Functional roles of the human ribonuclease A superfamily in RNA metabolism and membrane receptor biology

Abstract

The human ribonuclease A (hRNase A) superfamily is comprised of 13 members of secretory RNases, most of which are recognized as catabolic enzymes for their ribonucleolytic activity to degrade ribonucleic acids (RNAs) in the extracellular space, where they play a role in innate host defense and physiological homeostasis. Interestingly, human RNases 9–13, which belong to a non-canonical subgroup of the hRNase A superfamily, are ribonucleolytic activity-deficient proteins with unclear biological functions. Moreover, accumulating evidence indicates that secretory RNases, such as human RNase 5, can be internalized into cells facilitated by membrane receptors like the epidermal growth factor receptor to regulate intracellular RNA species, in particular non-coding RNAs, and signaling pathways by either a ribonucleolytic activity-dependent or -independent manner. In this review, we summarize the classical role of hRNase A superfamily in the metabolism of extracellular and intracellular RNAs and update its non-classical function as a cognate ligand of membrane receptors. We further discuss the biological significance and translational potential of using secretory RNases as predictive biomarkers or therapeutic agents in certain human diseases and the pathological settings for future investigations. ¹

1. Introduction

The metabolism of ribonucleic acids (RNAs) is carried out by many different ribonuclease (RNases), including endoribonuclease and exoribonucleases, and plays an important role in the regulation of fundamental genes in living organisms [1, 2]. The family of mammalian RNase A is one of the most extensively studied pancreatic-type endoribonucleases. Its bovine counterpart, bovine RNase A (bRNaseA), was first extracted from the bovine pancreas in high quantities [3]. In humans, the RNase A superfamily (hRNase A) is composed of thirteen members which are broadly classified into two subgroups: canonical RNases 1–8 and non-canonical RNases 9–13. All of the RNase genes are closely located on chromosome 14 and encode secretory proteins with a hydrophobic signal peptide at the N-terminus [4, 5]. Each RNase contains six to eight conserved cysteine residues that form three to four disulfide bonds to support the three-dimensional structure [6, 7]. Sequence analysis of the human genome revealed that the amino acid sequences of non-canonical RNases 9–13 are 15–30% identical to the canonical RNase subgroup; however, several residues and disulfide bonds critical for protein folding are highly conserved, implying that all of these RNases may share a common ancestry. Phylogenetic analyses suggested that the hRNase A superfamily originates from the fifth member, hRNase5 (also named angiogenin; hereinafter referred to as hRNase5/ANG), because only an RNase 5-like gene, but not others, was traced outside the Class Mammalia [5, 8].

A catalytic triad, formed by one lysine residue (Lys; K) within a consensus motif signature (CKXXNTF) and two histidine residues (His; H), is responsible for the endoribonuclease enzyme activity via acid-base catalysis and is conserved in bRNaseA and the hRNase A superfamily members [9]. For example, the catalytic triad of bRNaseA contains the essential catalytic residues His12, Lys41, and His119, which share sequence similarity with that of hRNase5/ANG containing residues His13, Lys40, and His114 [3, 10, 11]. The canonical RNase proteins are catalytically active with ribonucleolytic activity toward standard RNA substrates to varying degrees. Among them, the ribonucleolytic activity of hRNase5/ANG is extremely weak with 10⁵–10⁶ times less efficient than that of bRNaseA but remains critical for its angiogenic activity [12, 13]. Contrary to the canonical RNases, the non-canonical RNases 9–13 lose ribonucleolytic activity as certain active site residues are absent by insertions, deletions, or mutations [5, 14]. The present review summarizes the processes of RNA metabolism mediated by the hRNase A superfamily, such as the degradation of extracellular RNA, the cleavage of transfer RNA, and the transcription of ribosomal RNA. The former two have been reported to require ribonucleolytic activity. In addition, we describe an ribonucleolytic activity-independent role of hRNase5/ANG acting as a ligand of epidermal growth factor receptor (EGFR), a member of the cell membrane receptor tyrosine kinase (RTK) family [15], and further discuss the RNases’ potential crosstalk between RNA metabolism and membrane receptor biology (Figure 1).

Figure 1. A graphic summary of hRNase A superfamily in regulating RNA metabolism and membrane receptor biology.

Secretory hRNase1 and hRNase5/ANG are abundantly produced by endothelial cells. hRNase1 as an exRNA scavenger mainly contributes to blood vessel protection in some cardiovascular diseases. The clearance of excess exRNAs by hRNase1 administration has the potential to be a new regimen in clinical practice. Secretory hRNase5/ANG undergoes receptor-mediated endocytosis into cells by interacting with cell surface proteins, EGFR and plexin-B2. The internalized hRNase5/ANG plays a functional role in the metabolism of intracellular RNA in a ribonucleolytic activity-dependent or -independent manner. For instance, in response to cellular stress, cytoplasmic hRNase5/ANG participates in tRNA cleavage at positions close to the anticodon loop requiring ribonucleolytic activity and in turn mediates the production of 5′-tiRNA and 3′-tiRNA, leading to cell proliferation and survival. Moreover, hRNase5/ANG can be transported to the nucleolus, where it promotes rRNA transcription by binding to rDNA promoter in association with the formation of RNA Pol I pre-initiation complex. Nuclear hRNase5/ANG is also responsible for direct or indirect regulation mRNA transcription. The underlying mechanism is worthwhile to be further addressed, i.e., the involvement of the ribonucleolytic activity of hRNase5/ANG. Recently, a non-classical role of hRNase5/ANG in membrane receptor biology has been identified. Secretory hRNase5/ANG plays an oncogenic role as an EGFR ligand by binding to EGFR’s extracellular domain, activating EGFR (red stars) with tyrosine phosphorylation (p), and transmitting EGFR downstream signaling in the intracellular space. Notably, this EGFR ligand-like function of hRNase5/ANG occurs independent of its ribonucleolytic activity. Together, hRNase5/ANG-EGFR axis highlights a crosstalk of RNases between RNA metabolism and membrane receptor biology through cell surface receptor endocytosis and intracellular signal transduction. On the other hand, some RNases, such as hRNase2/EDN and hRNase3/ECP, are mainly involved in host defense against pathogens. Interestingly, among the host defense-related activities, some require the ribonucleolytic activity of RNases, such as the antiviral activity of hRNase2/EDN and hRNase3/ECP, whereas some do not, such as the antibacterial activity of hRNase3/ECP and hRNase7. Further details can be found in the main text. Not drawn to scale.

2. The hRNase A superfamily in RNA metabolism

RNA metabolism refers to the alteration of a series of distinct events in RNA molecules, including their splicing, modification, maturation, and degradation, and associates with the pathogenesis of many diseases, such as cancers and neurodegenerative diseases [16–18]. In protein synthesis of cellular organisms, messenger RNAs (mRNAs) convey genetic information to direct the assembly of proteins on the ribosomes, in which abundant and functional non-coding RNAs (ncRNAs) namely transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) are involved. Other ncRNA types comprising functional RNA molecules include small RNAs, e.g., microRNAs (miRNAs), tRNA-derived stress-induced small RNA (tiRNA), small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs), Piwi-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs) as well as long ncRNAs (lncRNAs), e.g., HOTAIR [19–21]. In addition to the intracellular functions, accumulating evidence demonstrates that a group of various types of RNAs, including mRNA and most of ncRNAs, are outside of cells either in free forms or within vesicles, termed extracellular RNAs (exRNAs), which are known to serve as intercellular communicators and biomarker for certain diseases, such as cancers and cardiovascular disease [22–25]. Because each newly synthesized RNase protein contains an N-terminal signal peptide that guides its biosynthesis within the endoplasmic reticulum toward the secretory route to the extracellular space [26, 27], secretory RNases exert ribonucleolytic activity toward free forms of exRNAs for clearance. In this section, we describe the role of RNA metabolism via secretory RNases in the extracellular environment and internalized RNases in the intracellular space (Figure 1).

2.1. Human RNase 1

Human RNase 1 (hRNase1) is conventionally thought as the direct ortholog of bRNaseA based on their sequence and structure similarity [28]. However, the distinct functions between hRNase1 and bRNaseA have been reported. Raines and colleagues showed that the functional bovine homolog of hRNase1 is the brain bovine RNase, not the pancreatic-type bRNaseA. Moreover, unlike bRNaseA, which is responsible for cleavage of single-stranded RNAs, hRNase1 harbors a remarkable ability to degrade single- and double-stranded RNAs, and RNA:DNA hybrids [29–32]. hRNase1 produced by the pancreas, previously known to be essential for the digestion of dietary RNA to aid with nutrition, can be detected in endothelial cells and in many tissues, including the gastrointestinal tract, male tissues, brain, appendix, and kidney [33, 34]. Notably, hRNase1 is secreted abundantly in the circulatory system, where it can modulate the content of exRNAs circulating in biological fluids, such as blood [7, 35, 36].

2.1.1. The role of hRNase1 in exRNA clearance

exRNAs act as natural procoagulant cofactors that induce blood coagulation and upstream mediators of vascular endothelial growth factor that enhances endothelial cell permeability [37, 38]. Furthermore, exRNAs released from stressed or injured cells can elevate proinflammatory cytokines to facilitate inflammation at the site of tissue damage [39–41]. A series of studies by Preissner and colleagues suggested hRNase1 as an exRNA scavenger for vessel protection in inflammatory and ischemic cardiovascular diseases, including myocardial infarction and atherosclerosis [25, 42, 43]. They performed an in vivo mouse model of myocardial ischaemia-reperfusion injury and found that tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, triggers exRNA release especially under hypoxia, leading to the decline of cardiomyocyte functions, which can be prevented by hRNase1 administration [44]. The authors further determined that increased vascular hRNase1 activity accompanied decreased levels of exRNAs and TNF-α in patients who received remote ischaemic preconditioning, a clinical strategy to protect the heart against ischaemia-reperfusion injury before cardiac surgery [45], supporting a protective role of hRNase1 in cardiovascular pathophysiology. Moreover, another group of researchers demonstrated that bRNaseA administration mitigates cognitive impairment caused by hepatic ischemia reperfusion through inhibition of proinflammatory cytokines in vivo [46]. In contrast, proinflammatory stimuli, such as TNF-α and interleukin-1β, are reported to downregulate hRNase1 secretion from endothelial cells, which may in turn interfere with exRNA/hRNase1 ratio in vascular homeostasis and aggravate proinflammatory activities mediated by exRNAs [47].

The application of hRNase1 as a protective role via exRNA degradation has also been discussed in other diseases. For instance, the release of exRNA from tumor cells has the capacity to promote tumor cell progression through liberation of the cytokine TNF-α from macrophages whereas the administration of hRNase1 reduces tumor growth [48], suggesting a new regimen for cancer treatment. In addition, exRNA reduction by hRNase1 treatment prevents adhesion of rheumatoid arthritis synovial fibroblast to cartilage [49], and hRNase1 as an antimicrobial agent that inhibits host-derived exRNA-triggered pneumococcal infection of lung epithelial cells [50]. Together, the exRNA/hRNase1 system plays an important role in human diseases involving proinflammatory cytokines, and the clearance of excess exRNAs by hRNase1 treatment demonstrates its potential clinical benefit.

2.2. Human RNases 2 and 3

Human RNase 2 (hRNase2) and human RNase 3 (hRNase3) are derived from a common ancestor and appeared in humans evolutionarily by gene duplication [51, 52]. Although they share 67% in amino acid and 88% in nucleotide sequence homology [53, 54], hRNase2 harbors at least 10 times higher ribonucleolytic activities than hRNase3 [55, 56]. In addition, hRNase2 and hRNase3, also named eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), respectively, are two of the major secretory proteins found in the specific granules of human eosinophil, a type of white blood cells, and released upon eosinophil activation by proinflammatory stimuli [57]. Notably, these two eosinophil granule proteins were the first ones among the hRNase A superfamily members to demonstrate the role of RNase in host defense system against pathogens and innate immune responses [58–61]. During pathogenic infections, e.g., parasites, bacteria, and viruses, eosinophilic granulocytes migrate toward the infected or damaged epithelial cells in response to inflammatory mediators produced and then release a number of effector proteins, including hRNase2/EDN and hRNase3/ECP, from their granules, a process called degranulation, which contributes to host defense by neutralizing pathogens through apoptosis. Moreover, elevated eosinophil levels also occur in certain diseases, such as asthma, allergic reactions, immunomodulatory responses, and cancers [62–65]. In addition to eosinophils, low levels of hRNase2/EDN and hRNase3/ECP proteins have also been detected in other types of white blood cells, such as neutrophil, basophils, and monocytes [66, 67].

One of the best characterized biological processes of hRNase2/EDN is its broad ribonucleolytic activity-dependent antiviral activity against RNA viruses, and its role as a chemoattractant to induce antigen-presenting dendritic cell maturation and activation [67–70]. Whether hRNase2/EDN is internalized into infected cells to degrade viral RNAs remains to be determined. Similar to hRNase2/EDN, hRNase3/ECP, which has the highest cationicity among the entire hRNase A superfamily (isoelectric point = 11.4), also harbors antiviral activity [6] and unique properties of antibacterial, antihelminthic, and cytotoxic activities, suggesting that hRNase3/ECP is tightly associated with innate host defense [35, 36]. Interestingly, the antiviral and helminthotoxin effects of hRNase3/ECP require its ribonucleolytic activity whereas the bactericidal and cytotoxic effects do not [70, 71]. hRNase3/ECP was reported to bind to the bacteria cell wall components with high affinity to destabilize and permeate the lipid bilayers of bacteria, leading to bacterial cell agglutination to facilitate the efficacy of bacterial elimination [72, 73]. It is worth noting that hRNase3/ECP participates in a range of airway or bowel inflammatory diseases, such as bronchial asthma and Crohn’s disease; the concentration of hRNase3/ECP in serum has been reported to serve as a potential biomarker for asthma [74, 75].

2.3. Human RNases 4 and 5

Human RNase 4 (hRNase4) and human RNase 5 (hRNase5) were first isolated from conditioned media of HT-29 human colon adenocarcinoma cells [76, 77]. The genes encoding hRNases 4 and 5 share the same promoter, and the two hRNases are products of differential splicing with a high sequence homology of 38.7% identity at the protein level [78–80]. Evolutionarily, hRNase4 is the most conserved gene within the hRNase A superfamily across different vertebrates [11, 81]. The three-dimensional structure of hRNase5 is unique with the presence of three instead of four disulfide bonds compared with that of other hRNase A superfamily members [6, 7]. While the two proteins both harbor ribonucleolytic activities, that of hRNase5 is significantly lower than hRNase4. The limited ribonucleolytic activity of hRNase5 is still essential for its role in angiogenesis and ncRNA cleavage [2, 8] (see next subsections). The expression of hRNase4 and hRNase5 are present in a wide range of biological fluids, e.g., blood, saliva, and seminal fluid as well as many tissues with the highest level detected in the liver [82]. Secretion of hRNase4 and hRNase5 mainly come from endothelial cells and have been reported to associate with cancer, neuronal, or immune cells [15, 83–85].

hRNase5 is also called angiogenin/ANG because of its role in promoting neovascularization and angiogenesis [86, 87]. In addition, hRNase5/ANG plays a role in innate immune response via its antibacterial and antiviral properties in host defense [27, 59]. Notably, various pathological conditions can be attributed to hRNase5/ANG [88]. For instance, a positive regulation of hRNase5/ANG in tumorigenesis has been demonstrated in many cancer types, including prostate and pancreatic cancers [8, 89]. Hu and colleagues further identified a functional role of hRNase5/ANG in hematopoietic regeneration by dichotomously promoting quiescence of stem cells and proliferation of progenitor cells [90]. Moreover, hRNase5/ANG acts a neurotrophic and neuroprotective factor in neurodegenerative diseases, such as amyotrophic lateral sclerosis and Parkinson’s disease [91–93]. In other nonmalignant scenarios, e.g., inflammatory bowel disease and diabetes, the serum level of hRNase5/ANG is elevated in patients compared with that in healthy individuals [94, 95]. Although only a few published studies evaluating the biological functions of hRNase4 are available, researchers have shown that it shares a similar role with hRNase5/ANG in angiogenic, neuroprotective, and anti-HIV activities, suggesting that their biological properties may be complementary or supplementary [79, 84]. In this section, we provide more insights into the physiological roles of hRNase5/ANG in regulating angiogenesis and RNA metabolism, which are related to its inherently weak ribonucleolytic activity.

2.3.1. The role of hRNase5/ANG in angiogenesis

The ribonucleolytic activity-dependent angiogenic effect of hRNase5/ANG was validated in experiments using the ribonucleolytic activity-deficient mutants in which residues His13 and His114 were mutated to alanine [10, 96]. Aside from the essential ribonucleolytic activity, several mechanisms underlying hRNase5/ANG-induced angiogenesis have been well established in the past three decades [88]. First, secretory hRNase5/ANG contributes to degradation of basement membrane and extracellular matrix by binding to cell surface actin, which leads to generation of plasmin from plasminogen and activation of several protease cascades, such as matrix metalloproteinase, that promote endothelial cell migration and invasion [97, 98]. Second, after endocytosis through a putative 170-kDa protein on the endothelial cell surface [99], the internalized hRNase5/ANG triggers signal transduction cascades, such as PLC, Akt, JNK, and ERK, which are involved in cell proliferation and further angiogenesis [100–102]. Third, nuclear translocation of hRNase5/ANG is reported to be required for hRNase5/ANG-dependent angiogenesis [103, 104]. Nuclear hRNase5/ANG in turns enhances rRNA transcription in endothelial and cancer cells [105, 106] and modulates mRNA transcription via unclear mechanisms [15, 88] (see next subsections). It would be of interest to further address whether intracellular and/or extracellular RNA fragments digested by hRNase5/ANG are involved in pathological angiogenesis.

2.3.2. The role of hRNase5/ANG in rRNA transcription

Upon translocation into the nucleus, hRNase5/ANG accumulates in the nucleolus [104, 107], the site of rRNA transcription and ribosome assembly responsible for protein synthesis [108]. A series of studies by Hu and colleagues demonstrated that nuclear hRNase5/ANG plays a role in the biogenesis of rRNA and subsequently promotes the corresponding proliferation of endothelial cells as well as several types of cancer cells [105, 106, 109–111]. The authors first performed in vitro studies in endothelial cells in which exogenous hRNase5/ANG enhances the production of 45S rRNA. Chromatin immunoprecipitation (ChIP) assay was then carried out through binding of hRNase5/ANG to the CT-rich repeats of ribosomal DNA (rDNA) named ANG binding elements (ABE) [105, 112]. They further demonstrated that nuclear hRNase5/ANG binds to the upstream core element (UCE) of the promoter of rDNA in vivo in hRNase5/ANG knockdown HeLa cells [113]. At the rDNA promoter, hRNase5/ANG increases the number of active rDNA copies and enhances the assembly of RNA polymerase I (Pol I) pre-initiation complex by epigenetic alteration via rDNA promoter methylation and histone modification [113]. Together, those findings supported one of the mechanisms by which hRNase5/ANG stimulates rRNA transcription through epigenetic activation of rDNA promoter. Although nuclear localization of hRNase5/ANG is essential for its angiogenic activity, which requires its ribonucleolytic activity, it remains unclear whether the enhanced rRNA transcription by hRNase5/ANG relies on its ribonucleolytic activity. Interestingly, hRNase5/ANG can catalyze the cleavage of both 28S and 18S rRNA into 100–500 nucleotides in length in vitro [114]. This suggested that hRNase5/ANG may be involved in the degradation of rRNA in addition to its function in rRNA transcription. Additional studies are warranted to further validate the role of hRNase5/ANG in rRNA degradation in vivo.

2.3.3. The role of hRNase5/ANG in mRNA transcription

Several lines of evidence to date point out a role of nuclear hRNase5/ANG in mRNA transcription. For instance, Hung and colleagues recently identified hRNase5/ANG as a new ligand of EGFR RTK (see next section) and demonstrated a global pattern of transcriptional changes induced by hRNase5/ANG treatment in pancreatic cancer cells [15]. The authors carried out next-generation RNA deep sequencing for whole transcriptome analysis to screen and identify mRNAs regulated by hRNase5/ANG. Among the top 5,000 genes, hRNase5/ANG upregulates or downregulates gene transcription similar to that of epidermal growth factor (EGF), a conventional EGFR ligand, with a high percentage of overlap (~80%) between hRNase5/ANG and EGF treatment. GSEA (Gene Set Enrichment Analysis) further showed these genes to be significantly enriched by both stimuli in several gene signatures, such as EGF signaling, RAS activation, hypoxia-inducible factor 1α target, and interleukin-1α response. Accordingly, hRNase5/ANG elicits signaling events that resemble EGF to control cellular functions through regulation of mRNA transcription in pancreatic cancer cells. Moreover, Sheng and Xu described that a total of 699 genes performed by ChIP-on-chip assay may be regulated by hRNase5/ANG and significantly associated with tumorigenesis, Wnt signaling, and transforming growth factor-β pathways using KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis [88]. Given that hRNase5/ANG can interact with histone H3 and remodel the histone modification [113], it may serve as a chromatin remodeling activator in regulating mRNA transcription, although the underlying mechanistic regulation needs to be further pursued.

2.3.4. The role of hRNase5/ANG in tRNA cleavage and tiRNA production

In addition to the nuclear function of hRNase5/ANG in RNA metabolism, two groups of researchers showed that hRNase5/ANG is also responsible for ribonucleolytic activity-dependent endonucleolytic cleavage of tRNA in the cytoplasm [115, 116]. Under certain insults of cellular stress, e.g., nutrition deficiency, oxidative stress, or hypothermia, the activated hRNase5/ANG stimulates the formation of cytoplasmic stress granules, cleaves tRNA at positions close to the anticodon loop, and mediates the production of tRNA-derived stress-induced small RNA (tiRNA) [87, 117, 118]. The tiRNA fragments in turn enhance damage repair and cellular survival by suppressing the formation of the translation initiation factor complex or associating with the translational silencer, leading to global protein translational arrest, which functions in a cytoprotective stress response program [116, 119, 120]. Interestingly, after respiratory syncytial virus infection, hRNase5/ANG mediates tRNA cleavage, which induces the production of tiRNA fragments that are important molecules for viral replication [121]. The levels of tiRNA are elevated in some cancer cells, such as breast and myeloma [122]. For example, in breast cancer cells, hRNase5/ANG produces tiRNA fragments to stimulate cell proliferation which is dependent on the sex hormones and their receptors [123]. Notably, ncRNA metabolism of tiRNA and rRNA contributes to hRNase5/ANG-activated hematopoietic regeneration by simultaneously enhancing tiRNA production in stem cells and rRNA biogenesis in progenitor cells [90].

The process of hRNase5/ANG-mediated tiRNA production is precisely controlled by its cytoplasmic endogenous inhibitor, ribonuclease/angiogenin inhibitor 1 (RNH1) [124–126]. Pizzo et al. demonstrated that under growth conditions, hRNase5/ANG tightly associates with RNH1 in the cytoplasm, preventing it from random cleavage of cellular RNA. In contrast, under stress conditions, cytoplasmic hRNase5/ANG dissociates from RNH1, activating its ribonucleolytic activity to degrade tRNA, leading to tiRNA production [127]. Furthermore, the stress-induced stress granule assembly by hRNase/ANG is abrogated in cells transfected with RNH1 [120]. Although the mechanism of stress-induced hRNase5/ANG dissociation from RNH1 requires further investigation, cytoplasmic hRNase5/ANG has been shown to evade RNH1 and nuclear transport via protein kinase C- and cyclin-dependent kinase-mediated phosphorylation [128].

Collectively, these studies have highlighted the contribution of cellular hRNase5/ANG to angiogenesis and RNA metabolism pathways by either a ribonucleolytic activity-dependent or independent manner (Table 1). Interestingly, researchers have found that motoneurons secrete hRNase5/ANG under stress, which leads to hRNase5/ANG uptake in a paracrine manner by astrocytes, where it induces cleavage of an unknown subsets of RNAs [129], suggesting that hRNase5/ANG may have other undiscovered RNA substrates for its biological function. Whether hRNase5/ANG participates in the metabolism of other RNAs is worthwhile to be further elucidated.

Table 1.

Summary of ribonucleolytic activity-dependent and -independent biological activities of RNases

RNase	Ribonucleolytic activity-dependent	Ribonucleolytic activity-independent	Undefined on ribonucleolytic activity
hRNase1	• exRNA degradation	n/a	n/a
hRNase2/EDN	• antiviral activity	n/a	• chemotactic activity
hRNase3/ECP	• antiviral activity • antihelminthic activity	• antibacterial activity • cytotoxic activity	n/a
hRNase4	n/a	n/a	• neuroprotective activity
hRNase5/ANG	• angiogenic activity • rRNA degradation • tRNA cleavage	• EGFR binding • EGFR signaling activation • EMT • plexin-B2 binding • rRNA transcription	• neuroprotective activity • mRNA transcription
hRNase6/k6	• antiviral activity	• antibacterial activity • antimicrobial activity	n/a
hRNase7	n/a	• antibacterial activity • antimicrobial activity • antifungal activity	n/a
hRNase8	n/a	n/a	• antimicrobial activity
hRNase9	n/a	• antibacterial activity	n/a
hRNases10–13	n/a	• potential host defense activities	n/a

2.4. Human RNases 6–8

Among the hRNase A superfamily members, human RNase 6 (hRNase6) is more evolutionarily closely related to human RNase 7 (hRNase7) and human RNase 8 (hRNase8); hRNase7 and hRNase8 have been suggested to originate from a recent gene duplication with a remarkable similarity in their amino acid sequences (78% identity) [7]. Although they are a related gene pair, hRNase7 and hRNase8 exert their physiological functions in a tissue-specific fashion [6, 12] (see description below). The ribonucleolytic activities of hRNases 6–8 against yeast tRNA are relatively weak compared with those of the canonical hRNases with the exception of hRNase5/ANG; among them, hRNase8 harbors the lowest ribonucleolytic activity [6, 12].

hRNase6, also named RNase k6, was first identified from a genomic search for a human ortholog of bovine kidney RNase (RNase k2), which shares 72% sequence identity with hRNase6 [130]. The crystal structure of hRNase6 has been determined [131], and most recently, it revealed a second catalytic triad proposed to favor endonuclease cleavage specificity that facilitates polynucleotide substrate catalysis [132]. Notably, hRNase6 is derived from the bone marrow by myeloid cells, such as monocytes and neutrophils, upon bacterial infection [130], suggesting a physiological role in host defense, which was recently validated to show that hRNase6 possesses antiviral activity by inhibiting HIV replication via viral RNA degradation [133]. hRNase6 also exhibits antimicrobial activity against different uropathogenic bacterial strains in the urinary tract via a membrane destabilizing mechanism of action similar to that of hRNase3/ECP [134, 135].

hRNase7 was first detected and purified as the most abundant RNase from the human skin [58] and is found in a range of other epithelial tissues, such as the kidney as well as the respiratory, urinary, and gastrointestinal tracts [26, 136, 137]. Although hRNase7 possesses limited antiviral activity, it harbors broad spectrum microbicidal activity against various microorganisms, including Gram-positive and Gram-negative bacteria as well as certain fungal species [138]. It is one of the major antimicrobial proteins secreted by skin cells, keratinocytes, which play a role in wound healing and tissue repair involving cutaneous host defense and epidermal protection [139–141]. While the mechanism underlying the antimicrobial properties of hRNase7 is not fully understood, it resembles hRNase3/ECP in that its microbicidal activity occurs independently of its ribonucleolytic activity, and instead relies on its ability to permeate the bacterial cell wall by acting as a membrane-disruptive antimicrobial peptide [73, 142]. It has been proposed that hRNase7 contains positively charged residues with clustered lysine at the N-terminus for interaction with negatively charged components on bacterial surface to facilitate its cell permeabilization and rapid leakage of cytoplasmic components [143]. Moreover, hRNase7 was reported to be bound with a bacterial cell surface receptor called outer membrane protein I which can be internalized into cells where it mediates the antimicrobial function of hRNase 7 by triggering cell death [144, 145]. It is worth noting that hRNase7 is upregulated in chronic inflammatory skin diseases, in which it selectively decreases the TH2 cytokines produced by activated T cells in lesioned skin of patients [35, 146–148], suggesting that hRNase7 may contribute to immunomodulation in cutaneous wound healing [140].

In contrast to hRNase7, hRNase8 is expressed primarily in the placenta [12] and exhibits significant antimicrobial activities against many pathogenic bacteria [149]. It has been proposed that hRNase8 plays a role in placental host defense in the amniotic fluid of pregnant women, where hRNase8 eliminates pathogenic infection from the maternal circulation to preserve amniotic cavity sterility for the fetus [7, 27]. Additional studies are required to further verify this proposed mechanism. Interestingly, hRNase8 displays an atypical structure among the hRNase A superfamily members due to a unique extension at its N-terminus, which does not represent a classical secretory mediator, suggesting that it may not undergo the secretion process [36, 150].

Taken together, the above three RNases are the last identified canonical members of the hRNase A superfamily, all of which harbor detectable ribonucleolytic activities as well as host defense-related antibacterial and antimicrobial properties. However, the precise physiological roles and mechanisms of actions involved remain unclearly defined. It would be worthwhile to further explore; for example, whether their ribonucleolytic activities participate in the processes of RNA metabolism that may link to their tissue-specific functions (Table 2).

Table 2.

Summary of main tissue-specific expression and functions of RNases

RNase	Main expression (tissues/cells)	Specific biological functions
hRNase1	• pancreas • endothelial cells	• anti-coagulation • anti-inflammation • vascular homeostasis
hRNase2/EDN	• bone marrow • eosinophils	• host defense/innate immunity
hRNase3/ECP	• bone marrow • eosinophils	• host defense/innate immunity • a potential biomarker in asthma
hRNase4	• liver • endothelial cells	• angiogenesis • neuroprotection
hRNase5/ANG	• liver • endothelial cells • cancer cells	• angiogenesis • neuroprotection • host defense/innate immunity • tumorigenesis • an EGFR ligand • a predictive biomarker for EGFR-targeted therapy • hematopoietic regeneration
hRNase6/k6	• bone marrow • monocytes • neutrophils	• host defense/innate immunity
hRNase7	• skin epidermis • keratinocytes • epithelial tissues/cells	• host defense/innate immunity • immunomodulation
hRNase8	• placenta	• placental host defense
hRNase9	• male tissues (epididymis)	• male reproductive functions • sperm maturation
hRNase10	• male tissues (epididymis)	• male reproductive functions
hRNase11	• male tissues (testis)	• male reproductive functions
hRNase12	• male tissues	• male reproductive functions
hRNase13	• male tissues	• male reproductive functions

2.5. Human RNases 9–13

The hRNase A superfamily expanded following the discovery of human RNases 9–13 and is classified into two subgroups: canonical (RNases 1–8) and non-canonical (RNases 9–13) [5]. The non-canonical RNases share the three most conserved disulfide bonds and one secretion signal peptide with the canonical members. Nevertheless, the non-canonical RNases do not contain the consensus signature motif (CKXXNTF) of the catalytic triad and likely fall short of ribonucleolytic activity because certain active site residues are encoded by insertions, deletions, or mutations [5, 14, 36]. It has been postulated that the non-canonical members, such as hRNase9, may play a role similar to the canonical RNases in innate host defense [151]. Indeed, researchers demonstrated that hRNase9 lacking intrinsic ribonucleolytic activity exhibits bactericidal activity against E. coli. in a dose-dependent manner [152]. Similar to the ribonucleolytic activity-independent antimicrobial function of other hRNases, e.g., hRNase3/ECP and hRNase7, the membrane destabilizing mechanism of action of hRNase9 to enter bacterial cells and induce cell death is attributed to the presence of its positively charged residues [152]. Notably, all of the non-canonical RNases are expressed in the male reproductive tract, specifically hRNase9 and hRNase10 in the epididymis, and hRNase11 in the testis, and associated with male reproductive functions [5, 14, 153, 154] (Table 2). The expression of hRNase9 and hRNase10 are regulated by androgens or other testicular factors, both of which have been suggested as putatively new members of epididymal proteins involved in host defense [155]. In addition, hRNase9 plays a critical role in sperm maturation as evidence by Rnase9-knockout mice resulting in impaired sperm maturation [156]. To date, little is known about the physiological functions of the non-canonical RNase subgroup. Further systematic studies are awaited to address whether these ribonucleolytic activity-deficient RNases play non-classical roles to associate with certain intrinsic functions other than ribonucleolytic activities. Based on the growing body of evidence, a ribonucleolytic activity-independent function of secretory RNases could be a general feature among the entire hRNase A superfamily. In the next section, we describe this ribonucleolytic activity-independent function using hRNase5/ANG in membrane receptor biology as an example in which hRNase5/ANG acts as a newly-identified ligand of EGFR RTK and serves as a serum biomarker for prediction of clinical response to EGFR-targeted therapy (Figure 2).

Figure 2. The proposed model of elevated ANG as an EGFR ligand in the sensitization to erlotinib therapy in pancreatic cancer.

In brief, higher levels of ANG induce its binding to EGFR and activate EGFR signaling, which in turn promotes tumorigenesis and increases erlotinib sensitivity in PDAC patients [15]. (This research was originally published in Cancer Cell. Wang YN, Lee HH, Chou CK, Yang WH, Wei Y, Chen CT, Yao J, Hsu JL, Zhu C, Ying H, Ye Y, Wang WJ, Lim SO, Xia W, Ko HW, Liu X, Liu CG, Wu X, Wang H, Li D, Prakash LR, Katz MH, Kang Y, Kim M, Fleming JB, Fogelman D, Javle M, Maitra A, and Hung MC. Angiogenin/ribonuclease 5 is an EGFR ligand and a serum biomarker for erlotinib sensitivity in pancreatic cancer. Cancer Cell. 2018; 33(4):752–769.e8. DOI: https://doi.org/10.1016/j.ccell.2018.02.012 © 2018 Elsevier Inc.)

3. Human RNase 5 in membrane receptor biology

In 1997, a putative 170-kDa cell membrane receptor was reported to facilitate the internalization of hRNase5/ANG into endothelial cells [99]. Recently, Hung and colleagues demonstrated that hRNase5/ANG functions as an EGFR ligand via a direct association with EGFR to convey EGFR downstream signaling intracellularly in cancer cells [15]. It is conceivable that EGFR may be the 170-kDa cell membrane receptor from endothelial cells as it has a molecular weight of 170-kDa. Notably, this EGFR ligand-like function of hRNase5/ANG does not require its ribonucleolytic activity. Their findings opened a direction toward our understanding of the non-classical roles of RNases independently of their ribonucleolytic activities.

3.1. The ribonucleolytic activity-independent role of hRNase5/ANG as an EGFR ligand

Wang and Lee et al. [15] initially sought to investigate whether exRNAs, which are known to function as pleiotropic molecules for intercellular communication to modulate biological functions, can regulate membrane receptor signaling cascades and cellular processes [22, 157]. They performed a pilot experiment by treating cells with bRNaseA in cultured media to broadly degrade RNA species in the extracellular space [3]. Interestingly, they found that bRNaseA treatment can increase cell viability, migration and invasion, and induce changes in epithelial-mesenchymal transition (EMT)-like phenotype. Cancer cell lines treated with bRNaseA displayed significant increases in the global tyrosine phosphorylation signals, particularly in the range of bands between ~150–250 kDa, which corresponds to molecular weight of an RTK. Using human antibody arrays for phospho-RTKs and phospho-kinases, they identified EGFR RTK as the dominant cell membrane target and several well-recognized EGFR downstream molecules, such as ERK1/2 and Akt, in response to bRNaseA. Through a series of biochemical and biological assays, the authors revealed that bRNaseA and its human counterpart, hRNase5/ANG, act as a ligand of EGFR by binding to EGFR’s extracellular domain and transmitting EGFR intracellular signaling. Moreover, the binding epitope of hRNase5/ANG to EGFR appeared to partially overlap with the EGFR binding region to EGF. It would be important and worthwhile to further pursue a more detailed structural determination, i.e., the direct contact between hRNase5/ANG and EGFR, for a better understanding of this newly-discovered ligand-receptor relationship [89].

Interestingly, the moonlighting ligand-like function of bRNaseA and hRNase5/ANG does not require their ribonucleolytic activities as exRNAs are not involved [15]. The authors generated ribonucleolytic activity-deficient mutants of bRNaseA in which residues Lys41 and His119 were mutated to alanine and observed similar patterns with the wild-type bRNaseA in morphological changes as well as EGFR activation and binding. Consistent results of comparable EGFR activation and binding were also shown in the wild-type and ribonucleolytic activity-deficient counterparts of hRNase5/ANG in which residues Lys40 and His114 were mutated to alanine. Those findings indicated a ribonucleolytic activity-independent role of bRNaseA and hRNase5/ANG as an EGFR ligand, and raised an interesting question of whether other members of the hRNase A superfamily may also have similar functions serving as a cognate ligand of cell membrane RTK, especially the non-canonical RNases 9–13 without ribonucleolytic activities [14]. A systematic study is required to uncover the potential ligand-like functions of the other RNases. Apart from EGFR, a cell surface protein termed plexin-B2 was indicated as a functional receptor of hRNase5/ANG in various cell types, including endothelial, cancer, neuronal, and normal and malignant stem and progenitor cells [85]. Blocking hRNase5/ANG interaction with plexin-B2 by competition with a plexin-B2 monoclonal antibody inhibits hRNase5/ANG-dependent physiological and pathological functions [85], suggesting that modulation of hRNase5/ANG activities by targeting plexin-B2 may have therapeutic potential [85]. Collectively, these recent studies highlight the importance of hRNase5/ANG in human disease progression and a critical role overlooked in membrane receptor biology.

3.2. The role of hRNase5/ANG as a serum biomarker for EGFR-targeted therapy

EGFR and its derived network are well known to play an oncogenic role in modulating tumor cell behavior, including pancreatic cancer. The initiation of KRAS-driven pancreatic tumorigenesis requires EGFR and its downstream signaling [158, 159]. The frequency of EGFR overexpression is about 30% to 95% in pancreatic cancer [160], and accumulating evidence shows that EGFR expression level and its activation are positively correlated with liver metastasis and pancreatic metaplasia [161–164]. Together, EGFR is closely associated with tumor initiation, development, and metastasis in pancreatic cancer. In clinical settings, EGFR has been considered an effective rational target for anticancer therapies [165, 166]. Nevertheless, even though an EGFR small-molecule tyrosine kinase inhibitor (TKI) erlotinib was approved to treat patients with pancreatic adenocarcinoma, only marginal improvement in a group of responsive population was observed [167]. An EGFR monoclonal antibody (mAb) cetuximab also did not improve the outcome in pancreatic cancer patients compared with those treated with gemcitabine alone [168]. Thus, pancreatic cancer continues to be one of the most lethal human malignancies without effective therapeutics, and the identification of any predictive biomarkers for a subpopulation of pancreatic cancer patients who may be more likely to respond to erlotinib to increase treatment efficacy is critically needed [169–171].

Elevated serum level of hRNase5/ANG has been shown to correlate with poorer survival in pancreatic cancer patients [172]. Consistently, Wang and Lee et al. [15] found that plasma hRNase5/ANG was significantly increased in pancreatic cancer patients compared with healthy individuals whereas no significant differences were observed between these two groups in levels of conventional EGFR ligands, EGF and transforming growth factor-α. The authors validated a pathological relevance of the hRNase5/ANG-EGFR relationship by a positive correlation between hRNase5/ANG level and EGFR activation in human pancreatic tissue microarrays. Moreover, increased level of murine RNase5 and EGFR activation are associated with tumor development in the Kras^G12D-driven transgenic mouse model of pancreatic cancer [15, 173]. They further demonstrated in vitro and in vivo that hRNase5/ANG through activation EGFR phosphorylation renders pancreatic cancer cells more addicted to the EGFR pathway and more sensitive to EGFR TKI erlotinib treatment. This oncogene addiction effect is further supported by a retrospective study of a cohort of pancreatic cancer patients in which patients with high concentrations of plasma hRNase5/ANG responded well to erlotinib treatment [15]. This implies that hRNase5/ANG^high status has the potential to serve as an effective serum biomarker to predict erlotinib response, which can stratify pancreatic cancer patients who would be more responsive to EGFR-targeted therapy, and improve precision medicine. In addition to pancreatic cancer, it would be worthwhile to further evaluate hRNase5/ANG as a predictive biomarker for EGFR-targeted therapy therapies in patients with other types of hRNase5/ANG^high-EGFR⁺ tumors, and apply to potential serum biomarker-guided treatment opinions (Figure 2).

4. Concluding remarks and future perspective

A general role of secretory RNases is to eliminate cellular self-RNA and/or pathogenic non-self RNA species in the extracellular space to prevent infection by pathogens and autoimmune responses [7, 35, 36]. On the other hand, through interaction with cell surface receptors, secretory RNases undergoing membrane engulfment and endocytosis can be transported into the cytoplasm and nucleus, and regulate both protein-coding and non-protein-coding RNA levels [2, 88]. The ribonucleolytic activity of intracellular RNases in mediating RNA metabolism is precisely controlled by the cytoplasmic endogenous inhibitor, RNH1 [127]. Notably, emerging evidence indicates that the ribonucleolytic activity of RNases is required for some but not all of their biological functions. Certain RNases, including the non-canonical RNases 9–13 lacking intrinsic ribonucleolytic activity, have been found to suppress pathogenic invasion and contribute to innate immunity independently of their ribonucleolytic activities [6]. In this review, we summarized the contribution of hRNase A superfamily to extracellular and intracellular RNA metabolism (Table 1), such as vessel protective role of hRNase1 by exRNA degradation, proliferation-promoting activity of hRNase5/ANG by rRNA transcription in the nucleus, and pro-survival activity by tiRNA production in the cytoplasm.

Here, we also summarized a newly identified ribonucleolytic activity-independent function of hRNase5/ANG as a cognate ligand of cell membrane EGFR RTK. Wang and Lee et al. demonstrated the translational potential of hRNase5/ANG to serve as a serum biomarker to stratify pancreatic cancer patients for effective treatment with EGFR TKI erlotinib [15]. In addition to hRNase5/ANG, hRNase3/ECP has also been extensively studied as a potential serum biomarker for patients with airway inflammation, such as asthma [74, 75], albeit the information regarding its mechanism and clinical usefulness is limited. The treatment of hRNase1 was reported to have potential clinical benefit through the clearance of excess exRNA for blood vessel protection in cardiovascular diseases [25]. It would be of interest to further investigate secretory RNases as non-invasive biomarkers or therapeutic agents in clinical practice.

It is worth noting that a unique subset of 11 out of the 20 cell surface RTK families, termed membrane receptors in the nucleus (MRIN), can be found in the nucleus, including members of the ErbB RTK family, e.g., EGFR and ErbB2 [174, 175]. Nuclear EGFR and ErbB2 are two of the best-studied RTKs in the MRIN field, known to act as a transcriptional co-activator, a protein kinase, and a protein interactor, and participate in different cellular processes, such as cell proliferation, DNA replication, DNA damage repair, and resistance to anticancer therapies [176–178]. A series of studies by Hung and colleagues showed that EGFR and ErbB2 play a role in the nucleus in cooperation with multiple nuclear proteins; for instance, EGFR transduces signal to chromatin by directly interacting with and phosphorylating histone H4 at a tyrosine residue Tyr72, which in turn regulates DNA synthesis and repair [179]. In addition, they found that nuclear ErbB2 enhances rRNA gene transcription through a physical association with rDNA, RNA Pol I, and β-actin, which promotes translation and cell growth [180]. Considering the nuclear roles of hRNase5/ANG in regulating histone modification and rRNA transcription [105, 106, 113], the two originally remoted studies seem to potentially link to each other. For instance, the association between EGFR and its ligand, hRNase5/ANG, may also form a complex in the nucleus and at least in part contribute to the above-mentioned biological functions. More studies toward this area may open a new era in cell biology to understand the functions of these ligand-receptor pairs in the nucleus.

Collectively, these findings highlight a crosstalk between RNases in regulating RNA metabolism, in particular hRNase5/ANG, and its newly-identified ligand-like role in membrane receptor biology through membrane receptor endocytosis and intracellular signal transduction (Figure 1). Furthering our understanding of classical and non-classical roles of the hRNase A superfamily will deepen our knowledge of the fundamentals of RNase biology and open a new avenue in the area of biomarker-guided treatment as well as the potential therapeutic implications using RNases.

List of abbreviations

RNase

ribonuclease

ANG

angiogenin

EDN

eosinophil-derived neurotoxin

ECP

eosinophil cationic protein

exRNA

extracellular RNA

tRNA

transfer RNA

rRNA

ribosomal RNA

rDNA

ribosomal DNA

ABE

ANG binding elements

UCE

upstream core element

tiRNA

tRNA-derived stress-induced small RNA

RNH1

ribonuclease/angiogenin inhibitor 1

RTKs

receptor tyrosine kinases

EGFR

epidermal growth factor receptor

EGF

epidermal growth factor

EMT

epithelial-mesenchymal transition

Pol I

polymerase I

ChIP

chromatin immunoprecipitation

mAb

monoclonal antibody

TKI

tyrosine-kinase inhibitor

MRIN

membrane receptors in the nucleus