key: cord-0269001-nvqnmhun
authors: Lu, Lu; Li, Jiarui; Wei, Ranlei; Guidi, Irene; Cozzuto, Luca; Ponomarenko, Julia; Prats-Ejarque, Guillem; Boix, Ester
title: Selective cleavage of ncRNA and antiviral activity by human RNase2/EDN in a macrophage infection model
date: 2021-08-30
journal: bioRxiv
DOI: 10.1101/2021.08.30.458223
sha: 21e722bfef32cf2764e3af0ba7587003d1f56178
doc_id: 269001
cord_uid: nvqnmhun

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.

Human RNase2 is a secretory protein expressed in leukocytes with a reported antiviral activity against single stranded RNA viruses [1, 2] . RNase2 is one of the main components of the eosinophil secondary granule matrix. The protein, upon its discovery, was named the Eosinophil Derived Neurotoxin (EDN), due to its ability to induce the Gordon phenomenon when injected into Guinea pigs [3] [4] [5] [6] [7] [8] .Apart from eosinophils, RNase2 is also expressed in other leukocyte cell types, such as neutrophils and monocytes, together with epithelial cells, liver and spleen [8] [9] [10] . The protein belongs to the ribonuclease A superfamily, a family of secretory RNases that participate in the host response and combine a direct action against a wide range of pathogens with diverse immunomodulation properties [2, 11] .

RNase2 stands out for its high catalytic activity against single stranded RNA and its efficiency against several viral types, such as rhinoviruses, adenoviruses and retroviruses, including HIV [12] [13] [14] . Recently, presence of eosinophils and their associated RNases has been correlated to the prognosis of COVID patients [15] [16] [17] . On the contrary, no action is reported against the tested bacterial species [12, 18, 19] . In particular, among respiratory viruses, which activate eosinophil recruitment and degranulation, the human Respiratory Syncytial Virus (RSV), which is the principal cause of death in infants [20] , is probably the most studied model for RNase2 antiviral action.

Indeed, RNase2 levels have predictive value for the development of recurrent wheezing post-RSV bronchiolitis [21] . RNase2 was proposed to have a role in the host response against the single stranded RNA virus [22] and early studies observed that RNase2 can directly target the RSV virion [12] . Interestingly, the protein ribonucleolytic activity is required to remove the RSV genome, but some structural specificity for RNase2 is mandatory, as other family homologues endowed with a higher catalytic activity are devoid of antiviral activity [23] .

In our previous work using a macrophage infection model, we observed that RNase2 is the most abundantly expressed RNaseA superfamily member in the monocytic THP1 cell line [24] . To broaden the knowledge of the immunomodulatory role and potential targeting of cellular RNA population by RNase2 in human macrophages, we built an RNase2-knockout THP1 monocyte cell line using CRIPSR/Cas9 (clustered regularly interspaced short palindromic repeats) gene editing tool. Transcriptome of the RNase2 knockout with the unedited THP1-derived macrophage cells revealed that the top differently expressed pathways are associated to antiviral host defence (Lu et al., in preparation) . Here, we explored the protein antiviral action by characterization of both THP1 native and RNase2-KO cell lines infected with RSV. The comparative infection study indicated that the knockout of RNase2 in THP1-derived macrophages resulted in a heavier RSV titre and reduced cell survival. Next, we analysed the total non-coding RNA (ncRNA) population by amplification of 2'3'-cyclic phosphate ends using the cp-RNAseq methodology and by screening a library array of tRNA-derived fragments.

Results proved that RNase2 expression in macrophage correlates to a selective ncRNA cleavage pattern. upregulated in a time-dependent manner upon RSV infection. The significant RNase2 gene levels upregulation could be detected as early as at 4h poi, with a 7-fold increase at 72 h poi. Furthermore, to determine whether the induction of RNase2 mRNA levels correlated with an increase in protein expression, ELISA and WB were conducted to detect intracellular and secreted RNase2 protein of THP1-derived macrophages. At indicated poi time, culture medium and whole-cell extracts of macrophages infected with RSV were collected for ELISA and WB, respectively. As indicated in Fig 1B, the secreted protein levels of RNase2 was detected in human macrophages stimulated with RSV and was enhanced in response to RSV in a time-dependent manner, while no significant change of secreted RNase2 was detected in control macrophages. However, the maximum concentration of secreted RNase2 protein in macrophage culture was detected at 48h poi, with a slight decrease at 72h poi. Likewise, a similar profile was obtained by WB ( Fig   1C) . Taken together, our results suggest that RSV induces both RNase2 protein expression and secretion in human THP1 induced macrophages. concentration of the cells was controlled as 10 6 /mL, secreted RNase2 in culture supernatant was measured by ELISA and normalized with alive cell number detected by MTT assay; C) The intracellular RNase2 protein in macrophage was detected by WB; "+" and "-" indicate with or without RSV infection, respectively; * and ** indicate the significance of p<0.05 and p<0.01, respectively.

To further characterize the protein mechanism of action, we knocked out RNase2 gene by the CRISPR/Cas9 methodology. RNase2 gene was successfully knocked out in 2 out of 32 single cells derived THP1 cell lines, named as KO18 and KO28. Both of KO18 and KO28 express a 15aa in length peptide comparing the wild type THP1, which encodes the full length 134aa protein (Fig 2A) .

After confirmation of successful gene deletion by Sanger sequencing, we ensured that the expression of functional RNase2 has effectively been abolished.

According to Western Blot (WB) assay, we can barely detect RNase2 in the KO18 cell lysate sample compared to the control sample and a total absence of signal is achieved in KO28 sample ( Fig 2B) . Moreover, the total absence of secreted RNase2 by THP1 cells was confirmed by ELISA assay in culture supernatant for KO28 line (Fig 2C) . Last, we conducted the ribonuclease activity staining assay to evaluate the ribonucleolytic activity of the samples from cell lysates and culture supernatants. According to the activity staining electrophoresis, two main activity bands can be visualized in wild-type macrophage lysate sample, with molecular weight sizes around 15 and 20kDa, as previously reported [2] . Compared to the WT control, both activity bands were missing in the RNase2 knockout cell lines ( Fig 2D) . In addition, as recent studies suggested that CRISPR/Cas9 frequently induces unwanted off-target mutations, we evaluated the offtarget effects on these transduced monocytes. Here, we examined the top 4 potential offtarget sites for the sgRNA1 (Table S1 ) and did not detect off-target mutation in the T7EI assay ( Fig S1) . Overall, we confirmed that the RNase2 gene has been both structurally and functionally knocked out. The KO28 THP1 cell line, which achieved full RNase2knockout, was selected for all the downstream experiments. Scheme of the mutation of RNase2 caused by sgRNA1, the sequence was validated by Sanger sequencing; replacement is indicated: red labelled sequence in wild type was replaced by the green labelled sequence, resulting in the coding frame change and stop codon insertion; B)

Western blot assay was applied to detect RNase2 protein; C) The secreted RNase2 in supernatant was measured by ELISA, the RPMI+10%FBS complete culture medium was used as a negative control, the supernatant was concentrated 50x; D) Ribonuclease activity staining assay was used to confirm the removal of catalytic function. Cells were collected and resuspended in water and sonicated, cell lysates were loaded in each well at the indicated quantity.

Next, we investigated whether RNase2 expression within macrophages contributes to the cell antiviral activity. First, THP1 cells (WT and RNase2 KO) were induced into macrophages as described above. Macrophages were then exposed to RSV at MOI=1 to investigate the kinetics of infection by monitoring both intracellular and extracellular RSV amplicon using probe RT-qPCR. Intracellularly, RSV increased at the beginning of the infection (24h) but decreased at longer infection periods (48-72h) , with a slow increase (0h-4h) followed by an exponential increase (4h-24h) and reaching a maximum at 24h (Fig 3A) . At 24h, RNase2 KO macrophages had significantly more intracellular RSV than WT macrophages. The extracellular RSV titre was also determined ( Fig 3B) . We observed that RSV increased in KO macrophage cell cultures until 48h and then stabilized at 48-72h. While in WT macrophages, RSV profile showed an increase that reached a peak at 48h and was followed by a decrease, significantly higher RSV levels were detected in KO macrophage cultures at 24h-72h. Moreover, we monitored cell death during RSV infection using MTT assay and our results confirmed that RSV infection increased cell death in either WT or KO macrophages. However, significant differences were detected, where KO macrophage cell death upon RSV infection was higher than in WT macrophage cells ( Fig 3C) . Altogether, we concluded that RNase2 KO macrophages burdening and cell death is significantly higher for RSV infection in comparison to WT macrophages. The present results confirm the direct involvement of the macrophage endogenous RNase2 in the cell antiviral activity. and calculated as the median tissue culture infectious dose (TCID50). C) Macrophage cell viability was monitored by registering the absorbance at 570 nm using the MTT assay; the star refers to the significance between KO+ and WT+, "+" and "-" indicate with or without RSV infection, respectively. Significance is indicated (* p < 0.05).

Following, we aimed to identify the potential changes in small RNA population associated to the expression of RNase2 by comparison of WT and KO cell lines.

Toward this end, we applied the cP-RNA-seq methodology that is able to exclusively amplify and sequence RNAs containing a 2'3′-cyclic phosphate terminus, product of an enzymatic endonuclease activity [26] . Total small RNA from WT and KO-THP1 cells was purified and the 20 to 100 nt fraction was extracted and processed as described in the methodology section. Sequence quality control indicated that for all samples more than 95% sequences achieved an average value > 30M Reads. Following RNAseq amplification, the sequence libraries were inspected by differential enrichment analysis. Principal Component Analysis (PCA) confirmed good clustering within WT and KO and appropriate discriminant power between the two groups ( Fig S2) .

Results revealed that RNase2 expression in THP1 cells is mostly associated to Table S2 and Fig   S4) and miRNAs (Table S3 and Fig S5) .

Analysis of differential sequence coverage between WT and KO samples (Table   S2 ) indicated that the preferential cleavage sites for RNase2 on tRNAs were CA and UA (Fig 4) . Fig 5A illustrates the base preferences for B1 and B2 deduced from the differential sequence coverage analysis of bam files. Results highlighted a selectivity for pyrimidines at B1 and preference for purines at B2, with a U/C ≥ 1 at the 5' side of the cleavage site, and a pronounced predilection for A at the 3' side.

We also explored whether the cleavage preference was dependent on the RNA adopted secondary. We can see how RNase2 preferentially cuts at tRNA loops, mostly at the anticodon loop and secondarily at the D-loop, as well as stem regions near the anticodon loop ( Fig 5B) . Table S3 ). Overall, we can infer for RNase2 a slight preference for U and C at B1 site, followed by G, and no defined consensus for B2. In parallel, we decided to explore the changes in tRNA-derived fragments population associated with RNase2 expression in THP1 cells by screening a tRNAderived fragment library. The nrStar TM Human tRF&tiRNA PCR array includes a total of 185 regulatory tRNA-derived fragments, of which 110 are taken from the tRF and tiRNA databases and 84 have been recently reported in the bibliography.

Using the nrStar TM Human tRF&tiRNA library, we found that out of a total of 185 tRNA fragments, only 5 were significantly decreased in RNase2 KO macrophage in comparison to the WT control group in uninfected samples and 22 under RSV infection: 6 tiRNAs, 4 itRFs, 9 tRF-5, 4 tRF-3, 1 tRF-1 (see Table 1 ). Overall, the most significant changes associated to RNase2 in both infected and non-infected cell cultures (p < 0.01) are observed in release products from few parental tRNAs.

Upon inspection of sequence of the putative cleavage sites we observed most sites at or near any of the tRNA loops, with predominance of anticodon loop ( 50%), followed by D-loop (Table S4) . Moreover, we observed that the preferred target sequences in WT samples were significantly enriched with U at the 5' position of cleavage site, although not enough data is available for a proper statistical analysis. On the other hand, when analysing all the conditions, with and without infection, we also observed overall a significant preference for U at B1 at loop sequences. Moreover, the most significant tRNA fragments associated to RNase2 presence showed a U/C cleavage target for B1 at the anticodon loop (Table S4) . On the contrary, the main base at B1 was G when the cleavage site was located at a stem region. As for the base located at the 3' side of the cleavage target, we did not observe a clear distinct preference, with only a slight tendency for U, followed by A.

Overall, most of the parental tRNAs precursors identified by the array screening matched the identified by the Cp-RNAseq assay (> 70%), although some differences were observed in the identity of the accumulated fragments and their relative fold change. To note, few of the top listed precursors by the library screening (Lys CTT and Met CAT ) were also present in the amplified sequences by the Cp-RNAseq, but with a lower abundance (Additional file 2). On the contrary, few of the top listed fragments spotted by the later methodology were absent from the commercial library array. Notwithstanding, we must bear in mind that the results obtained from the tRNA array screening are determined by the intrinsic library composition. The tiRNA&tRFs list is based on the previously reported tRNA fragments, which have been identified mainly by the characterization of other RNases [27] . On the contrary, by Cp-RNAseq method only the RNA by-products of an endonuclease enzymatic cleavage are amplified.

The present study is the first specific characterization of the catalytic activity of RNase2 on ncRNAs. Therefore, our data is the first report of the specific release of tRNA fragments associated to RNase2 expression.

Expression of human RNase2 is widely distributed in diverse body tissues such as liver and spleen together with leukocyte cells [2] . Among the blood cell types, RNase2

is particularly abundant in monocytes [11] , which are key contributors to host defence against pathogens. A number of host defence-associated activities have been proposed for RNase2, mostly associated to the targeting of single stranded RNA virus infection [1] . In particular, the protein has been reported to reduce the infectivity of the human respiratory syncytial virus (RSV) in cell cultures [2, 12, 28] .

Here, we studied RNase2 expression in THP1-derived macrophage upon RSV infection. Previous work indicated that RNase2 is the most abundant RNaseA family member expressed in this human monocytic cell line [24] (https://www.proteinatlas.org/).

Viruses can manipulate cell biology to utilize macrophages as vessels for dissemination, long term persistence within tissues and virus replication [29] . In our working model, we increase of the secreted protein is detected by ELISA after 24h of infection, reaching a peak at 48h (Fig 1) . It was previously reported that human monocyte-derived macrophages challenged with a combination of LPS and TNF- produced RNase2 in a time-dependent manner [30] . However, we did not find any significant change of transcriptional expression of RNase2 upon Mycobacteria aurum infection [24] .

Discrepancy of expression induction is also found for RNase2 secretion by eosinophils upon distinct bacterial infections. For example, Clostridium difficile and Staphylococcus aureus infection caused release of RNase2, while Bifidobacteria, Hemophilus, and

Prevotella species infection did not [2] . In agreement, our previous work on THP1 derived macrophages infected by mycobacteria also discarded any induction of RNase2 expression [24] . In contrast, using the same infection model and experimental protocol, we observe here how RSV infection significantly activate both the expression and protein secretion of RNase2 in THP1 macrophage derived cells (Fig 1) . The present study corroborates previous reports on RNase2 involvement in host response to viral infections [1, 2] . In particular, our work highlights the protein role in macrophage cells challenged by RSV infection. More importantly, we observe how the knockout of RNase2 in macrophage derived cells results in heavier RSV infection and cell death (Fig 2) .

in the protein antiviral activity [12] and the evidence that RSV infection alters the cellular RNA population, including the specific release of regulatory tRFs [31] [32] [33] , we decided here to analyse the contribution of macrophage endogenous RNase2 on cellular small

RNAs. Increasing data demonstrates that small noncoding RNAs (ncRNAs) play important roles in regulating antiviral innate immune responses [34] [35] [36] . In particular, ncRNAs derived from tRNAs, such as tRNA halves (tiRNAs) and tRNA-derived fragments (tRFs), have been identified and proven as functional regulatory molecules [37] . RSV infection together with other cellular stress processes can regulate the population of tiRNAs and tRFs. For example, it has been demonstrated that RSV infection and hepatitis viral infection can induce the production of tRFs and tRNA-halves, and their release has been related to RNase5 activity [32, 38] . RNase5, also called Angiogenin (Ang) due to its angiogenic properties, is one of the most well-known ribonucleases that are responsible for endonucleolytic cleavage of tRNA [39] [40] [41] . Surprisingly, the release of a specific tRF, tRF5-Glu CTC , which targets and suppresses the apolipoprotein E receptor 2 (APOER2), can also promote the RSV replication [32, 33] . In the present study, although the tRF5-Glu CTC was not significantly changed upon RSV infection alone, we observed a significant reduction in the RNase2 knockout cell line challenged with RSV infection (Table 1) . Besides, tiRNA-5034-Val CAC -3, the 5'half originated from Val CAC , is identified both in the present work (Table S4 ) and the previous mentioned study associated to RSV infection [32] . To note, we found that RSV infection on WT cell line induced the production of 4 tRFs in macrophages, in agreement to the previous study that indicated that RSV infection induced the release of tRFs in A549 epithelial cells [32] , although most of the tRNA products differ, which may be attributed to the specific basal composition of each source cell line [42] . Moreover, tRFs production is also observed to be dependent on the specific viral infection type; for example, human metapneumovirus did not induce tRFs [32] . Interestingly, release of tRNA products is mostly associated to an antiviral defence mechanism [43] . For example, the tRF3 from tRNALys TTT , which stands out among the identified tRFs by our library array screening ( Tables S2 and S3). In addition, a higher frequency of cleavage takes place at tRNA single stranded regions, with predilection for the anticodon loop, followed by the D arm (Fig 5) .

Exhaustive analysis of differential sequence coverage in WT and RNase2-KO THP1 cells suggests that RNase2 preferentially targets at UA and CA sequences at tRNA loops.

Recently, Bartok and co-workers reported a RNase2 selectivity for U at B1 site in synthetic RNAs [47] . 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. This discrepancy is also evident for the other two RNaseA family members described to release specific tRFs [51] [52] [53] [54] , 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 [49, 50] . Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites respectively. Interestingly, RNase 2 shows a marked preference for U at B1 and A at B2 on synthetic oligonucleotides [49, 55] , which mostly corresponds to the observed preference identified by Cp-RNAseq for tRNA in this study ( Fig 5) . Nevertheless, our analysis on tRNA cleavage sites would suggest a U/C ratio for B1 a bit lower than the estimated for some synthetic substrates ( Table 2) . The present study on cellular ncRNA also highlights the key role of the RNA 3D structuration. Overall, our data reveal a cleavage preference by RNase2 at single stranded sequences and secondarily at stem adjacent to loop regions. Besides, we also observe that location of the targeted site also influences the cleavage base preference.

Interestingly, previous kinetic and structural studies on RNaseA highlighted the unusual enhancement of the protein affinity to a dinucleotide probe by addition of a phosphate linkage insert that can adopt a contorted conformation close to the cleavable 3'5' phosphodiester bond [56] [57] [58] . Likewise, previous work on Onconase nucleotide base selectivity also encountered significant differences in vitro among di and tetranucleotides [59] and tRNA [53] .

In addition, the cleavage of tRNAs by RNases would probably be modulated by the presence of regulatory proteins within the cell [60] . For example, Onconase selectivity for specific tRNAs was attributed to the presence in vivo of RNA binding proteins that might protect RNA regions from RNase activity [52, 61] . In this context, we should consider the formation of regulatory complexes, such as the RISC formation, the binding of Argonaute (AGO) subunits [62] or interactions with the RNHI. On its turn, the released tRNA products would regulate the formation of cellular complexes. For example, tRF3

interaction with AGO2 mediates the cleavage of complementary Priming Binding Sequences (PBS) in retroviruses and thereby can avoid the replication of endogenous virus elements [63, 64] .

Among the ncRNA population mostly altered upon RNase2 knockout, we find, together with tRFs, miRNAs. Interestingly, the identified miRNAs subproducts come frequently from the same group of parental miRNAs. Inspection on information related to these miRNAs entities reveals a predominance of miRNAs associated to cancer and neurological disorders, although few are also related to virus replication. However, caution should be taken when extracting conclusions, as miRNA databases are strongly biased from a predominance of previous clinical studies. We must also bear in mind that our working model, THP1, is a leukaemia cell line. Accumulation of miRNAs can be toxic to the cells, due to their potential interference with the translation of essential proteins. Raines and co-workers correlated miRNA release by Ang with potential toxicity to cells [41] . In any case, in our working model we do not observe any change on the viability of both WT and KO cell lines.

On the other hand, we should be aware not to over interpret our results that can be also somehow biased by the applied methodologies. The RNAseq methodology might lead to underrepresentation of some fragments, due to their relative size, short half-life or presence of posttranscriptional modifications. Therefore, caution must be taken when conclusions are drawn from the analysis of tRNA cleavage product population. Another important source of variability comes from the presence of posttranscriptional modifications, which can influence both the RNase selectivity and the product amplification step [26, 65] . Unfortunately, the current ncRNA databases are still incomplete and lack full information on the precise post-transcriptional modifications that take place in vivo and might intervene in the RNases recognition target.

More importantly, the array screening technique is prone to be biased by the selection criteria used to build the tiRNA&tRF array; a library composed on previously available experimental data, i.e. products by RNases such as Dicer, Angiogenin, RNaseP or RNaseZ. This might explain some of the differences observed in the identified fragments when comparing the screening of the tRFs array and the amplified sequences by the Cp-RNAseq methodology, which only amplifies the products of an endonuclease cleavage.

On the other hand, we should also take into account the protein traffic and accessibility to the distinct subcellular compartments in the assayed experimental conditions. For example, in contrast to RNase5, mostly located at the nucleus, RNase2 is associated to the endolysosomal compartment [47, 48, 62] . In addition, cleavage of cellular mature tRNAs might occur during stress conditions, when leakage of the RNases to the cytosol is favoured. We should bear in mind that the cell cytosolic RNA is in normal conditions protected by the presence of the RNHI, which would lose its functional conformation under stress conditions due to oxidation of surface exposed Cys residues [66] . Accordingly, in the case of RNase5/Ang, it has been described that the selective tRNA cleavage only takes place in oxidative conditions [51, 67, 68] . This might also explain the much higher number of tRNA fragments obtained in the present study in the RSV infected versus non-infected cells (Tables 1 and S4) .

Interestingly, under certain cell conditions, such as nutrition deficiency or oxidative stress, RNase5/Ang is reported to stimulate the formation of cytoplasmic stress granules and produce tRNA-derived stress-induced RNAs (tiRNAs) [69] [70] [71] . The released tiRNAs functionally enhance damage repair and cellular survival through suppressing the formation of the translation initiation factor complex or associating with the translational silencer [40, 63] . Ivanov and co-workers recently characterized the structural determinants that guide release of tiRNA population during stress conditions [54, 72] . Accessibility of tRNAs will also depend on their potential entrapment in Tbox riboswitch or RNA granules, which are abundant in starvation situation and have an unequal propensity to protect tRNA from cleavage. Besides, proteomic analysis revealed the presence of RNHI within stress granules [73] , an inhibitor protein that can complex to RNase5/Ang and other regulatory proteins to control cell translation [62] .

Another important source of variability comes from the assayed cell type.

Although it is widely accepted that the levels of parental tRNAs differ significantly upon cell conditions and tissues [27] and a very unequal tissue distribution is observed for the more than 500 tRNAs listed in our genome, little is still known of their relative expression rates.

Despite the inherent limitations of the present study, our results confirm that RNase2 targets ncRNA and releases specific miRNAs and tRFs. Particular interest should be drawn to the new identified tRNA fragments associated to RNase2 and absent from the commercial library array, which represent potential new regulatory elements for future studies. A growing evidence emphasizes the key role of tRNA halves and tRFs in regulating cellular functions [43, [74] [75] [76] . Deciphering the contribution of the distinct RNases to shape the cell ncRNA population should be key to analyse the cell response to adapt to distinct stress conditions, such as viral infection.

This is the first report of RNase2 selective targeting of ncRNA. We have 

For long term consideration, we used the two-plasmid system to run the CRISPR gene editing experiments instead of using all-in-one CRISPR system. Thus, we Table S6 .

sgRNA design and clone into pLentiGuide (pLenti239R) vector N20NGG motifs in the RNase2 locus were scanned, and candidate sgRNAs that fit the rules for U6 Pol III transcription and the PAM recognition domain of Streptococcus pyogenes Cas9 were identified. From CRISPOR (http://crispor.tefor.net/) and CRISPR-ERA, the top 2 sgRNA were selected for knockout RNase2. Using the same procedure, potential OT sites were also predicted. The sequences are listed in Table   S1 . Oligonucleotides were annealed and cloned into BbsI-digested pLenti-239R. The resulting plasmids containing sgRNAs were further confirmed by Sanger sequencing.

HEK293T cell line was kindly provided by Raquel Pequerul Pavón (UAB). medium with 10% heat-inactivated FBS at 37°C in humidified 5% CO2 conditions. The culture media was replaced every 3 days.

To make the cell reach 90% confluence for transfection, 7.5×10 6 of HEK293T cells were seeded in T75 culture flask with 15ml DMEM + 10% FBS complete medium one day before transfection. The lentiviral plasmids were transfected into HEK293T cells using calcium phosphate protocol [77] . 

Briefly, the genomic DNA of THP1 cells was extracted using GenJET Genomic DNA purification kit (ThermoFisher, K0721) and was further used to amplify RNase2 using NZYTaq II 2×Green master mix (Nzytech, MB358). Genomic DNA was subjected to PCR (BioRad) using primers listed in 

A lentiviral system was selected to deliver CRISPR components into the THP1 monocytic cell line. Cas9 and sgRNAs lentiviral particles were produced in HEK293T cells by Calcium phosphate precipitation method as previously reported [77] [78] [79] . THP1 cells were then transduced with the concentrated lentiviral particles with 10 µg/mL of polybrene, the overall transduction efficiency is about 7% (Fig S6) . We designed 2 single guide RNAs (sgRNAs) ( Table S1 ) targeting the RNase2 locus to generate double strand breaks (DSBs) (Fig S7A) . T7EI assay was employed to select the more active guide RNA, achieving the knockout efficiencies of about 40% (Fig S7B and S7C) . 

For the western blot assays, 5 ×10 5 cells with or without transduction and their supernatants were harvested with RIPA buffer and the protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225).

Equal amounts of protein (50 µg) for each sample were loaded and separated by 15% SDS-PAGE, transferred to polyvinylidene difluoride membranes. Then the membrane was blocked with 5% non-fat milk in TBST for 1 h at room temperature, and incubated with rabbit source anti-RNase2 primary antibody (abcam, ab103428) overnight at 4°C.

After washing, the membranes were treated with horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (Sigma Aldrich, 12-348) for 1 h at room temperature (RT). Finally, the membranes were exposed to an enhanced chemiluminescent detection system (Supersignal West Pico Chemiluminescent Substrate, ThermoFisher Scientific, 32209). As a control, GAPDH was detected with chicken anti-GAPDH antibodies (Abcam, ab9483) and goat anti-chicken secondary antibody (Abcam, ab6877).

Secretory RNase2 in cell culture was detected by using human RNASE2 ELISA Kit (MyBioSource, MBS773233). Beforehand, the supernatant of the culture was concentrated 50 times using 15kDa cut-off centrifugal filter unit (Amicon, C7715).

Following, the standard and the concentrated culture supernatants were loaded to wells pre-coated with anti-RNase2 antibody, then the HRP-conjugated reagent was added.

After incubation and washing for the removal of unbound enzyme, the substrate was added to develop the colourful reaction. The colour depth or light was positively correlated with the concentration of RNase2. Triplicates were performed for all assays.

Zymograms were performed as previously described [81] . 

Human respiratory syncytial virus (RSV, ATCC, VR-1540) stock was ordered from ATCC. Hela cells were used to produce RSV under biosafety level II conditions [82] . to remove the cell debris. The virus suspension without cell debris were either frozen immediately and stored at -80°C as seeding stock and concentrated before use with Ultra15 Amicon 100 kDa cut-off filters. The produced viruses were titrated using the median tissue culture infectious dose (TCID50) method in HEK293T cells [83] .

Before RSV infection, THP1 cells were induced to macrophage by 50 nM of PMA treatment for 48h as previously described [24] . Cells were washed three time with pre-warmed PBS and replaced with fresh RPMI+10%FBS medium for 24h incubation.

After that, macrophages were washed and incubated with RSV, mixing at every 15 min for the first 2h. All virus treatment tests were performed using RSV at a MOI of 1 TCID50/cell. Kit (Bio-Rad, 170-8891). The synthesis was performed using random hexamers, starting with 1 g of total cell RNA. The RT-qPCR was performed using ddPCR TM Supermix for Probes (Bio-Rad, 1863024). Samples with a cycle threshold value of more than 40 were recorded as negative. A standard curve was prepared using serially diluted RNA extracts from a known quantity and used to quantify RSV as TCID50/mL. In parallel with the RSV probe assays, an endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control was used for relative quantification of the intracellular virus. The relative expression of GAPDH and RNase2 gene in macrophages was quantified by realtime PCR using iTaq Universal SYBR Green Supermix (Bio-Rad, 1725120). The primers and probe [84] used were listed in Table S6 .

Cell viability assay THP1 monocytes (wild type or RNase2 knockout) were seeded at 5×10 4 cells/well in 96 well plates and differentiated into macrophages as described [24] . After infection with RSV under MOI=1 for different times, dynamic cell viability was measured using MTT assay.

Selective amplification and sequencing of cyclic phosphatecontaining RNA (cP-RNA-seq) and data analysis Selective amplification and sequencing of cyclic phosphate-containing RNAs was performed as previously reported [26] . Briefly, small RNAs (<200nt) were extracted using mirVanaTM miRNA Isolation Kit (Ambion, Life Technologies, AM1560) as described by the manufacturer. Following RNA extraction, 20-to -100nt RNAs were purified from 8% TBE-PAGE gel. Then, the purified RNAs were treated by calf intestinal alkaline phosphatase. After phenol-chloroform purification, the RNAs were oxidized by incubation in 10mM NaIO4 at 0 ℃ for 40 min in the dark, followed by ethanol precipitation. The RNAs were then treated with T4 PNK. After phenol-chloroform purification, directed ligation of adapters, cDNA generation, and PCR amplification were performed using the Truseq Small RNA Sample Prep Kit for Illumina (NewEngland Biolabs, E7335S) according to the manufacturer's protocol. The amplified cDNAs were sequenced using Illumina hiSeq2500 system at the Centre for Genomic Regulation, CRG, Barcelona).

For small RNA-seq analysis, skewer (v0.22) was used to remove the 5'adaptor sequences and discard low-quality reads [85] . Reads have been size selected before being aligned to the reference genome (GRCh38) with shortStack based on bowtie1 aligner [86, 87] . The mapped reads were counted with HTSeq-count [88] using the annotation from miRBase version 22.1 ((http://www.mirbase.org/) [89] . For differential analysis, DESeq2 [90] was used on count matrices of tRNA-derived fragments and miRNAs.

Quantification of differential abundance of small RNAs was estimated by tRAX 

Host defence RNases as antiviral agents against enveloped single stranded RNA viruses

Eosinophil-derived neurotoxin (EDN/RNase 2) and the mouse eosinophil-associated RNases (mEars): expanding roles in promoting host defense

Eosinophil granule proteins: form and function

Eosinophils in health and disease: the LIAR hypothesis

Functions of tissue-resident eosinophils

Eosinophil cytokines, chemokines, and growth factors: emerging roles in immunity

Biochemical and functional similarities between human eosinophil-derived

Neutrophils as a novel source of eosinophil cationic protein in IgE-mediated processes

Immune modulation by human secreted RNases at the extracellular space

Recombinant human eosinophil-derived neurotoxin/RNase 2 functions as an effective antiviral agent against respiratory syncytial virus

Respiratory viruses and eosinophils: exploring the connections

Antimicrobial activity of human eosinophil granule proteins: involvement in host defence against pathogens

Eosinophil response against classical and emerging respiratory viruses: COVID-19

Eosinophils and COVID-19: diagnosis, prognosis, and vaccination strategies

Role of eosinophils in the diagnosis and prognostic evaluation of COVID-19

Respiratory syncytical virus-induced chemokine expression in the lower airways: eosinophil recruitment and degranulation

Diversity among the primate eosinophil-derived neurotoxin genes: a specific C-terminal sequence is necessary for enhanced ribonuclease activity

Respiratory syncytial virusa comprehensive review

Eosinophil-derived neurotoxin levels at 3 months post-respiratory syncytial virus bronchiolitis are a predictive biomarker of recurrent wheezing

Eosinophils, ribonucleases and host defense: solving the puzzle

An insertion in loop L7 of human eosinophil-derived neurotoxin is crucial for its antiviral activity

Human antimicrobial RNases inhibit intracellular bacterial growth and induce autophagy in mycobacteria-infected macrophages

Human RNase3 immune modulation by catalytic-dependent and independent modes in a macrophage-cell line infection model

Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method

tRNA fragments in human health and disease

Ribonuclease is partly responsible for the HIV-1 inhibitory effect activated by HLA alloantigen recognition

Monocytes and macrophages as viral targets and reservoirs

Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation

Identification of two novel functional tRNA-derived fragments induced in response to respiratory syncytial virus infection

Identification and functional characterization of tRNA-derived RNA fragments (tRFs) in respiratory syncytial virus infection

Respiratory syncytial virus utilizes a tRNA fragment to suppress antiviral responses through a novel targeting mechanism

Viruses, microRNAs, and host interactions

Small silencing RNAs: an expanding universe

Non-coding RNAs and their role in respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections

The noncoding RNA revolution-trashing old rules to forge new ones

Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C

Stress induces tRNA cleavage by angiogenin in mammalian cells

Angiogenin cleaves tRNA and promotes stress-induced translational repression

Human angiogenin is a potent cytotoxin in the absence of ribonuclease inhibitor

Differential expression of human tRNA genes drives the abundance of tRNA-derived fragments

Infection-induced 5'-half molecules of tRNA HisGUG activate Toll-like receptor 7

Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys)

Role of host tRNAs and aminoacyl-tRNA synthetases in retroviral replication

LTR-retrotransposon control by tRNA-derived small RNAs

Immune sensing of synthetic, bacterial, and protozoan RNA by Toll-like receptor 8 requires coordinated processing by RNase T2 and RNase 2

TLR8 is a sensor of RNase T2 degradation products

The eight human "canonical" ribonucleases: molecular diversity, catalytic properties, and special biological actions of the enzyme proteins

Nucleotide binding architecture for secreted cytotoxic endoribonucleases

Angiogenin generates specific stressinduced tRNA halves and is not involved in tRF-3-mediated gene silencing

Transfer RNA cleavages by onconase reveal unusual cleavage sites

Ribonucleases as potential modalities in anticancer therapy

In lysate RNA digestion provides insights into the angiogenin's specificity towards transfer RNAs

Evolutionary trends in RNA base selectivity within the RNase A superfamily

Toward rational design of ribonuclease inhibitors: high-resolution crystal structure of a ribonuclease A complex with a potent 3',5'-pyrophosphate-linked dinucleotide inhibitor

Influence of naturally-occurring 5'-pyrophosphate-linked substituents on the binding of adenylic inhibitors to ribonuclease a: an X-ray crystallographic study

Nucleoside tetra-and pentaphosphates prepared using a tetraphosphorylation reagent are potent inhibitors of ribonuclease A

Structural basis for catalysis by onconase

tRNA-derived fragments: mechanisms underlying their regulation of gene expression and potential applications as therapeutic targets in cancers and virus infections

Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family

Angiogenin (ANG)-ribonuclease inhibitor (RNH1) system in protein synthesis and disease

Angiogenin-induced tRNA fragments inhibit translation initiation

The importance of AGO 1 and 4 in post-transcriptional gene regulatory function of tRF5-GluCTC, an respiratory syncytial virus-induced tRNA-derived RNA fragment

Detection of RNA modifications

Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival

The cell and stress-specific canonical and noncanonical tRNA cleavage

Oxidative stress enhances the expression of 2',3'-cyclic phosphate-containing RNAs

Emerging role of angiogenin in stress response and cell survival under adverse conditions

Single-cell analysis of the impact of host cell heterogeneity on infection with foot-and-mouth disease virus

Small RNA modifications: integral to function and disease

Isolation and initial structurefunctional characterization of endogenous tRNA-derived stress-induced RNAs

ATPasemodulated stress granules contain a diverse proteome and substructure

Generation of 2',3'-cyclic phosphatecontaining RNAs as a hidden layer of the transcriptome

Genome-wide identification of short 2',3'-cyclic phosphate-containing RNAs and their regulation in aging

The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis

Optimized production and concentration of lentiviral vectors containing large inserts

Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors

Production and purification of lentiviral vectors

Generation of gene-modified mice via Cas9/RNA-mediated gene targeting

The first crystal structure of human RNase 6 reveals a novel substratebinding and cleavage site arrangement

An in vitro model to study immune responses of human peripheral blood mononuclear cells to human respiratory syncytial virus infection

Immunostimulatory defective viral genomes from respiratory syncytial virus promote a strong innate antiviral response during infection in mice and humans

Development of a quantitative TaqMan RT-PCR for respiratory syncytial virus

Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads

Ultrafast and memory-efficient alignment of short DNA sequences to the human genome

ShortStack: comprehensive annotation and quantification of small RNA genes

HTSeq-a Python framework to work with highthroughput sequencing data

miRBase: microRNA sequences, targets and gene nomenclature

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

We thank Yundong Peng (Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany), Manuel Kaulich (Goethe University Frankfurt, Germany) for technical supporting with CRIPSR design. We would also like to show our gratitude to Laura Tussel, Anna Genesca, Marina Rodriguez Muñoz, and David Soler from UAB for