*2.2. CircRNA Biogenesis and Structure*

CircRNAs are a unique subtype of lncRNAs that are covalently closed, single-stranded circular transcripts without 50 caps or 30 poly(A) tails. Although circRNAs were first described in the late 1970s, research in the past decade has dramatically improved our understanding of the expression and various biological functions due to the application of new technologies, mainly deep RNA sequencing [36]. The single-stranded, closed RNA molecules originate from precursor mRNAs (pre-mRNAs) and are usually from splicing within a protein-coding gene. CircRNAs have several biological functions in normal cells, including acting as sponges to efficiently subtract microRNAs and proteins [36].

CircRNAs can be classified into intronic circRNAs, exonic circRNAs, and exon-intron circRNAs (EIcirRNAs) (Figure 1). Multiple mechanisms have been used to describe the biogenesis of exonic circRNAs, including lariat-driven circularization, RNA binding protein (RBP) mediated circularization, and intron pairing-driven circularization [36]. During splicing, an exon skips, resulting in the back-splicing of RNA folding regions. EIciRNA is formed when the spliced intron lariat, or loop-like structure, remains. The loop-like structure can be mediated by an RBP or be intron-pairing-driven. The final structure is an exonic circRNA when the intron sequence is removed. The intronic circRNAs are formed both upstream and downstream of introns and are mainly accumulated in the nucleus. By contrast, exonic circRNAs without introns are most wielded in the cytoplasm to regulate past-transcriptional gene regulation [37].

**Figure 1.** Biogenesis of circRNAs. Three types of circRNAs: exonic-intronic circRNA (EIciRNA), exonic circRNA, and intronic circRNA. EIcirRNAs and intronic circRNAs are abundant in the nucleus. Exonic circRNAs are exported from the nucleus to the cytoplasm and play functional roles in gene transcription and post-transcription. Adapted from [36]. **Figure 1.** Biogenesis of circRNAs. Three types of circRNAs: exonic-intronic circRNA (EIciRNA), exonic circRNA, and intronic circRNA. EIcirRNAs and intronic circRNAs are abundant in the nucleus. Exonic circRNAs are exported from the nucleus to the cytoplasm and play functional roles in gene transcription and post-transcription. Adapted from [36].

contrast, exonic circRNAs without introns are most wielded in the cytoplasm to regulate

#### **3. Approaches for Discovering lncRNAs 3. Approaches for Discovering lncRNAs**

expression and function.

past-transcriptional gene regulation [37].

Most lncRNAs and circRNAs are challenging to discover due to their low expression levels [38]. With the development of innovative technologies, an increasing number of novel linear lncRNAs and circRNAs have been identified by computational analysis of the transcriptomic datasets, high throughput sequencing, and experimental validation. Table 1 lists the techniques readily available for studying linear lncRNA and circRNA Most lncRNAs and circRNAs are challenging to discover due to their low expression levels [38]. With the development of innovative technologies, an increasing number of novel linear lncRNAs and circRNAs have been identified by computational analysis of the transcriptomic datasets, high throughput sequencing, and experimental validation. Table 1 lists the techniques readily available for studying linear lncRNA and circRNA expression and function.

**Table 1.** Key experimental approaches for identifying and validating lncRNA expression and function.

