Orthologs of the Makorin RING finger protein (MKRN) have been found in fungi, plants, and mammals (1). The mkrn gene family encodes proteins with unique zinc finger arrays, including C3H motifs, a new Cys-His motif, and a RING finger. The ancestral founder of this gene family is mkrn1. Another component, mkrn2, may have evolved 450 million years ago due to mkrn1 gene duplication (2). mkrn3 is an intronless gene found on chromosome 15’s long arm in the Prader-Willi syndrome (PWS) crucial area (3). mkrn4 has been previously known as MKRNB (4).

Ubiquitination is a post-translational modification mechanism involved in several biological processes, including cell survival, differentiation, innate and adaptive immunity. Ub is covalently coupled to the target protein with single or multiple 76 amino acid globular protein by activating (E1), conjugating (E2), and ligating (E3) enzymes (5). These three types of enzymes work in a certain order. E1 activates Ub before it is passed to an E2 conjugating enzyme. Following that, E3 ubiquitin ligases attach to E2 and the substrate, principally supplying the Ub chain to the substrate and promoting isopeptide synthesis. Finally, the 26S proteasome degrades the target protein into tiny peptide fragments (6, 7). Indeed, the presence of the RING finger domain is the most noticeable structural feature of MKRNs, not only because this region is found in proteins that form repressive complexes to reduce target gene activity (8), but also because it is a signature domain of E3 ubiquitin ligases (9). This review will concentrate on the function of E3 ubiquitin ligases to mediate substrate degradation by UPS of MKRN1, MKRN2, MKRN3, and MKRN4. Special attention is also focused on the potential role of MKRNs in physiological functions and disease regulation.

Makorin RING finger protein (MKRN) is produced by the mkrn gene family, distinguished by the intron-containing founder of the intronless and a high level of sequence conservation in taxa spanning from invertebrates to vertebrates. Nine mkrn family loci spread throughout the human genome (2). So far, four functional mkrn genes mkrn1, mkrn2, mkrn3 and mkrn4 have been elaborated in literature. mkrn1 is ancestral gene of this family and characterized in humans, mice, wallabies, chickens, pigs drosophila, nematode and plants (10, 11). MKRN1 is substantially and ubiquitously expressed in human organs, including the hypothalamus and the amygdala, according to expression assessments. When transfected into different cell types, MKRN1 is expressed in the nucleus and cytoplasm.

The identification and characterization of the mkrn2 locus in yellowtail fish aided research into the makorin gene family. mkrn2 orthologs are found in humans, mice, and zebrafish. mkrn2 is assumed to have evolved from an ancestral mkrn1 by gene duplication 450 million years ago, and it (mkrn2) partially overlaps with the raf1 protooncogene in an antisense transcriptional direction. MKRN2 is found in all human tissues and cell lines, according to expression studies (12). MKRN2 expression was greater in primary leukemia samples than in age-matched normal BM cells. However, no significant association was found between MKRN2 expression levels in any leukemia subtypes (13).

mkrn3 is specific to therian mammals, and it is an intronless retrocopy of mkrn1 produced by reverse transcription of an mkrn1 mRNA molecule. The reverse transcriptase encoded by autonomous retrotransposable elements catalyzes the production of such retrogenes. Several more mkrn1 retrocopies have been found in mammalian genomes, with the majority of these most likely belonging to pseudogenes (14). The presence of mkrn3 in the dog, mouse, and human genomes, together with its lack in the chicken, fish, and platypus genomes, suggests that Prader-Willi syndrome (PWS) acquired mkrn3 critical area about 80-90 million years before (15). MKRN3 is ubiquitously expressed in adult tissues in both mice and humans, with the greatest level in the testis. MKRN3 was found in high concentrations in the brain and lung of human embryonic tissues. mkrn3 gene expression has been identified in mice from the blastocyst stage and embryonic days 8 to 17 and in ESCs (3).

However, mkrn4, a novel member of the makorin gene family, was discovered in poeciliidae fish. MKRN4 has been discovered as having gonad-specific expression in vertebrates (4). MKRN4 shares 81% of its amino acid identity with MKRN1, 46% with MKRN2, and 52% with MKRN3 (16). mkrn4 gene has been previously known as MKRNB, although it is also present in the human genome, labelled as a pseudogene (4). MKRN4 expression investigation in medaka, zebrafish (ray-finned fishes), and amphibians revealed a substantially gonad-biased expression pattern, as did MKRN1 and MKRN2, with particularly strong expression in the ovaries (2, 17).

The gene structures of the members of the makorin protein family (human mkrn1, mkrn2, mkrn3, mkrn4) were analyzed in Figure 1 to show their similarities and differences. mkrn1 and mkrn2 have the same exons, but mkrn3 has only one exon, mkrn4 has five exons. All of them possessed different exons about location and sizes.

The mkrn gene family encodes different proteins with diverse zinc-finger motif composition and structure, including numerous C3H motifs, a RING finger motif, and a Cys-His motif (2, 3). C3H zinc fingers, which are present in various ribonucleoproteins and may serve as RNA-binding proteins, affect post-transcriptional RNA processing at numerous levels, including alternative splicing, mRNA stability, mRNA localization, and translation efficiency (18, 19). Most E3 ubiquitin ligases have the RING finger domain, which mediates the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to target protein substrates (20). Figure 2 demonstrated the functional domains of human MKRN1, MKRN2, MKRN3, and MKRN4. It showed that MKRN1, MKRN2, and MKRN4 have the same number of C3H-type zinc fingers, but MKRN3 has one less. They all share the identical Cys-His (CH) and C3HC4-type RING finger domains as makorins ( Figure 2 ). Human MKRN1 has four isoforms that encoded by a single mkrn1 gene and emerge via alternative splicing and variable polyadenylation. MKRN1-long has four C3H-type zinc fingers, a Cys-His-type motif, and a highly conserved C3HC4-type RING finger domain. MKRN1-short1/MKRN1-short3 human MKRN1 transcript variants are missing the C-terminal ZF and the final 6 amino acids (aa) of the RING finger domain (RFCC), which are required for binding the second zinc ion, or the N-terminal segment (64 aa). Previous research found that the pattern of pig MKRN1 expression is similar to that of human MKRN1-short 2, which lacks the N-terminal 64 aa seen in MKNR1-long (11) ( Figure 2 ). Three C3H zinc fingers flank the RING finger domain on its N-terminal side and one C3H zinc finger on its C-terminal side in most plants and invertebrates (but not in drosophila). The cysteine and histidine residues motif (the Cys-His motif) is located between the third C3H zinc finger and the RING domain. These domains are associated with their function, as discussed below.

MKRN2’s possible physiological activities have recently been identified. MKRN2 knockout mice, for example, have been shown to have problems in male fertility as well as abnormalities in testis function (51). Furthermore, MKRN2 has been implicated as a negative regulator of a variety of cellular and physiological pathways, including neurogenesis in Xenopus laevis (52, 53), NF-kB signaling in human cells (54), and non-small-cell lung cancer cell metastasis via the PI3K/Akt signaling pathway (55). MKRN2 inhibits the p53 apoptosis effector related to PMP22 (PERP) expression, and levels of the protein in sperm samples have an inverse correlation with infertility levels, implying that MKRN2 is important for protecting germ cells from excessive apoptosis and implicating MKRN2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility (56). In addition, specific gene missense mutation of MKRN2 is associated with degenerative lumbar spinal stenosis (DLSS), the major variant type was single nucleotide polymorphism (SNP), and C > T was the most common single nucleotide change (57), and genetic differences in microRNA 154-binding sites, as well as MKRN2, reduce or abolish microRNA-mediated regulation of genes related with cardiometabolic abnormalities (58).

MKRN2 also has E3 ligase activity associated with the RING finger domain, which is bound to p65 and promotes polyubiquitination, proteasome-dependent degradation of p65 via the MKRN2 RING finger domain, which inhibits p65-mediated NF-κB transactivation and inflammatory responses (54). Furthermore, MKRN2 controlled melanoma cell proliferation via interacting with and ubiquitylating p53, implying that MKRN2 might be a therapeutic target for melanoma (59). Previous research found that IGF2BP3 is a novel ubiquitylating substrate for MKRN2 and that MKRN2 reduces central nervous system tumors by regulating CD44 and PDPN in an IGF2BP3-dependent way. As a result, MKRN2 may be a promising therapeutic target for neuroblastoma (60).

The RING finger domain of MKRN3 is thought to be a potential E3 ubiquitin ligase that suppresses Nptx1 expression by polyubiquitination before puberty. Genetic variations in and near the MKRN3 gene have been linked to instances of familial and non-familial central precocious puberty (61–64). As a result, MKRN3 has been found to inhibit trigger protein maturation in juvenile puberty delay. The genetic treatment for accurate MKRN3 compensation may be a promising strategy for central precocious puberty (CPP) therapy.

Because of the lack of functional promoters, integrated, processed pseudogenes have been assumed to be untranscribed and utterly non-functional since their discovery (65). Processed pseudogenes may occasionally gain promoter activity and be transcribed (66, 67). Interestingly, a recent study reported that the novel E3 ligase MKRN4 was identified that induces GLK protein degradation, which suppresses GLK protein overexpression and has a positive SLE function. For the first time, MKRN4 is shown as an E3 ubiquitin ligase instead of a pseudogene (16).

mkrn gene family has similar the RING finger domain required for E3 ubiquitin ligases of the RING finger class, despite a different array of zinc-finger motifs. However, as multiply function proteins, MKRNs served as E3 ubiquitin ligase play a leading role in degrading many substrates and regulating several diseases ( Figure 3 ). Thus, in the future, novel therapeutic techniques that target protein-protein interactions and interfere with the binding of MKRNs and their substrates may be developed, and these tactics may create fresh therapeutic options for the treatment of illnesses or pathogens infection.

TW and LL conceived and wrote the manuscript. WL, CW, MA, and YL revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Natural Science Foundation of China (32002248) and the Natural Science Foundation of Shandong Province (ZR2020QC016, ZR2020QC017).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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