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![(A) Sequence alignment of human RNase A superfamily members. Secondary structure elements of RNase A are depicted at the top. Strictly conserved residues are boxed in black and conserved residues, as calculated by a similarity score, are boxed in white. Coloured residues in RNase 1, RNase 2 (EDN), RNase 3 (ECP) and RNase 4 refer to those identified in protein complexes (see Table 2), and ascribed to phosphate/ribose (blue), pyrimidine (green) and purine (red) bases. Cysteine pairings for disulfide bridges are numbered below. The figure was created using the ESPript software [100]. (B) Representation of the superimposed three-dimensional structures of the RNases showing the subsites location and corresponding residue side chains for RNase A, coloured according to the same criteria as above.](profile/Ester-Boix-2/publication/234010789/figure/fig3/AS:667856047964167@1536240698608/A-Sequence-alignment-of-human-RNase-A-superfamily-members-Secondary-structure-elements.png)
(A) Sequence alignment of human RNase A superfamily members. Secondary structure elements of RNase A are depicted at the top. Strictly conserved residues are boxed in black and conserved residues, as calculated by a similarity score, are boxed in white. Coloured residues in RNase 1, RNase 2 (EDN), RNase 3 (ECP) and RNase 4 refer to those identified in protein complexes (see Table 2), and ascribed to phosphate/ribose (blue), pyrimidine (green) and purine (red) bases. Cysteine pairings for disulfide bridges are numbered below. The figure was created using the ESPript software [100]. (B) Representation of the superimposed three-dimensional structures of the RNases showing the subsites location and corresponding residue side chains for RNase A, coloured according to the same criteria as above.
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Vertebrate secreted RNases are small cationic protein endowed with an endoribonuclease activity that belong to the RNase A superfamily and display diverse cytotoxic activities. In an effort to unravel their mechanism of action, we have analysed their nucleotide binding recognition patterns. General shared features with other nucleotide binding prot...
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... secreted RNase superfamily comprises small secreted proteins showing very diverse catalytic and biological properties [1]. A variety of biological functions have been attributed to some family members, ranging from angiogenesis to host defence [2e6]. Mammalian homologues are grouped in eight lineages which are referred as the canonical RNases [5] (Fig. 1A). The family members were first gathered together as pancreatic type RNases, in honour to the family reference prototype, the bovine pancreatic RNase A, conforming the so-called "RNase A superfamily". The catalytic mechanism of RNase A was already proposed in the 60s decade prior to the three-dimensional struc- ture knowledge [7]. RNase ...
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... RNase A, is nowadays one of the best studied enzyme and represents an ideal model to understand the endor- ibonuclease catalytic mechanism and polymeric substrate binding mode [8e11]. The protein active site architecture reveals several phosphate binding subsites adjacent to the main catalytic site which contributes to align the RNA substrate (Fig. 1B). Together with a primary role in RNA digestion in ruminants for RNase A, a variety of non-catalytic biological properties were described for the other family members [2,12,13]. Other RNases were identified in many organs and tissues, and found not only in mammals, but in reptiles, birds, amphibians and fishes, setting the basis to ...
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... better interpret the statistical significance, the number of studied protein structures and complexes are indicated below each amino acid. For each of the best binding amino acid, the ligand interacting atom was plotted against its frequency ( Figure S1). ...
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... with the main phosphate binding site (p1), other secondary subsites facilitate the RNA binding and would contribute to the enzyme endonuclease cleavage pattern [10,46,47]. Residues ascribed to each defined site are labelled in the sequence alignment, where conservation of the main binding sites is visualized (Fig. 1). Variability at the secondary binding sites could explain the RNase homologues distinct catalytic efficiencies and substrate specificities [5,12,48]. Table 3 summarizes the available experimental data to Table 1 List of protein complexes with nucleotide-type ligands from the RNase A superfamily. Residues involved in potential hydrogen ...
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... the catalytic rates [71]. A double pairing in a coplanar orientation is provided by Asn71 (Fig. 3) binding to the N1 and N6 adenine atoms (Table S2) that can contribute as acceptor and donor groups. The strategy matches again the overall preferred binding mode for adenine [28]. Asn71 is conserved by all human RNase homologues except for RNase 5 (Fig. 1). On the other hand, we observe more variability at the other two purine binding residues (Gln69 and Glu111) (Figs. 1 and 4). Gln69 is only found in RNase A and human RNase 1 (Fig. 5A). Cationic residues at equivalent positions in other homo- logues may offer a distinct paring. While Gln OE1 atom can fix the N6 donor group in adenine ...
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... is conserved by all human RNase homologues except for RNase 5 (Fig. 1). On the other hand, we observe more variability at the other two purine binding residues (Gln69 and Glu111) (Figs. 1 and 4). Gln69 is only found in RNase A and human RNase 1 (Fig. 5A). ...
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... as zebrafish RNase, where a Lys residue is oriented towards the B2 pocket. A more conserved substitution is found at Glu111 counterparts (Fig. 4), although Asp substitution was attributed to a lower binding efficiency at B2 for eosinophil RNases 2 and 3. The only exception at position 111 for human family members is observed for RNase 7 ( Fig. 1), where a Lys residue close to the N3 acceptor atom would facilitate the purine binding. Unfortunately, no kinetic studies are available to check the substrate base specificity for some of the recent new family members. Kinetic data highlight that canonical RNases have a clear pref- erence for adenine for the secondary base site (Table ...
Similar publications
Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functi...
Citations
... In addition, in some RNase A family members, other additional subsites have been described at both 5 0 and 3 0 ends, which can help to anchor extended substrates ( Figure 5A). 9,30 To further explore the RNase 2 interaction mode to RNA and identify which protein residues can contribute to each binding site, protein-hairpin complexes were modeled by docking followed by molecular dynamics. Thus, we screened all the paired combinations between RNase 2 and the corresponding anticodon loop of tRNA AspGTC , together with its substitution variants as detailed in previous section (see Figure 4A). ...
... They belong to the human canonical RNases (hcRNases) that are vertebrate exclusive nucleases [8]. It was suggested that this family has a host defense ancestral origin from which it evolved, as antibacterial activity was observed for the members with distant relation to the ancestral RNases [96]. Another suggestion is that these RNases started off as proteins with angiogenesis function [3]. ...
... Interestingly, according to Hornung and co-workers, the release of U > p ends by RNase2 would participate in the activation of TLR8 at the endolysosomal compartment and will contribute to sense the presence of pathogen RNA [52]. To note, we find a good agreement between RNase2 substrate specificity identified in the present cell assay study on tRNAs and the previously reported for synthetic single stranded oligonucleotides [53,54] (see Table 2). However, some differences are evidenced at the miRNAs cleavage and in particular at the B2 site specificity, which does not fully match the reported on synthetic substrates. ...
... This discrepancy is also evident for the other two RNaseA family members described to release specific tRFs [55][56][57][58], i.e., RNase5/Ang and Onconase, an RNase purified from Rana pipiens with antitumoral properties (Table S5). Previous kinetic studies on RNaseA family cleavage preference using single stranded RNA substrates revealed a specificity for pyrimidines at the main B1 site and preference for purines at B2 [53,54]. Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites, respectively. ...
RNase2 is the member of the RNaseA family most abundant in macrophages. Here, we knocked out RNase2 in THP-1 cells and analysed the response to Respiratory Syncytial Virus (RSV). RSV induced RNase2 expression, which significantly enhanced cell survival. Next, by cP-RNAseq sequencing, which amplifies the cyclic-phosphate endonuclease products, we analysed the ncRNA population. Among the ncRNAs accumulated in WT vs KO cells, we found mostly tRNA-derived fragments (tRFs) and second miRNAs. Differential sequence coverage identified tRFs from only few parental tRNAs, revealing a predominant cleavage at anticodon and d -loops at U/C (B1) and A (B2) sites. Selective tRNA cleavage was confirmed in vitro using the recombinant protein. Likewise, only few miRNAs were significantly more abundant in WT vs RNase2-KO cells. Complementarily, by screening of a tRF & tiRNA array, we identified an enriched population associated to RNase2 expression and RSV exposure. The results confirm the protein antiviral action and provide the first evidence of its cleavage selectivity on ncRNAs.
Graphical abstract
... Interestingly, according to Hornung and co-workers, the release of U > p ends by RNase2 would participate in the activation of TLR8 at the endolysosomal compartment and will contribute to sense the presence of pathogen RNA [52]. To note, we find a good agreement between RNase2 substrate specificity identified in the present cell assay study on tRNAs and the previously reported for synthetic single stranded oligonucleotides [53,54] (see Table 2). However, some differences are evidenced at the miRNAs cleavage and in particular at the B2 site specificity, which does not fully match the reported on synthetic substrates. ...
... This discrepancy is also evident for the other two RNaseA family members described to release specific tRFs [55][56][57][58], i.e., RNase5/Ang and Onconase, an RNase purified from Rana pipiens with antitumoral properties (Table S5). Previous kinetic studies on RNaseA family cleavage preference using single stranded RNA substrates revealed a specificity for pyrimidines at the main B1 site and preference for purines at B2 [53,54]. Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites, respectively. ...
... In particular, we observe a shift from cytidine to uridine preference at B1 in the last two variants, which could be attributed to a gradual predominance of RNase 3 traits. Previous kinetic data highlighted the uridine versus cytidine preference of RNase 3 in contrast to RNase 1 [9,46,47]. Further work is currently in progress to analyse the chimera structural peculiarities that explain their differential substrate preferences (Fernández-Millán et al., in preparation). ...
Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs’ ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants’ capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics.
... Interestingly, according to Hornung and co-workers, the release of U>p ends by RNase2 would participate in the activation of TLR8 at the endolysosomal compartment and will contribute to sense the presence of pathogen RNA [48]. To note, we find a good agreement between RNase2 substrate specificity identified in the present cell assay study on tRNAs and the previously reported for synthetic single stranded oligonucleotides [49,50] (see Table 2). However, some differences are evidenced at the miRNAs cleavage and in particular at the B2 site specificity, which does not fully match the reported on synthetic substrates. ...
... RNase5/Ang and Onconase, an RNase purified from Rana pipiens with antitumoral properties (Table S5). Previous kinetic studies on RNaseA family cleavage preference using single stranded RNA substrates revealed a specificity for pyrimidines at the main B1 site and preference for purines at B2 [49,50]. Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites respectively. ...
RNase2, also named the Eosinophil derived Neurotoxin (EDN), is one of the main proteins secreted by the eosinophil secondary granules. RNase2 is also expressed in other leukocyte cells and is the member of the human ribonuclease A family most abundant in macrophages. The protein is endowed with a high ribonucleolytic activity and participates in the host antiviral activity. Although RNase2 displays a broad antiviral activity, it is mostly associated to the targeting of single stranded RNA viruses. To explore RNase2 mechanism of action in antiviral host defence we knocked out RNase2 expression in the THP1 monocyte cell line and characterized the cell response to human Respiratory Syncytial Virus (RSV). We observed that RSV infection induced the RNase2 expression and protein secretion in THP1 macrophage-derived cells, whereas the knockout (KO) of RNase2 resulted in higher RSV burden and reduced cell viability. Next, by means of the cP-RNAseq methodology, which uniquely amplifies the RNA 2'3'cyclic-phosphate-end products released by an endonuclease cleavage, we compared the ncRNA population in native and RNase2-KO cell lines. Among the ncRNAs accumulated in WT versus KO cells, we found mostly tRNA-derived fragments and secondly miRNAs. Analysis of the differential sequence coverage of tRNAs molecules in native and KO cells identified fragments derived from only few parental tRNAs, revealing a predominant cleavage at anticodon loops and secondarily at D-loops. Inspection of cleavage region identified U/C and A, at 5' and 3' sides of cleavage sites respectively (namely RNase B1 and B2 base binding subsites). Likewise, only few selected miRNAs were significantly more abundant in WT versus RNase2-KO cells, with cleavage sites located at the end of stem regions with predominance for pyrimidines at B1 but following an overall less defined nucleotide specificity. Complementarily, by screening of a tRF/tiRNA PCR array we identified an enriched population of tRNA-derived fragments associated to RNase2 expression and RSV infection. The present results confirm the contribution of the protein in macrophage response against virus infection and provide the first evidence of its cleavage selectivity against ncRNA population. A better understanding of the mechanism of action of RNase2 recognition of cellular RNA during the antiviral host defence should pave the basis for the design of novel antiviral drugs.
... The distinct activities of the RNases were also apparent in the degradation of homo-and heterooligomers (Figure S3D). RNase 2 had limited activity on homooligomers and the purine oligomer (GA) 10 G, in line with previous reports (Boix et al., 2013;Sorrentino and Libonati, 1994). In contrast, RNase T2 could degrade U 21 , A 21 , GU 10 G, CU 10 C, UA 10 U, and GA 10 G, as previously shown (Campomenosi et al., 2006;Luhtala and Parker, 2010). ...
... We then examined the B1 and B2 preferences of both RNases. RNase 2 had no activity at B1 purines, consistent with previous work on the RNase A superfamily (Boix et al., 2013;Lu et al., 2018), but exhibited strong activity after B1 uridines. In contrast, for RNase T2, most publications have not reported a strong B1 or B2 preference (Luhtala and Parker, 2010), but our data demonstrated a strong preference for cleavage before a B2 uridine ( Figure 4D). ...
... study, provides information beyond the core sequence tested by Greulich et al. (2019). For example, though we found that RNase 2 could digest U-G heterooligomers, which was also observed by others (Boix et al., 2013), the same group (Boix and colleagues) has recently reported that RNase 2 is capable of cleaving a U-A dinucleotide but not U-G (Prats-Ejarque et al., 2019). These findings demonstrate important differences in dinucleotide and ORN degradation. ...
Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2−/− or RNASET2−/− but not RNASE2−/−RNASET2−/− cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.
... The enzyme cleaves the 3′5′ phosphodiester bonds with specificity for pyrimidines at the main anchoring site (B1) and preference for purines at the secondary site (B2) (Richards and Wyckoff, 1971;Raines, 1998). In a previous work, we analyzed the enzyme residues that were reported to participate in the specific binding of adenine (A) and guanine (G) bases at the B2 site among the RNase A superfamily members (Boix et al., 2013). A high evolutionary conservation was observed for B1, whereas a significant variability was visualized for the secondary base selectivity. ...
... A high evolutionary conservation was observed for B1, whereas a significant variability was visualized for the secondary base selectivity. Interestingly, the observed structural differences at the secondary base site correlate with their substrate specificity and catalytic efficiency (Tarragona-Fiol et al., 1993;Sorrentino, 1998;Boix et al., 2013). Likewise, the analysis of the protein conformational changes induced upon nucleotide binding by NMR and molecular dynamics highlighted an evolutionary trend in base interaction selectivity (Gagné and Doucet, 2013;Narayanan et al., 2017;Narayanan et al., 2018a). ...
... On the other hand, when we analyze the kinetic characterization of other family members available in the literature, we can infer a shift at the substrate secondary base predilection, from lower to higher order vertebrates, from guanine to adenine (Boix et al., 2013). Basically, the characterized fish, amphibian and reptile RNases show a marked preference for G at B2 site (Hsu et al., 2003;Ardelt et al., 2008), while mammalian prefer A (Richards and Wyckoff, 1971;Zhao et al., 1998;Prats-Ejarque et al., 2016). ...
There is a growing interest in the pharmaceutical industry to design novel tailored drugs for RNA targeting. The vertebrate-specific RNase A superfamily is nowadays one of the best characterized family of enzymes and comprises proteins involved in host defense with specific cytotoxic and immune-modulatory properties. We observe within the family a structural variability at the substrate-binding site associated to a diversification of biological properties. In this work, we have analyzed the enzyme specificity at the secondary base binding site. Towards this end, we have performed a kinetic characterization of the canonical RNase types together with a molecular dynamic simulation of selected representative family members. The RNases’ catalytic activity and binding interactions have been compared using UpA, UpG and UpI dinucleotides. Our results highlight an evolutionary trend from lower to higher order vertebrates towards an enhanced discrimination power of selectivity for adenine respect to guanine at the secondary base binding site (B2). Interestingly, the shift from guanine to adenine preference is achieved in all the studied family members by equivalent residues through distinct interaction modes. We can identify specific polar and charged side chains that selectively interact with donor or acceptor purine groups. Overall, we observe selective bidentate polar and electrostatic interactions: Asn to N1/N6 and N6/N7 adenine groups in mammals versus Glu/Asp and Arg to N1/N2, N1/O6 and O6/N7 guanine groups in non-mammals. In addition, kinetic and molecular dynamics comparative results on UpG versus UpI emphasize the main contribution of Glu/Asp interactions to N1/N2 group for guanine selectivity in lower order vertebrates. A close inspection at the B2 binding pocket also highlights the principal contribution of the protein ß6 and L4 loop regions. Significant differences in the orientation and extension of the L4 loop could explain how the same residues can participate in alternative binding modes. The analysis suggests that within the RNase A superfamily an evolution pressure has taken place at the B2 secondary binding site to provide novel substrate-recognition patterns. We are confident that a better knowledge of the enzymes’ nucleotide recognition pattern would contribute to identify their physiological substrate and eventually design applied therapies to modulate their biological functions.
... The past studies have suggested substrate preferences at the B 1 pyrimidine binding site for some of the human RNases: hRNase1 showed a preference for cytosine (C) over uridine (U), while hRNases 2, 3, 4 and 6 showed a preference for U over C [6,9]. In contrast, the B 2 purine binding site was shown to prefer adenosine (A) in these hRNases [6,9,28]. Substrate preferences for the other hRNases remains unclear [29]. ...
... More specifically, favorable interactions with the catalytic His119 (bRNaseA numbering) were observed for both the substrates with all RNases. Most RNases also showed favorable interactions with Thr45 (bRNaseA numbering) in the β1 strand, a residue shown to bind to the pyrimidine nucleotide [28]. bRNaseA residue Lys7 further showed favorable interactions with both the substrates and Lys66 (from loop L4) with substrate ACAC. ...
... Similar interactions were observed for hRNase1, which showed additional favorable interactions by Asn71 and Lys7 with AUAU. In case of hRNase2 residues Arg133 and Trp8 show additional interactions in case of substrate AUAU; the latter residue is previously reported to interact with the ribose group [28]. In contrast to hRNase2 where a significant number of interactions are present in the region 39-43, hRNase3 showed significantly fewer interactions with the two substrates. ...
Human genome contains a group of more than a dozen similar genes with diverse biological functions including antiviral, antibacterial and angiogenesis activities. The characterized gene products of this group show significant sequence similarity and a common structural fold associated with binding and cleavage of ribonucleic acid (RNA) substrates. Therefore, these proteins have been categorized as members of human pancreatic-type ribonucleases (hRNases). hRNases differ in cell/tissue localization and display distinct substrate binding preferences and a wide range of ribonucleolytic catalytic efficiencies. Limited information is available about structural and dynamical properties that influence this diversity among these homologous RNases. Here, we use computer simulations to characterize substrate interactions, electrostatics and dynamical properties of hRNases 1-7 associated with binding to two nucleotide substrates (ACAC and AUAU). Results indicate that even with complete conservation of active-site catalytic triad associated with ribonucleolytic activity, these enzymes show significant differences in substrate interactions. Detailed characterization suggests that in addition to binding site electrostatic and van der Waals interactions, dynamics of distal regions may also play a role in binding. Another key insight is that a small difference in temperature of 300 K (used in experimental studies) and 310 K (physiological temperature) shows significant changes in enzyme-substrate interactions.
... Another possible reason for the seemingly different degradation rate is that the ndufa and rl17 amplicons are longer than the cyp1a amplicon. Moreover, there are observations that RNases may prefer certain sequences over others when they degrade RNA-even the most abundant form, RNase A (Boix et al. 2013;Wang et al. 2008). To avoid the possible biasing effect of differences created by RNA degradation, it is best to use samples with as intact RNA as possible, and with similar RINs, in transcriptomic analyses. ...
As transcriptomic studies are becoming more and more common, it is important to ensure that the RNA used in the analyses is of good quality. The RNA integrity may be compromised by storage temperature or freeze-thaw cycles, but these have not been well studied in poikilothermic fishes. This work studied the effects of tissue storage time and temperature, and freeze-thaw cycles of tissue and extracted RNA on RNA integrity in brown trout (Salmo trutta L.) liver. The storage time and temperature had an effect on RNA integrity, but RNA suitable for quantitative reverse transcription PCR (RT-qPCR) (RIN > 7) was still obtained from samples preserved at − 20 °C for 6 months. Freeze-thaw cycles of tissue or RNA did not compromise the integrity of RNA. RNA degradation had an effect on RT-qPCR results, and the effect depended on gene. The RT-qPCR analysis of historical samples from a bleached kraft pulp mill effluent exposure in 1984 revealed no significant cyp1a induction. Recommendations are given for the preservation and handling procedures of samples designated for transcriptomic analyses.
