Selective cleavage of ncRNA and antiviral activity by human RNase2/EDN in a macrophage infection model

TL;DR: In this article, the authors investigated the role of RNase2 in antiviral host defence against RSV infection and showed that the cleavage sites at the anticodon loops and secondarily at D-loops are the most prominent sites of cleavage.

Abstract: 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 2939cyclic-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 59 and 39 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.

Summary

Introduction

• Human RNase2 is a secretory protein expressed in leukocytes with a reported antiviral activity against single stranded RNA viruses [1,2].
• Recently, presence of eosinophils and their associated RNases has been correlated to the prognosis of COVID patients [15–17].
• 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].

Results

• RSV infection activated the expression of RNase2 in macrophages RSV virus stock was obtained at a titration of 2.8×106 TCID50/mL, as previously described [25] and THP1 macrophages were exposed to the RSV at a selected MOI of 1:1 up to 72h post of infection (poi).
• Moreover, the total absence of secreted RNase2 by THP1 cells was confirmed by ELISA assay in culture supernatant for KO28 line (Fig 2C).
• At 24h, RNase2 KO macrophages had significantly more intracellular RSV than WT macrophages.
• Following RNAseq amplification, the sequence libraries were inspected by differential enrichment analysis.
• Therefore, their data is the first report of the specific release of tRNA fragments associated to RNase2 expression.

Discussion

• Expression of human RNase2 is widely distributed in diverse body tissues such as liver and spleen together with leukocyte cells [2].
• In contrast, using the same infection model and experimental protocol, the authors observe here how RSV infection significantly activate both the expression and protein secretion of RNase2 in THP1 macrophage derived cells (Fig 1).
• In addition, the cleavage of tRNAs by RNases would probably be modulated by the presence of regulatory proteins within the cell [60].
• 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.
• Their results confirm that RNase2 targets ncRNA and releases specific miRNAs and tRFs.

Conclusions

• This is the first report of RNase2 selective targeting of ncRNA.
• Comparison of native and knockout THP1-derived macrophages in a RSV infection model confirms the RNase involvement in the cell host antiviral defence.
• By amplification of 2’3Cp end RNA products, the authors have identified the tRNA fragments and miRNAs associated to RNase2 cleavage.
• The analysis of RNA recognition regions reveals the RNA base composition at the 5’ and 3’ of cleavage site.
• Further work is mandatory for an unambiguous pattern assignment towards the understanding of how RNase2 can shape the ncRNA population and its role to fight viral infection.

Figures

Fig 3. Knocking out of RNase2 resulted in macrophages suffering heavier RSV burden and more cell death. The intracellular (A) and extracellular (B) RSV was quantified by probe qPCR 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).

Fig 6. Representative miRNA overrepresented in WT versus RNase2-KO samples (log2fold >2 and p < 0.05) and predicted cleavage sites on precursor miRNA to release mature miRNAs. Cleavage sites in the precursor miRNA were predicted based on the 3’-terminal positions of each miRNA in bam files.

Fig 1. RSV infection activates the expression of RNase2 in THP1 induced macrophages. 106 THP1 cells were seeded in 6-well tissue culture plate per well and induced by 50 nM of PMA treatment. After induction, macrophages were infected with RSV under MOI=1 for 2h and then cells were washed and replaced with fresh RPMI+10%FBS (0h time point post of infection). At each time point post of infection, the supernatant and cells were collected to quantify the expression of RNase2. A) qPCR detection of relative expression of RNase2 gene; B) The concentration of the cells was controlled as 106/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.

Table 2. Comparison of RNase2 cleavage specificity on ncRNA in THP1-derived macrophages (this study) with synthetic RNA substrates in vitro.

Fig 5. Overall cleavage preference of RNase2 on tRNAs in THP1 macrophage cells of WT vs RNase2-KO identified by cP-RNA-seq. A) Estimated base preference at 5’ and 3’ of cleavage site (B1 and B2 respectively) deduced from analysis of differential sequence coverage. B) The percentage of predicted cleavage location is depicted from low (white) to high (blue) values.

Fig 4: Predicted cleavage sites of the most significantly abundant tRNA-derived fragments identified by cp-RNAseq. Parental tRNAs with significant coverage differences between WT and RNase2-KO macrophage cells are depicted and the identified tRFs are marked in red. The possible cleavage sites are based on the 3’-terminal positions of the five prime fragments (blue arrows) and the 5’ terminal positions of three prime fragments (green arrows) according to the different coverage. Only sequences with a log2fold >0.5 and adjusted p value < 0.05 are included.

Table 1. tRNA fragment population changes upon RNase2 knockout and/or RSV infection.

Fig 2. CRISPR/Cas9 mediated knock out of RNase2 gene in THP1 derived macrophages. A) 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.

Full text

1
Selective cleavage of ncRNA and antiviral activity by human
RNase2/EDN in a macrophage infection model
Lu Lu
1,2#*
, Jiarui Li
1*
, Ranlei Wei
3
, Irene Guidi
1
, Luca Cozzuto
4
, Julia
Ponomarenko
4
, Guillem Prats-Ejarque
1
and Ester Boix
1#
1
Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat
Autonomous de Barcelona, Cerdanyola del Vallès, Spain
2
College of Animal Science and Technology, Sichuan Agricultural University, Chengdu,
Sichuan, China
3
Center of Precision Medicine& Precision Medicine Key Laboratory of Sichuan Province,
West China Hospital, Sichuan University, Chengdu, China
4
Bioinformatic Unit. Centre de Regulació Genòmica (CRG), Barcelona, Spain
*Both authors contributed equally to this work
#
Corresponding authors: Ester Boix, Ester.Boix@uab.es ; Lu Lu, lu.lu@sicau.edu.cn
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 31, 2021. ; https://doi.org/10.1101/2021.08.30.458223doi: bioRxiv preprint

2
Abstract
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.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 31, 2021. ; https://doi.org/10.1101/2021.08.30.458223doi: bioRxiv preprint

3
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.
Short title: RNase2-induced specific ncRNA cleavage
Keywords: RNase2, EDN, macrophage, antiviral, RSV, ncRNA, tRNAs, tRFs
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 31, 2021. ; https://doi.org/10.1101/2021.08.30.458223doi: bioRxiv preprint

4
Introduction
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 [3–8].Apart from eosinophils,
RNase2 is also expressed in other leukocyte cell types, such as neutrophils and
monocytes, together with epithelial cells, liver and spleen [8–10]. 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 [12–14]. Recently, presence of eosinophils and their
associated RNases has been correlated to the prognosis of COVID patients [15–17]. 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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 31, 2021. ; https://doi.org/10.1101/2021.08.30.458223doi: bioRxiv preprint

5
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.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 31, 2021. ; https://doi.org/10.1101/2021.08.30.458223doi: bioRxiv preprint

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Selective cleavage of ncrna and antiviral activity by human rnase2/edn in a macrophage infection model" ?

In this paper, the authors showed that RNase2 expression in macrophages correlates with a selective ncRNA cleavage pattern. 

Q2. What have the authors stated for future works in "Selective cleavage of ncrna and antiviral activity by human rnase2/edn in a macrophage infection model" ?

Further work is mandatory for an unambiguous pattern assignment towards the understanding of how RNase2 can shape the ncRNA population and its role to fight viral infection. 

Q3. What is the role of RNase2 in the host defence?

Among the blood cell types, RNase2 is particularly abundant in monocytes [11], which are key contributors to host defence against pathogens. 

Q4. What is the role of RNase2 in the host response?

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]. 

Q5. What is the main reason for the cleavage of miRNAs?

Accumulation of miRNAs can be toxic to the cells, due to their potential interference with the translation of essential proteins. 

Q6. What are the potential regulatory elements for future studies?

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. 

Q7. What role does ncRNA play in regulating antiviral innate immune responses?

Increasing data demonstrates that small noncoding RNAs (ncRNAs) play important roles in regulating antiviral innate immune responses [34–36]. 

Q8. How did the RSV infection affect the expression of RNase2 in macrophages?

RSV infection activated the expression of RNase2 inmacrophagesRSV virus stock was obtained at a titration of 2.8×106 TCID50/mL, as previouslydescribed [25] and THP1 macrophages were exposed to the RSV at a selected MOI of 1:1 up to 72h post of infection (poi). 

Q9. What is the abundant RNase2 protein in the THP1 cell line?

In their previous work using a macrophage infection model, the authors observed thatRNase2 is the most abundantly expressed RNaseA superfamily member in the monocytic THP1 cell line [24]. 

Q10. Under what conditions is RNase5/Ang reported to stimulate the formation of cytoplasmic?

under certain cell conditions, such as nutrition deficiency oroxidative stress, RNase5/Ang is reported to stimulate the formation of cytoplasmic stress granules and produce tRNA-derived stress-induced RNAs (tiRNAs) [69–71]. 

Q11. What was the significant tRNA fragments associated with RNase2 presence?

the most significant tRNA fragments associated to RNase2 presence showed a U/C cleavage target for B1 at the anticodon loop (Table S4). 

Q12. What is the abundant RNaseA family member in human monocytic cell line?

Previous work indicated that RNase2 is the most abundant RNaseA family member expressed in this human monocytic cell line [24] (https://www.proteinatlas.org/). 

Q13. What is the main reason why the tiRNA&tRF array is biased?

More importantly, the array screening technique is prone to be biased by theselection 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. 

Q14. How many tiRNAs were significantly decreased in RNase2 KO macrophage?

Using the nrStarTM Human tRF&tiRNA library, the authors found that out of a total of185 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). 

Q15. What was the effect of ELISA on the expression of RNase2 in THP1?

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.