pre-mRNA can also be alternatively spliced by SRSF1, and its aberrant splicing leads to an alteration in the ratio of the pro−/anti-angiogenic isoforms in GBM [ 108 ]. In addition to disrupting the splicing factor network, circPABPN1 sequesters HuR that stabilizes poly-adenosine binding protein nuclear 1 (PABPN1) mRNA, consequently lowering PABPN1 levels and inhibiting HeLa cell proliferation [ 109 ]. It also prevents HuR from binding to autophagy-related 16 like 1 mRNA and causes subsequent autophagy defects, triggering inflammatory bowel diseases [ 110 ]. Similar examples in which circRNAsterm·dom indirectly regulate mRNA stability or translation include circZKSCAN1 [ 111 ], circMTO1 [ 112 ], circMMP9 [ 113 ] and circPPM1F [ 115 ]. CircANRIL appears to play an atheroprotective role against its linear counterpart linANRIL. The nucleolar protein pescadillo ribosomal biogenesis factor 1 (PES1) strongly interacts with circANRIL. By occupying the C-terminal lysine-rich domain of PES1, circANRIL prevents exonuclease-mediated pre-rRNA maturation and impairs ribosome biogenesis and assembly, leading to nucleolar stress. As a result, p53 is activated and accumulates in the nucleus, thus inducing apoptosis in atherosclerotic plaques [ 114 ]. Blocking proteins from other proteins The third type has similarities to “Dissociating interactions between proteins” in the last section. In both of them, protein contact is broken. For example, circGSK3β promotes ESCC migration and invasion by decreasing β-catenin phosphorylation by GSK3β and its sequential ubiquitination [ 116 ]. CDR1as can block p53 from MDM2, thus relieving p53 ubiquitination and protecting cells from DNA damage [ 22 ]. Similar instances include circ102171 [ 117 ], circH19 [ 118 ] and circECE1 [ 119 ]. Recently, a breakthrough concerning mitochondrial genome-encoded circRNAsterm·dom reported that SCAR (steatohepatitis-associated circRNAterm·dom ATP5B regulator) can bind to ATP synthase subunit b (ATP5B) that resides in the mitochondrial permeability transition pore (mPTP) complex, and block mPTP from cyclophilin D, which shuts down mPTP and inhibits mitochondrial ROS output and fibroblast activation. However, endoplasmic reticulum stress induced by lipid overload (mimicking liver fibroblasts isolated from nonalcoholic steatohepatitis patients) represses PGC-1α mediated by C/EBP homologous protein (CHOP), then downregulating SCAR [ 50 ]. However, the majority of current studies have merely observed the effects of circRNAterm·dom sequestering proteins and have barely explored how circRNAterm·dom influences proteins. Little is known about what happens to both molecules in this pattern (conformation, stability, abundance, distribution, modification). Recruiting proteins to chromatin In this section, several studies displayed novel patterns in which circRNAsterm·dom bind to cis-elements and control TFs or modulate the epigenome, altering gene expression. We categorized them into three types: TFs, DNA or histone modifying enzymes and chromatin remodelers. Recruiting TFs Four examples showed that circRNAsterm·dom recruit transcription activators to promoter regions. CircRHOT1 knockout suppresses hepatocellular carcinoma (HCC) proliferation, migration and invasion. It recruits Tat interactive protein 60 kDa (TIP60) to the nuclear receptor subfamily 2 group F member 6 (NR2F6) promoter to initiate transcription [ 47 ]. Additionally, circAnks1a is upregulated in dorsal horn neurons following spinal nerve ligation and increases central sensitization and behavioral hypersensitivity. Mechanistically, cytoplasmic circAnks1a augments the transportin-1-mediated nuclear translocation of YBX1. Then, nuclear circAnks1a recruits YBX1 to the VEGFB promoter and activates transcription. Cytoplasmic circAnks1a also sponges miR-324-3p that targets VEGFB mRNA. Elevated VEGFB contributes to neuron excitability and pain behavior caused by nerve injury [ 120 ]. Circ0005276 [ 121 ] and circPOK [ 122 ] are also in this category. Recruiting modifying enzymes In regard to the epigenome, covalent modifying enzymes are recruited to DNA (methylation) or histones (methylation and acetylation) to alter chromatin accessibility and turn gene expression on or off. CircFECR1 enhances breast cancer (BC) invasion by recruiting ten–eleven translocation 1 (TET1) to the promoter of its parental gene Friend leukemia virus integration 1 (FLI1), inducing DNA demethylation and activating transcription [ 123 ]. Additionally, circMRPS35 suppresses GC proliferation and invasion by recruiting lysine acetyltransferase 7 (KAT7) to the FOXO1/3a promoter, which elicits H4K5 acetylation [ 124 ]. In contrast, circAGFG1 [ 125 ] and circLRP6 [ 126 ] both recruit enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) to target gene promoters, inducing methylation and deactivating transcription. Recruiting chromatin remodelers CircDONSON promotes GC proliferation, migration and invasion by recruiting nucleosome remodeling factor (NURF, a chromatin remodeler complex) to the SRY-box transcription factor 4 (SOX4) promoter to activate its transcription [ 127 ]. In contrast, circKcnt2 inhibits the innate colitis by recruiting the nucleosome remodeling deacetylase (NuRD) complex to the Batf promoter to inhibit its transcription [ 128 ]. How cells acquire new identities or undergo reprogramming in response to various environmental cues has attracted attention. The interplay between TFs and three-dimensional genome architecture triggers cell-fate decisions [ 164 ]. It will be of interest to determine how circRNAsterm·dom shape the chromatin landscape and to examine the relationship between circRNAsterm·dom and the transcriptome. Forming circRNA-protein-mRNA ternary complexes Ternary complexes are quite common in circRNA-protein interactionsterm·dom, and we mentioned circRNA-protein A/B and circRNA-protein-chromatin complexes in the first and third sections, respectively. In this section, we focus on circRNA-protein-mRNA ternary complexes, which regulate mRNA stability or directly modulate translation. It is noteworthy that in circRNA-protein-nucleic acid (DNA or RNA) ternary complexes, three (or more) elements are combined together. Regulating stability The former case is conventional; circRNAsterm·dom facilitate RBPs to combine with mRNAs, thus stabilizing mRNAs and increasing translation (no example of destabilizing so far). CircNSUN2 combines with IGF2BP2 and high mobility group A (HMGA2) mRNA, forming a ternary complex that stabilizes mRNA. Then, HMGA2 upregulation induces epithelial-mesenchymal transition and promotes colorectal cancer aggressiveness [ 43 ]. Additionally, circPOK functionsterm·dom antithetically to its linear counterpart that encodes Pokemon, which suppresses tumors. CircPOK interacts with interleukin enhancer binding factor 2/3 (ILF2/3) complex and supports the stabilization of IL-6 and VEGF mRNA by ILF2/3. It also potentiates the occupancy of ILF2/3 on the IL-6 promoter. Collectively, circPOK regulates tumor cell secretome at both the transcriptional and posttranscriptional levels [ 122 ]. Similarly, the circFNDC3B-IGF2BP3-CD44 mRNA ternary complex stabilizes mRNA and upregulates CD44 [ 129 ]. Regulating translation The latter case is more fascinating. One study described an unprecedented mode in which circRNAterm·dom becomes stuck between mRNA and ribosome, acting like a brake and retarding translation. CircMALAT1 forms a ternary complex with paired box 5 (PAX5, a tumor suppressor) mRNA and ribosome through 11-complementary-bases and IRESs, respectively, leading to mRNA braking. It also sponges miR-6887-3p and activates the JAK2/STAT3 signaling. Both pathways promote HCC stem cells self-renewal [ 130 ]. Another study introduced a circRNAterm·dom that interposes the translation initiation machinery. CircYap combines not only with its linear counterpart Yap mRNA, but also with eIF4G and PABP, which attach to the 5′-cap and 3′-tail, respectively. This tetramer obstructs the interaction between PABP and eIF4G, thus preventing Yap translation initiation [ 131 ]. Both studies depict how circRNAterm·dom controls translation; however, one interrupts the elongation process, while another suppresses the assembly of initiation machinery. In contrast, circMYBL2 exacerbates acute myeloid leukemia by strengthening the interaction between PTBP1 and FMS-like tyrosine kinase 3 (FLT3) mRNA and promoting translation [ 132 ]. However, this study did not decipher how these molecules combine with each other (through binding domain, dynamic structure or sequence recognition?) and how this interaction improves translation efficiency. Therefore, circRNAsterm·dom exert biological roles at both the transcriptional/epigenetic and translational levels. Nevertheless, exact structures of the circRNA-protein-nucleic acid ternary complexes and precise processes of assembly, interconnection and possible disassembly have not been completely analyzed. Translocating or redistributing proteins The most common intracellular translocation events occur between the nucleus and cytoplasm. In this section, according to the circRNAterm·dom localization and protein redistribution guided by circRNAsterm·dom, we placed the situations into four categories: (1) nuclear circRNAsterm·dom cause the nuclear retention of proteins; (2) cytoplasmic or shuttling circRNAsterm·dom facilitate the nuclear import of proteins; (3) cytoplasmic circRNAsterm·dom cause the cytoplasmic retention of proteins; and (4) nuclear or shuttling circRNAsterm·dom facilitate the nuclear export of proteins. The former two lead to nuclear translocation, while the latter two result in the cytoplasmic translocation of proteins. Increasing nuclear distribution CircAmotl1 promotes BC proliferation and invasion, and inhibits apoptosis. Mechanistically, nuclear circAmotl1 directly binds to c-myc, increasing its nuclear retention and stability, and then enhances the affinity of c-myc to downstream gene promoters [ 46 ]. Another study showed that circAmotl1 interacts with STAT3 and facilitates its nuclear translocation. Nuclear STAT3 binds to the DNMT3a promoter and activates transcription. DNMT3a can methylate the miR-17 promoter, thus upregulating miR-17-5p targets, including fibronectin, DNMT3a and STAT3. These factors form a positive feedback loop and promote fibroblasts proliferation, survival, adhesion, and migration, which together accelerate wound repair [ 133 ]. Similarly, circDNMT1 overexpression inhibits cellular senescence, which is mediated by stimulating autophagy. CircDNMT1 promotes the nuclear translocation of p53 and heterogeneous nuclear ribonucleoprotein D (hnRNPD, AUF1), inducing autophagy (by elevating LC3B level) and increasing DNMT1 translation (by stabilizing DNMT1 mRNA), respectively. Then, DNMT1 inhibits p53 transcription and decreases its total and cytoplasmic abundance while increasing its nuclear abundance [ 134 ]. Previous studies reported a dual role of p53 in controlling autophagy (cytoplasmic p53 represses autophagy, while nuclear p53 enhances autophagy) [ 165 ]. In turn, autophagy increases circDNMT1 levels, thus forming a complicated positive feedback network that suppresses senescence and promotes tumor progression [ 134 ]. Furthermore, circABCC1 [ 135 ] and circSOX4 [ 136 ] both increased the nuclear translocation of β-catenin and expedited tumor progression via the Wnt pathway. Increasing cytoplasmic distribution CircFoxo3 overexpression induces cardiac senescence and aggravates doxorubicin-induced cardiomyopathy by retaining the anti-senescence proteins inhibitor of DNA binding 1 (ID-1) and E2F1, as well as the anti-stress proteins tyrosine kinase 2 (FAK) and HIF1α in the cytoplasm, disrupting the functionsterm·dom of these TFs, which mainly work in the nucleus (FAK in the mitochondria) [ 137 ]. Additionally, circFOXP1 promotes the Warburg effect and gallbladder progression by increasing the cytoplasmic translocation of PTBP1 that binds to the 3′-UTR and coding region of pyruvate kinase L/R (PKLR) mRNA, protecting it from decay [ 138 ]. Similarly, circSTAG1 ameliorated astrocyte dysfunction and depressive-like behaviors induced by chronic unpredictable stress by capturing ALKBH5 in the cytoplasm and increasing m6A levels of fatty acid amide hydrolase (FAAH) mRNA [ 139 , 166 ]. CircBACH1 [ 140 ] and circZFP609 [ 141 ] also fall in this situation. A recent study showed that circCCAC1 promotes cholangiocarcinoma (CCA) tumorigenesis by sponging miR-514a-5p that targets YY1. Additionally, it can be packed into EVs and transferred to endothelial monolayer cells, resulting in disrupted barrier integrity and enhanced angiogenesis. Mechanistically, circCCAC1 retains EZH2 in the cytoplasm, demethylating the SH3 domain containing the GRB2-like 2 (SH3GL2) promoter. Subsequently, intercellular junction proteins are decreased, and cell permeability is increased. However, this effect is absent in CCA cells. Therefore, circCCAC1 plays dual roles in CCA cells and endothelial cells by sponging miR and redistributing protein, respectively [ 24 ]. Other intracellular regions Three studies illustrated that circRNAsterm·dom transport proteins to the nucleolus, membrane and mitochondria, respectively. CircERBB2 promotes gallbladder cancer proliferation and mainly accumulates in the nucleolus. Ribosome synthesis is crucial for malignancy, and cancer is characterized by abnormal ribosomal DNA (rDNA) transcription by Pol I [ 167 ]. CircERBB2 increases the nucleolar localization of proliferation-associated 2G4 (PA2G4), promoting PA2G4-TIFIA (Pol I transcription factor) interaction and thereby recruiting Pol I to the rDNA promoter [ 168 ]. However, a more detailed mechanismterm·dom of how circERBB2 moves PA2G4 and whether circERBB2 anchors other proteins involved in rDNA transcription remains unknown [ 142 ]. CircSKA3 enhances BC invasion and metastasis by recruiting Tks5 to the membrane. This induces the formation of invadopodia where circSKA3 co-localizes with actin, Tks5 and integrin β1. Tks5 does not directly bind to integrin β1, but they precipitate each other, which indicates that a ternary complex bridged by circSKA3 forms [ 143 ]. A recent study found that mitochondria-encoded circRNAsterm·dom (mecciRNAs) mecciND1 and mecciND5 can serve as molecular chaperones for replication protein A2 (RPA32) and hnRNPA1, respectively, and facilitate their entry into mitochondria by interacting with translocase of outer mitochondrial membrane 40 (TOM40). However, this effect in the posttranslational system is much weaker than that in the co-translational system, which suggests that mecciRNAs may only help newly synthesized peptides adopt structures that favor mitochondrial importation but have little impact on mature proteins [ 52 ]. MecciRNAs are distributed both inside and outside the mitochondria and may shuttle dynamically. Polynucleotide phosphorylase (PNPASE, a major mitochondrial RNA importation factor) interacts with most mecciRNAs and controls their mitochondrial levels [ 52 ]. In conclusion, protein redistribution is usually accompanied by enhanced or dampened activity and enabled or hampered access to targets, thus leading to boosted or arrested functionsterm·dom and corresponding downstream variations. However, the relationship between the transportation of proteins and circRNAsterm·dom themselves has scarcely been studied. Other manners and summary Several other studies also verified circRNA-protein interactionsterm·dom. However, because detailed mechanistic research is lacking, we are unable to classify them into any manners (Table 1 ) [ 144 – 160 ]. Of note, the techniques of identifying and characterizing the dynamic circRNA-protein interactionsterm·dom have been systematically reviewed, which mainly include RBP immunoprecipitation (RIP), RNA pull-down, RNase protection assay (RPA), crosslinking immunoprecipitation (CLIP), electrophoretic mobility shift assay (EMSA), fluorescence in situ hybridization (FISH) and immunofluorescence. RIP followed by RNA-sequencing/polymerase chain reaction profiles/verifies circRNAsterm·dom that bind to specific proteins, while RNA pull-down followed by mass spectrometry/western blotting identifies/verifies proteins that bind to specific circRNAsterm·dom. RPA and CLIP can map the sites of interactions, while EMSA can verify the formation of circRNA-protein complexes. FISH and immunofluorescence can detect the co-localization of circRNAsterm·dom and proteins [ 8 , 87 – 89 ]. Some circRNAsterm·dom, such as circFOXO3 and circAmotl1, may implement diverse but similar functionsterm·dom by interacting with distinct proteins under different or dynamic circumstances. Some, such as circCcnb1 and circCCAC1, can even exert dual roles in different cell types. The underlying reason for this remains unknown and may be explained by external stimuli (specific microenvironmental or instructive factors) or internal features (dynamic tertiary structures or physicochemical properties). Some, such as circSMARCA5 and circPABPN1, may interact with the same protein that possesses various targets and thus be multifunctional. It is necessary to state that five manners are not strictly exclusive, and occasional overlap appears among them. We distinguished them depending on the predominate effects observed and emphasized by researchers. Apart from the abovementioned ubiquity of ternary complexes, protein retention by circRNAterm·dom is a sort of sequestration in some ways. Sometimes the formation of complexes occurs after the transportation of circRNPs, while sometimes alterations in protein activities or modifications lead to redistribution. Another aspect worth considering is the correlation between circRNAterm·dom functionterm·dom and that of its parental gene. Nearly 50% circRNAsterm·dom that interact with proteins act as positive regulators of parental genes, while approximately 20% act as negative regulators. The rest are unrelated or unknown yet. This phenomenon seems to not be subject to any specific rules. Some circRNAsterm·dom directly interact with or indirectly regulate their cognate genes, linear counterpart transcripts or host gene products (DNA, mRNA and protein, respectively). However, most of them tread totally different pathways that lead to similar, opposite or unrelated outcomes compared to parental genes. Interacting with the chief executors of life processes endows circRNAsterm·dom with multiple biological functionsterm·dom. Among them, approximately 80% correlate with tumor progression or repression. Specifically, circRNAsterm·dom engage in the overlapping modulation of tumorigenesis (proliferation and apoptosis), metastasis (invasion and migration), cell cycle, angiogenesis, metabolic reprogramming, senescence, autophagy, stemness, chemosensitivity, etc. The rest mainly include cardiovascular and neurological functionsterm·dom, inflammation and autoimmunity, wound repair and regeneration, myogenesis and adipogenesis, and undefined downstream effects. Though these studies involve extensive areas, they do not obey any other specific rules thus far, which indicates a large gap between basic research and clinical practice and reminds us that further explorations are indispensable. Conclusions and perspectives In this reviewterm·mth, we briefly summarize recent progress in circRNAterm·dom metabolismterm·dom and functionsterm·dom. Among these, circRNA-protein interactionsterm·dom hold climbing appeal to scientists and still lack a comprehensive research framework. Therefore, we collected almost all relevant studies to date and classified them into five patterns according to the direct effects that circRNAsterm·dom exert on proteins: (1) altering interactions between proteins; (2) tethering or sequestering proteins; (3) recruiting proteins to chromatin; (4) forming circRNA-protein-mRNA ternary complexes; and (5) translocating or redistributing proteins. Despite fruitful progress, challenges and difficulties posed by theoretical deficiencies and technological restrictions exist. For example, what factors determine whether circRNAsterm·dom act as scaffolds or decoys to support or impede proteins? Are there transcription repressors, other cis-elements (enhancers), chromatin modifiers or remodelers that are involved in circRNAterm·dom regulation upon transcription? What upstream intrinsic properties or extrinsic factors elicit the formation of complexes from several specific molecules? Are there ncRNAs or other molecules (e.g., metabolites and ionic compounds) that combine with circRNPs? Are circRNAsterm·dom involved in protein translocation to other organelles (e.g., the endoplasmic reticulum, Golgi apparatus and lysosome), subcellular compartments (e.g., euchromatic or heterochromatic territories, the nuclear lamina and membrane-less organelles formed by phase separation) or extracellular spaces (e.g., the matrix and adjacent or distant cells)? Do circRNAsterm·dom affect the translocation of other molecular species? How can we profile circRNAsterm·dom located in other organelles or deliver circRNAsterm·dom to specific subcellular compartments? How can we analyze the exact structure of circRNPs beyond the binding sites to interpret the conformational and functional changes of proteins? How can we improve the canonical techniques (e.g. RIP, RNA pull-down, RNA-sequencing) in view of the low abundance and special structure of circRNAsterm·dom? How can we capture the real-timecircRNA-protein interactions in vivo and display the dynamic processes? How can we design the animal models and conduct pre-clinical studies targeting circRNPs? At present, the study on circRNA-protein interactionterm·dom is still staying at the very beginning stage and far away from translational medicine. Though their unique structures and resistance to RNA decay machinery enable them to act as ideal diagnostic biomarkers and therapeutic targets, regrettably, not a single circRNA-based medical application has been approved so far. However, some excellent studies that we have mentioned above have shown promising results and achievements. For example, in the study of circRNAterm·dom SCAR, a mitochondria-targeting nanoparticle (mito-NP) platform that specifically delivers encapsulated vectors to the mitochondria was designed and this enables mitochondria-targeted therapy to tackle metaflammation associated diseases [ 50 ]. In the study of circPan3, cia-cGAS and circKcnt2, the researchers generated circRNA-knockout mice by targeting the ICSs in flanking introns of circRNAsterm·dom with CRISPR/Cas9 technology, which avoids affecting corresponding parental genes [ 78 , 106 , 128 ]. This provides convenience for basic research and possibilities for translational practice. CircCcnb1 has totally inverse roles in different cells, which makes it a potential therapeutic agent for p53 mutant tumors because it causes less damage to adjacent benign p53 wild-type tissues [ 100 ]. Proteins control the biological activities in all aspects. CircRNA-protein interactionsterm·dom make changes to proteins and thus make a difference in life processes, which sparks our interest in devising novel clinical strategies that manipulate (interfere or utilize) circRNAsterm·dom based on circRNA-protein interactionterm·dom.