Circular RNAs’ cap-independent translation protein and its roles in carcinomas

According to the latest global cancer burden data released by the International Agency for Research on Cancer of the World Health Organization in 2020, 19.29 million new cancer cases and 9.96 million deaths are observed worldwide in 2020. Malignant tumors seriously threaten people’s health. Advances in biotechnology in recent years have deepened our understanding of the complex genetic and non-genetic heterogeneities within individual tumors. The combination of proteomics and genomics may reveal the most accurate information about the activity status of a single gene. Molecular diagnosis and treatment can help improve the poor prognosis of malignant tumors [1]. Therefore, the molecular mechanism of the occurrence and development of malignant tumors should be deeply explored.

CircRNAs are a type of single-stranded covalently closed RNA molecule, and produced by non-canonical reverse splicing events [2]. CircRNAs are important in the occurrence and development of human malignant tumors [3, 4]. Given the lack of typical mRNA characteristics, CircRNAs are generally considered to be a subtype of non-coding RNA (ncRNA) [5, 6]. However, some characteristics of CircRNAs suggest translational potential. For example, most CircRNAs are composed of exon sequences, are mainly located in the cytoplasm, and can even carry a translatable open reading frame (ORF) containing a start codon. Therefore, since the 1970s, scholars devoted to reveal the translation potential of CircRNAs. Early reports indicated that most CircRNAs are not related to multimers [7]. Later, studies show that CircRNAs can be used as translation templates in viruses [8]. A study in 1995 showed that the synthetic circular RNA can recruit 40S ribosomal subunits and translate detectable polypeptides in human cells through internal entry sites [9]. Some subsequent studies focused on the endogenous CircRNA molecules, but failed to find evidence to support the translation of CircRNAs in cells [6, 10, 11], but the research is continuing. In recent years, some studies provided strong evidence for the translation of CircRNAs in cells [12‐14]. New studies also questioned the translation capabilities of CircRNAs. Stagsted et al. identified and characterized a new subset of CircRNAs, i.e., AUG circRNAs, encompassing the annotated translational start codon from protein-coding host genes. Thorough cross-species analysis, extensive ribosome analysis, proteomics analysis and related experimental data on a selected group of AUG circRNAs, showed CircRNAs have no sign of translation [15]. The translation of CircRNAs is still a controversial issue, and is worthy of further exploration. Recently, more studies based on ribosome maps and improved mass spectrometry showed that some CircRNAs have translation functions. CircRNAs can achieve cap-independent translation through the internal ribosome entry site (IRES), N6-methyladenosine (m6A), or a unique rolling circle amplification (RCA) method, and the protein produced by CircRNAs may be involved in the occurrence of tumors. CircRNAs play an important biological function in development. The translation function of CircRNAs has remarkably enriched genomics and proteomics and provides a new perspective for tumor diagnosis and treatment to improve status quo.

CircRNAs are a type of RNA with unique structure and unknown function. In 1976, Sanger et al. first discovered CircRNAs in RNA viruses [16]. In 1979, Hsu et al. observed CircRNAs in eukaryotic cells by using an electron microscope [17]. For the biosynthesis of CircRNAs, two models, namely, “exon skipping” and “direct reverse splicing circularization”, has been proposed. Both methods involve mechanically catalyzed reverse splicing of the spliceosome [18, 19]. The closed-loop structure formed by reverse splicing results in the lack of cap structure and Poly A tail in CircRNAs and is more resistant to exonuclease degradation compared with linear RNAs [10]. In accordance with the origin of the genome and way of splicing, CircRNAs are usually divided into three types, i.e.,intron CircRNAs (CiRNAs) [20], exon CircRNAs (EcircRNAs) [21] and exon–intron CircRNAs (EIciRNAs) [22]. Recently, some new forms of CircRNAs including fusion CircRNAs (f-circRNAs) [23, 24], read-through CircRNAs (rt-CircRNAs) [25, 26] and CircRNAs derived from mitochondrial DNA (mecciRNAs), have been reported [27] (Table 1). CircRNAs containing introns are isolated in the nucleus, whereas EcircRNAs are exported to the cytoplasm or exosomes [28, 29]. Initially, CircRNAs are considered to be meaningless, low-abundance splicing byproducts [30]. Given the development of sequencing technology and bioinformatics, CircRNAs can be produced by thousands of genes, and the same gene can also produce different CircRNAs through alternating cycles [10, 31]. Many CircRNAs are found in all known eukaryotes, and their expression in different species is often highly conserved [32, 33]. Increasing studies showed that CircRNA plays an important role in the occurrence, development and prognosis of various diseases especially tumors [34, 35] (Table 2). In addition, CircRNAs is related to the process of neurodevelopment [36], autoimmune response [37] and infertility [38]. Considering their abundance, high stability, tissue-specific expression pattern and wide distribution in various body fluids, CircRNAs have remarkable potential as a biomarker for the liquid biopsy of human diseases [39‐41]. Currently known CircRNAs can act as miRNA sponges [5, 42, 43] (Fig. 1a) or interact with proteins [44] (Fig. 1b) and can regulate the expression of parental genes [22] (Fig. 1c) and participate in the occurrence and development of the disease. Recently, some scholars discovered that some CircRNAs can be effectively translated into detectable peptides and play a regulatory role in disease. Translation is the hidden function of CircRNAs, and its underlying mechanism is still unclear and requires in-depth exploration to a large extent.

Generally speaking, the translation of RNA in eukaryotic cells requires the eukaryotic translation initiation factor eIF4F. eIF4F is a complex composed of helicase eIF4A, cap-binding subunit eIF4E, and scaffold protein eIF4G. eIF4G can attract the 43S pre-initiation complex (PIC, loaded with eIF1, eIF1A, eIF3, eIF5 and the 40 s subunit of the ternary complex eIF2xMet–tRNAiMetx GTP) by interacting with eIF3 [64, 65]. When eIF4F recognizes the 5′-end 7-methylguanosine (m7G) cap structure of mRNA and recruits the 43S complex, the translation process begins. This mechanism is called the the cap-dependent translation pathway [66, 67], and the main mechanism of eukaryotic translation initiation, but is not the only mechanism. Under certain circumstances, such as stress, a cap-independent translation mechanism is present in the body [68‐70]. The cap-independent translation pathway is the underlying mechanism of CircRNA translation.

The earliest report of CircRNAs as a translation template is the study of viral nucleic acid in 1986 [8]. However, the translation ability of CircRNAs has always been controversial, requires different methods to study the structural and functional characteristics of circular RNA, and clearly demonstrate its coding potential in vivo. For RNA with translation capability, ORF is essential, and long, and conservative ORFs are likely to be encoded [103, 104]. However, for CircRNAs, a short open reading frame (sORF) that is usually less than 300 nt in length seems to be important [105‐107], and ORF spanning splice sites are also a significant feature of CircRNA-encoding peptides [81]. Aiming at the characteristics of CircRNAs with translation potential and their potential translation mechanism, current research ideas include reading frame prediction, translation initiation component prediction, conservative analysis, translationomics, proteomics, and many other aspects. Research methods also include the methods of bioinformatics predictive analysis and experimental verification.

Recently, specialized databases that integrate multiple methods and data are gradually developed. The RiboCIRC analyzes 3168 publicly available Ribo-seq and 1970 matched RNA-seq data sets. These data cover 314 studies of 21 different species, and established a comprehensive translatable CircRNA database that is predicted by calculations and verified by experiments [108]. The TransCirc is also recently established a comprehensive database [109]. The TransCirc contains information on more than 300,000 CircRNAs and has obtained multi-omics evidence to support CircRNA translation from published literature. Through a combination of direct and indirect evidence, The TransCirc predicts the potential of all CircRNAs in encoding functional peptides.

After calculation and evaluation, experimental methods should be used to identify the translation of CircRNAs. The Ribosome Atlas, the deep sequencing of mRNA fragments protected by ribosomes, is a powerful tool for the global monitoring of protein translation in the body. The Ribosome Atlas can discover the regulation of gene expression behind various complex biological processes, important aspects of protein synthesis mechanisms, and even new proteins [110, 111]. The sequencing of the ribosome footprint provides an accurate record of the position of the ribosome when translation stops, revealing what transcript is being translated by the ribosome. The overall density of the ribosome footprint reflects the rate of translation that occurs on different transcripts, allowing a direct and quantitative measure of the rate at which each protein is produced by the cell [112]. Polysome profiling is based on the high sedimentation coefficient of ribosomes, and the sucrose density gradient centrifugation can be used to separate polyribosomes. Analyzing the separated components can evaluate the coding potential of ncRNA and directly determine the translation efficiency of CircRNAs in the whole genome [113, 114]. Ribosome profiling and polymer separation are two complementary methods that can realize genome-wide translation analysis. Proteomics can be used to discover and directly detect the micropeptides encoded by circRNAs, and the biological mass spectrometry is a common identification and analysis method for these micropeptides [115, 116].

Thus far, identified translatable CircRNAs are lacking. For the function of the protein obtained by CircRNA translation, Zhao et al. proposed the protein bait hypothesis. The protein encoded by CircRNAs competes with its homologous linear splicing protein isoforms for binding molecule, thereby preventing the normal function of the isomer [117]. CircRNA disorders are common in tumors including basal cell tumors, and the unbalanced expression of CircRNA-encoded proteins may contribute to the occurrence and development of tumors [25]. Based on the abnormal expression pattern of CircRNAs in human malignant tumors, CircRNA-encoded proteins can provide new specific targets for tumor diagnosis and treatment.

CircRNAs achieve protein translation through a cap-independent translation mechanism, which blurs the definition between coding RNA and ncRNA, and makes us realize that the translation mechanism in eukaryotic cells is much more complicated than we understand. Most of the CircRNA-encoded proteins found in most studies play an anti-tumor or tumor-promoting role through different signal transduction pathways. This finding indicates the importance of CircRNA-encoded protein in tumorigenesis and development and fully proves its potential development and clinical application values. Given the deepening theoretical and clinical research, CircRNAs and its encoded protein may be applied to tumor diagnosis and treatment through a variety of ideas. Examples include using these peptides/proteins with classic anti-cancer drugs, vaccinating synthetic peptides or viral vector vaccines encoding related peptide sequences, and developing new drugs on the basis of potential targets in the mechanism. However, most CircrNAs seem to not be related to ribosomes. Moreover, the efficiency of cap-independent translation initiation is low, and the abundance of polypeptides translated by CircrNAs is limited. At present, the research on CircRNA translation is still in its infancy. Many issues, such as refined mechanism, regulation, extension, and termination process, are unknown, and further research and exploration are needed.