- Email: [email protected]
Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism
ACCEPTED ARTICLE PREVIEW
Accepted Article Preview: Published ahead of advance online publication Respiratory syncytial virus utilizes a tRNA fragment to suppress antiviral responses through a novel targeting mechanism
t p ri
Junfang Deng, Ryan N Ptashkin, Yu Chen, Zhi Cheng, Guangliang Liu, Thien Phan, Xiaoling Deng, Jiehua Zhou, Inhan Lee, Yong Sun Lee, and Xiaoyong Bao
c us
Cite this article as: Junfang Deng, Ryan N Ptashkin, Yu Chen, Zhi Cheng, Guangliang Liu, Thien Phan, Xiaoling Deng, Jiehua Zhou, Inhan Lee, Yong Sun Lee, and Xiaoyong Bao, Respiratory syncytial virus utilizes a tRNA fragment to suppress antiviral responses through a novel targeting mechanism, Molecular Therapy accepted article preview online 09 July 2015; doi:10.1038/mt.2015.124
d e t
an m
This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
p e c
c A
Received 26 January 2015; accepted 27 June 2015; Accepted article preview online 09 July 2015
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Respiratory syncytial virus utilizes a tRNA fragment to suppress antiviral responses through a novel targeting mechanism Junfang Denga,b#, Ryan N Ptashkinc,d#, Yu Chena, Zhi Chenga, Guangliang Liua,e, Thien Phana , Xiaoling Denga, Jiehua Zhoua, Inhan Leec, Yong Sun Leef, and Xiaoyong Baoa,g * Running Title: RSV regulates host gene expression via a tRF Author Affiliations Department of Pediatricsa, University of Texas Medical Branch, Galveston, TX; Department of
t p ri
Hepatobiliary Surgeryb, The First Affiliated Hospital, College of Medicine, Zhejiang University, China; miRcorec, Ann Arbor, MI; Department of Computational Medicine and Bioinformaticsd, University of
c us
Michigan, MI; Department of Otorhinolaryngologye, Sixth Affiliated Hospital, Sun Yat-Sen University,
an m
China; Department of Biochemistry and Molecular Biologyf and Institute for Translational Scienceg, University of Texas Medical Branch, Galveston, TX.
d e t
#Equal contribution
p e c
*Correspondence should be addressed to: Yong Sun Lee, PhD and Xiaoyong Bao, PhD,
c A
Department of Pediatrics, Division of Clinical and Experimental Immunology & Infectious Diseases, 301 University Boulevard, Galveston, TX 77555-0372 Phone: (409) 772-1777; Fax: (409)772-0460; Email: [email protected] and [email protected] Key Words: RSV, sncRNA, tRFs and APOER2.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Abstract Target identification is highly instructive in defining the biological roles of microRNAs. However, little is known about other small non-coding RNAs; for example, tRNA-derived RNA Fragments (tRFs). Some tRFs exhibit a gene-silencing mechanism distinctly different from that of typical microRNAs. We recently demonstrated that a respiratory syncytial virus (RSV)-induced tRF, called tRF5-GluCTC, promotes RSV replication. RSV is the single most important cause of lower respiratory tract infection in children. By using biochemical screening and bioinformatics analyses, we have identified apolipoprotein E receptor 2 (APOER2) as a target of tRF5-GluCTC. The 3′-portion of
t p ri
tRF5-GluCTC recognizes a target site in the 3′-untranslated region of APOER2 and suppresses its
c us
expression. We have also discovered that APOER2 is an anti-RSV protein whose suppression by tRF5GluCTC promotes RSV replication. Our report represents the first identification of a natural target of a
an m
tRF and illustrates how a virus utilizes a host tRF to control a host gene to favor its replication.
d e t
p e c
c A
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Introduction In addition to microRNAs (miRNAs) many new types of small non-coding RNAs (sncRNAs) are continuously emerging, whose functions and underlying mechanisms are largely unknown 1. tRNAderived RNA Fragments (tRFs) are a recently identified family of sncRNAs. They are present in diverse eukaryotes 2-5, often cleaved from a specific subset of tRNAs (transferRNAs) by specific nucleases under certain biological conditions 4-8. So, tRF production is under tight control. Although some tRFs regulate gene expression, the regulatory mechanisms are largely unexplored.
t p ri
Respiratory syncytial virus (RSV) is the most common cause of life-threatening lower respiratory
c us
tract infection in children, responsible for several hundred thousand deaths annually 9. However, therapeutic strategies and vaccines for RSV are unavailable 10, largely due to the lack of comprehensive
an m
understanding of host-RSV interaction. We have recently discovered that the most dominant sncRNAs 8
induced by RSV are tRFs . Moreover, a specific tRF derived from the 5'-end of tRNA-GluCTC (tRF5-
d e t
GluCTC) demonstrates a trans-silencing activity and promotes viral replication. Herein, we have
p e c
elucidated a molecular mechanism(s) underlying tRF5-GluCTC’s functions by identifying its cellular targets. Because tRFs have distinct trans-silencing mechanism(s) from miRNAs 8, instead of simply
c A
using algorithms for miRNA target prediction we took an unbiased approach combining biochemical screening, analysis of gene expression data, and in silico prediction to identify tRF targets. As a result, we found apolipoprotein E receptor-2 (APOER2) as an endogenous target of tRF5-GluCTC and also a new anti-RSV protein. This is a novel example for a virus controlling host genes through the induction of tRNA cleavage and the first identification of an endogenous target of a tRF.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Results Identification of direct targets of tRF5-GluCTC. According to our recent paper, tRF5-GluCTC is localized exclusively in the cellular cytoplasm and the cis-acting element for its trans-silencing activity lies within the transcribed region of a luciferase sensor gene 8. Therefore, it is reasonable to posit that tRF5-GluCTC’s gene trans-silencing activity is mediated by its interaction with the target mRNA. Therefore, we sought to identify RNAs physically associated with tRF5-GluCTC as the first step for target identification. A synthetic biotinylated tRF5-GluCTC oligonuleotide (tRF5-GluCTC mimic) was transfected into A549 human lung epithelial cells as a bait to capture associated RNAs. The tRF-
t p ri
containing complex was isolated from a cell lysate by pull down with streptavidin beads and the
c us
associated RNAs therein were identified by HIgh-Throughput Sequencing of RNAs Associated with Biotinylated tRF (HITS-ABt).
an m
In prioritizing HITS-ABt-captured targets, we took advantage of published mRNA array data upon RSV infection 11, 12. Given the gene trans-silencing activity of tRF5-GluCTC8, tRF5-GluCTC’s targets
d e t
were expected to be suppressed by RSV. Promising target candidates were the 44 genes identified not
p e c
only associated with tRF5-GluCTC but also inhibited by RSV infection (Fig. 1A and Supplementary
c A
Table I).
A genuine direct target of tRF5-GluCTC was presupposed to harbor a reverse complementary sequence to interact with tRF5-GluCTC; therefore, we searched by the RNAhybrid tool for such sequences in those 44 genes 13. This search was done by scanning the whole transcripts, because tRFs might recognize a target site in the untranslated regions (UTRs) as well as the coding sequences (CDS). Twenty genes, with a minimum free energy hybridization (∆G) value less than -30 kcal/mol or lower were selected (Table I). Among those 20 genes, we chose APOER2 as a primary candidate because it has a target site in its 3'-UTR (Fig. 1B) with a ∆G value (-42.5 kcal/mol) superior to the other genes. Reverse transcription (RT)-PCR revealed that APOER2 mRNA was associated with biotinylated tRF5-GluCTC mimic.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
However, β-actin was not, suggesting a binding specificity of APOER2 to tRF5-GluCTC mimic (Fig. 1C). The input lanes in Fig. 1C showed that APOER2 mRNA began to decrease at 6 hr posttransfection, but was enriched in the complex, ascertaining the association was real. APOER2 protein was also decreased by the tRF5-GluCTC mimic (Fig. 1D). To corroborate the above data we investigated APOER2 expression in the context of RSV infection. Upon infection, both mRNA and protein levels of APOER2 were significantly decreased. This was reversed by an anti-tRF5-GluCTC oligonucleotide (oligo), demonstrating that the suppression is caused specifically by tRF5-GluCTC (Fig. 1E-F). Since the tRF5-GluCTC is in the cytoplasmic compartment, its effect on APOER2 mRNA should
t p ri
be at a post-transcriptional stage. Therefore, the mRNA decrease of APOER2 was most likely due to
c us
mRNA destabilization.
The regulation of APOER2 by tRF5-GluCTC. As depicted in Fig. 1B, the base pairing between
an m
tRF5-GluCTC and APOER2’s 3'-UTR is imperfectly complementary. However, their overall interaction has a ∆G value of -42.5 kcal/mol, much lower than ∆G (~-30 kcal/mol) for common miRNA-target
d e t
interactions 13. We reasoned that tRF5-GluCTC targets APOER2 via this site and tested the hypothesis
p e c
by a luciferase report system that we recently used to demonstrate tRF’s trans-silencing function 8. In brief, the target site of APOER2 was inserted into the 3'-UTR of the firefly (“Pp”) luciferase gene
c A
(designated as “Pp-APOER2-WT” and simplified as “Pp-WT” in Fig. 2A). Cells were then cotransfected with Pp-WT together with a plasmid expressing renilla luciferase (“Rr”) for normalization. The relative luciferase activity (Pp/Rr values in y-axis) was compared among treatments. Compared to mock-infection, RSV infection significantly decreased the relative luciferase activity of Pp-WT (left two bars in Fig. 2B). This decrease was reversed by the co-transfection of “anti-tRF5-GluCTC” (right two bars in Fig. 2B). Our luciferase data demonstrated that the target site alone was sufficient for the suppression by tRF5-GluCTC.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
In addition to decreased luciferase expression, real-time PCR (qRT-PCR) indicated that the Pp mRNA level of Pp-WT was decreased by tRF5-GluCTC (the Supplementary Fig. 1), which was consistent with the decrease of endogenous APOER2 mRNA upon RSV infection (Fig. 1E). These data further supported that tRF5-GluCTC facilitated mRNA destabilization. We further scrutinized the sequence requirement by mutagenesis experiments. Point mutations at 36 bases were introduced in the 3'-, middle- (M-), or 5'-region of the target site to abrogate potential regulation by tRF5-GluCTC (illustrated in Fig. 2A). It should be clarified that the Pp-APOER2-Mut series refers to the regions in tRF5-GluCTC; for example, Pp-Mut3 actually has a mutation at the 5'-
t p ri
portion at the target site and so its interaction to the tRF 3'-portion is abrogated (Fig. 2A). Compared to
c us
Pp-WT, Pp-Mut3 was completely unresponsive to RSV infection (Fig. 2C). To further investigate which nucleotides (nts) are important for the 3'-end-mediated APOER2 suppression, we also constructed Pp
an m
plasmids containing partial mutations at the 5'-portion of the target site (illustrated as Pp-Mut3-A and Pp-Mut3-B in Fig. 2A) and found that these nts were critical for the gene regulatory function of tRF5-
d e t
GluCTC (Fig.2C). In contrast, Pp-Mut5 responded very moderately and Pp-MutM barely responded
p e c
upon RSV infection (Fig. 2C). Overall, our results demonstrates that the 3'-end of tRF5-GluCTC plays a dominant role in suppressing APOER2 and the 5'-end also contributes to the AOPER2 suppression. The
c A
same results were obtained when we compared luciferase activities of Pp-WT and Pp-mutants in response to tRF5-GluCTC WT-mimic. Compared to Pp-WT, Pp-Mut3 failed to response to WT-mimic, while Pp-MutM and -Mut5 had comparable and moderately reduced responses respectively (Fig. 2D). In addition, a mimic harboring mutations in the 3'-portion (Mut3-mimic) failed to suppress the luciferase activity of Pp-WT (Fig. 2E). All these data unequivocally support that the 3'-portion of tRF5-GluCTC is the most critical motif for targeting. This is an intriguing result, given that the base pairing in the 3'portion is poorer than those in the 5'- and M-portions (see Fig. 2A). Compared to eight or nine consecutive canonical base pairings within the 10 nt block in the 5'- or M- portion, there are only four
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
canonical and one wobble G:U base pairings at the 3'-end. Overall, we identified APOER2 as a genuine direct target of tRF5-GluCTC. Antiviral function of APOER2. The next question was the functional significance of the tRF5GluCTC-APOER2 axis in RSV infection. Since tRF5-GluCTC promoted RSV replication, its target APOER2 was expected to be antiviral. Therefore we examined the effect of APOER2 suppression upon RSV replication. Compared to control siRNA (si-ctrl), siRNA against APOER2 (si-APOER2) efficiently decreased APOER2 protein (Fig. 3A), leading to enhanced production of RSV particles and genome copies (Figs. 3B and C). The antiviral role of APOER2 was also investigated in ectopic expression. It
t p ri
should be noted that the exogenous APOER2 lacked its 3'-UTR and therefore was unresponsive to RSV-
c us
induced tRF5-GluCTC (Fig. 3D). APOER2 overexpression significantly decreased RSV replication and viral genome copies (Figs. 3E and F) in a dose-dependent manner (data not shown), confirming its antiRSV function.
an m
Antiviral mechanism of APOER2. APOER2 is a low-density lipoprotein receptor and these
d e t
receptors promote the replication of some Flaviviruses via mediating the endocytosis of viruses 14. The
p e c
role of APOER2 in Paramyxoviruses, including RSV, has not been previously reported. However, our data proved its anti-RSV role (Fig. 3D), suggesting that its impact on viral replication is virus specific.
c A
Since previous studies on RSV entry suggested that RSV, like most other Paramyxoviruses, fuses its membrane directly with the plasma membrane of target cells 15, it is less likely for APOER2 to control RSV replication via receptor-mediated endocytosis. On the other hand, it is well-known that all lowdensity lipoprotein receptor family proteins contain a cytoplasmic tail with at least one motif important for binding intracellular proteins 16. As shown in Fig. 3, APOER2 affected the viral genome synthesis, a process controlled by viral RNA-dependent RNA polymerases (RDRP), which is a cytoplasmic enzyme composed of multiple subunits including phosphoprotein P and large protein L 17, 18. Therefore, it is possible that APOER2 inhibits RSV replication via interacting with those RSV protein(s). As shown by the immunoprecipitation assay (Fig. 4A), RSV P protein was indeed co-precipitated with APOER2,
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
demonstrating their physical interaction, which may lead to sequestration of P protein away from the formation of the functional RDRP complex and consequently to suppression of RSV replication. In support of this scenario, supplement of exogenous P protein offset the inhibitory effect of APOER2 on RSV replication (Fig. 4B). Overall, we have shown here a novel regulatory network between RSV and host genes. In our model (Summarized in Fig. 4C), RSV infection induces tRNA cleavage to produce tRF5-GluCTC which targets APOER2 to make more P proteins available for RSV replication.
t p ri
Discussion
c us
In our previous work, we found that tRF5-GluCTC is induced by RSV and promotes RSV replication. Here, we signally substantiated this by identifying its natural cellular target genes. Because a
an m
target prediction algorithm for tRFs is not available, we used experimental and computational tools and identified several candidate targets. Among them, we selected APOER2 for experimental validation and
d e t
found that tRF5-GluCTC suppresses APOER2 mRNA using its 3′-portion to recognize a target site in
p e c
the APOER2 3′-UTR.
Our target identification here is an important initial process in understanding the basic biological
c A
role of tRFs. The knowledge regarding the effects of tRF5-GluCTC and its target(s) on RSV replication will provide insights critical for the development of new therapeutic strategies to control RSV replication. It should be highlighted that our scheme was prudently designed in a logical and unbiased manner. We first clarified that tRF5-GluCTC is different from miRNAs in target recognition. It is known that the 5′-end of miRNAs is generally important for its gene silencing function 19. However, in the case of tRFs, we found that the 3′-portion of tRF5-GluCTC is critical for gene targeting. Then we combined a biochemical approach (HITS-ABt), global expression data from mRNA array, and in silico prediction of RNA-RNA interaction for target identification. Our search turned out to be influential, because all our data proved that the best candidate, APOER2, is a real direct target. We plan to test more candidates as
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
well as to apply the same strategy to identify targets of other tRFs besides tRF5-GluCTC. As the list of genuine target genes increases, we will be able to decipher a target recognition rule more accurately. Once established, the rule will greatly facilitate the studies on tRFs, like the miRNA studies of the last decade. We have also discovered that APOER2 is an anti-RSV protein. According to our experimental data, the antiviral ability of APOER2 was surprisingly strong; it seemed as if the decrease of APOER2 by tRF5-GluCTC accounted for the entire effect of tRF5-GluCTC on viral replication. However, this does not necessarily mean that APOER2 is the sole functional target for the tRF5-GluCTC effect because the
t p ri
degree of suppression of APOER2 by siRNA is not necessarily equivalent to that of APOER2 decrease
c us
by tRF5-GluCTC in the context of RSV infection. Overall, our RNA pull-down assay demonstrated several other potential candidates of tRF5-GluCTC. We will delineate the contribution of other targets to RSV replication in future studies.
an m
The tRF study is at a beginning stage and the whole tRF-target network is sketchy at present. We
d e t
plan to expand this network by adding more tRFs and more targets. Although our study on tRFs is
p e c
currently limited to infectious diseases, strategies on tRFs’ function characterization and target identification should be useful in other biological settings given the fact tRFs are found in events
c A
associated with cell proliferation, cellular stress responses and development 5, 8, 20-24. In addition, our results on antiviral target identification and the targeting mechanism will provide new insights important for the design of preventive and therapeutic strategies involving RSV replication. Materials and Methods Cell lines, virus and antibody. HEp-2, HEK-293 and A549, human alveolar type II-like epithelial cells (all from ATCC, Manassas, VA) were maintained as we previously described 8, 26. RSV A2 strain was grown in HEp-2 cells and purified by sucrose gradient as described 8, 26. Viral titer was determined by immunostaining in HEp-2 cells using polyclonal biotin-conjugated goat anti-RSV antibody (Ad
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Direct, Barberton, OH) and streptavidin peroxidase polymer (Sigma-Aldrich, St Louis, MO) sequentially, as described 8, 25. RNA pull-down. Biotinylated tRF5-GluCTC mimic is a synthetic oligoribonucleotide containing a biotin moiety at the 5'-end 8. 100 nM biotin-tRF5-GluCTC or its control (Sigma-Aldrich) were transfected into uninfected A549 cells in 10-cm dishes using Lipofectamine 2000 (Life Technologies, Grand Island, NY) according to the manufacturer’s instruction. Mimics at 100 nM did not induce a significant amount of antiviral mediator, such as IFN-β, or cellular toxicity as determined by lactate dehydrogenase release (data not shown). At 6 hr post transfection, cells were treated with UV
t p ri
crosslinking (UV Stratalinker 1800, Stratagene/Agilent, Santa Clara, CA) and then harvested using cell
c us
lysis buffer (Roche, Cat. No. 11719394001, Indianapolis, IN) supplemented with RNase inhibitors. Streptavidin beads (Pierce, Rockford, IL) were added to pull down biotinylated oligos. The beads were
an m
washed with detergent-free lysis buffer twice and then treated with protease K (New England BioLabs, Ipswich, MA). Associated RNAs in pull-downed biotinylated tRF5-GluCTC mimic were extracted by
d e t
TRIzol® (Life Technologies) for RNA sequencing.
p e c
Library construction and RNA sequencing. The library construction and RNA sequencing were performed by the Next Generation Sequencing Core at the University of Texas Medical Branch,
c A
Galveston, TX. In brief, ribosomal RNAs (rRNAs) were removed from 1 ug of total RNA using RiboZero biotinylated, target-specific oligos (Epicentre, Madison, WI). Poly-A+ RNA was removed using poly (dT)-magnetic beads and the bound RNA containing predominantly adenylated mRNAs were fragmented by incubation at 94°C for 8 min in 19.5 ul of fragmentation buffer (Cat. No.15016648, Illumina, San Diego, CA). First strand synthesis was performed using reverse transcriptase (Superscript II, Life Technologies) and random primers. Second strand synthesis using DNA polymerase I and RNAse H was performed using a deoxyribonucleotide mixture that had dUTP substituted for dTTP. End polishing, addition of 5' phosphates and 3' adenylation and adapter ligation were performed as prescribed by the Illumina TruSeq Stranded protocol. Samples were tracked using “index tags”
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
incorporated into the adapters. Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Quantitation of library DNA templates was performed using qPCR and a known-size reference standard. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Template input was adjusted to obtain a cluster density of 700-850 K/mm2. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000 using protocols defined by the manufacturer.
t p ri
Genome alignments for tRF5-GluCTC-bound RNAs. The raw paired-end sequence reads for
c us
RNAs bound to biotin-labeled tRF5-GluCTC or control mimic were first aligned to a custom database of rRNA sequences. Those read pairs that aligned concordantly to the rRNA database were excluded from
an m
the proceeding analysis. The remaining read pairs were aligned to human reference (hg19). All alignments were performed with Bowtie2 using default parameters26. Read pairs that mapped
d e t
concordantly to human reference were converted to BED file format. These reads were further
p e c
characterized by searching for overlap to databases for the following genomic regions downloaded from UCSC table browser: coding sequences (CDS), 5UTR, 3UTR, introns, and intergenic. Comparisons
c A
were made to the above databases via the intersectBed function of BEDTools 27. We used a parameter of minimum overlap of 0.5 as a fraction of the first bed file. The resulting alignment files for mRNAs were then processed with Cuffdiff to determine gene and transcript level expression and test for differential expression 28, 29. Isoform-level abundances were estimated using a maximization likelihood function (Cufflinks) and upper quartile normalized to account for differences in library sizes29. Gene counts were calculated as the summed counts of transcripts sharing each gene identification (ID). We further compared mRNAs which have enhanced binding to tRF5-GluCTC (tRF5-GluCTC vs control oligo, with fold changes in binding >10 and P<0.05) to mRNAs shown to be decreased from previous microarray data. The overlap of mRNAs from these two data sets were then identified
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
(Supplementary Table I). The tRF5-GluCTC sequence was also queried against whole gene sequences of common genes using RNAhybrid. Genes with a hybridization energy value (∆G value) less than -30 kcal/mole were listed in Table I. Northern blot. Northern hybridization for sncRNAs was performed as we previously described 8, 30. Briefly, RNA was separated in 15% denaturing polyacrylamide gel with 7M urea and then transferred to a positively charged nylon membrane (Amersham Biosciences, Piscataway, NJ). The membrane was hybridized with 32P-labled probes in ULTRAhyb®-Oligo solution (Life Technologies), followed by washing according to the manufacturer’s instruction.
t p ri
Western blot. The total cellular extracts of uninfected and infected cells were prepared using RIPA
c us
buffer (New England BioLabs) supplied with protease inhibitor cocktail (Roche). After protein quantification, the lysates were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride
an m
membranes. Membranes were blocked with 5% milk in TBS-Tween 20 and incubated with the proper primary antibodies according to manufacturer's instruction. Anti-APOER2 and β-actin antibodies were
d e t
obtained from Abcam (Cambridge, MA) and Sigma-Aldrich respectively.
p e c
Construction of luciferase sensor plasmids and luciferase assays. The sensor plasmid “Pp-
c A
APOER2-WT” (simplified as “Pp-WT”) was constructed by inserting a tRF5-GluCTC-targeting sequence complementary to APOER2’s three prime untranslated region (3'-UTR) into EcoRI/XhoI sites of pcDNA3.1-Zeo(+)-Pp as described 31 (also illustrated in Figs. 1B and 2A). Paired primers used for insertion were: 5'-AATTGTTCTGCCACTTACTCCCACTAGACAACCAGGGA-3' and 5'- TCGATCCCTGGTTGTCTAGTGGGAGTAAGTGGCAGAAC-3' (bold letters represent extra nts to generate EcoRI/XhoI overhangs). Five mutant plasmids were constructed in the same manner (their mutated sequences shown in Fig. 2A).
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
To investigate the effect of RSV-induced tRF5-GluCTC on APOER2 mRNA expression A549 cells were co-transfected with Pp-WT sensor plasmids (firefly plasmids), pRL-CMV plasmids expressing Rr (renilla luciferase), and anti-tRF5-GluCTC oligo or its control (anti-ctrl), using Lipofetamine 2000 according to the manufacturer’s instruction (Invitrogen, Grand Island, NY). Synthetic anti-tRF5GluCTC oligo used in this study contained a backbone phosphorothioate and had 5 nts on each end substituted with 2'-O-methyl ribonucleotides which have been shown to be effective in suppressing RSV-induced tRF5-GluCTC 8. After 2 hr of transfection, the cells were infected with mock or RSV. At 15 hr post infection (p.i.) cells were lysed for luciferase assays using a dual-luciferase kit (Promega,
t p ri
Madison, WI). Data processing and normalization were described in Fig. 2 legend.
c us
Pp-WT sensor plasmids were mutated as shown in Fig. 2A to identify the targeting specificity of tRF5-GluCTC. A549 cells were co-transfected with each individual mutant and Rr expressing plasmids,
an m
followed by mock or RSV infection with a multiplicity of infection or MOI of 1. Cells were lysed for luciferase assays at 15 hr p.i. (Fig. 2C). Similarly, cells transfected with Pp-WT or its mutant were co-
d e t
transfected with WT-mimic for tRF5-GluCTC or its control (ctrl-mimic) to confirm the targeting
p e c
specificity (Fig. 2D). Specificity was further confirmed by comparing the luciferase activity of Pp-WT in response to WT-mimic and a mimic containing mutations at the 3'-end of tRF5-GluCTC (Mut3-
c A
mimic). In brief, A549 cells were co-transfected with Pp-WT sensor plasmids, Rr expressing plasmids, and WT- or Mut3-mimic for 15 hr, followed by luciferase assays (Fig. 2E). All other descriptions, such as the concentrations of oligos and plasmids were described in the legend of Fig. 2. RNA interference. siRNAs were purchased from Sigma-Aldrich. 100 nM of siRNA was transfected into A549 cells, by using Lpofectamine 2000. After 48 hr, A549 cells were mock- or RSVinfected for 6 hr at a MOI of 1. Quantitative real-time PCR (qRT-PCR). Total cellular RNAs were extracted using TRIzol® reagents. qRT-PCR for viral replication was performed by using SYBR as we previously described 8, 25.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Co-immunoprecipitation (Co-IP). Logarithmically growing A549 cells in 6-well plates were cotransfected with 2 µg of pcDNA3 containing Flag-tagged APOER2 or empty vector. Cells were then mock infected or infected with RSV at MOI of 1 for 6 hr, followed by cell lysis and immunoprecipitation (IP kit from Roche, Cat. no. 11719386001), similarly as previously described 32, 33. In brief, 6×106 cells were lysed using 1.5 ml of lysis buffer, followed by a preclearing. Precleared samples were exposed to 5 µg of antibody against Flag for 1 hr at 4°C. 50 µl of the protein A/G-agarose were added to the samples and incubated overnight at 4°C. The IP complexes were then recovered by centrifugation and washed three times using buffers provided by the kit. The IP complexes were eluted
t p ri
from the beads and subjected to SDS-PAGE followed by Western blot using an anti-RSV antibody. The
c us
membrane was stripped and reprobed with an anti-Flag antibody to ensure the proper IP process. Statistical Analysis. Statistical significance for sequencing data was analyzed using analysis of
an m
variance (ANOVA) or student’s t-test. P value of <0.05 was considered significant. Mean ± standard error (SE) is shown.
d e t
p e c
Acknowledgements
c A
The authors concur there are no conflicts of interest associated in this published work. This work was supported by grants from the National Institutes of Health-National Institute of Allergy and Infectious Diseases 1 R56 AI107033-01A1, the American Thoracic Society Unrestricted Research Grants, Institute for Translational Science Novel Method Grant (UTMB) to X.B. We also thank Betty H. Johnson for her assistance with manuscript editing.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Reference:
1.
Esteller,M. Non-coding RNAs in human disease. Nat. Rev. Genet. 12, 861-874 (2011).
2.
Lee,S.R. & Collins,K. Starvation-induced cleavage of the tRNA anticodon loop in Tetrahymena thermophila. J. Biol. Chem. 280, 42744-42749 (2005).
3.
Haiser,H.J., Karginov,F.V., Hannon,G.J., & Elliot,M.A. Developmentally regulated cleavage of tRNAs in the bacterium Streptomyces coelicolor. Nucleic Acids Res. 36, 732-741 (2008).
4.
Thompson,D.M., Lu,C., Green,P.J., & Parker,R. tRNA cleavage is a conserved response to oxidative stress in eukaryotes. RNA. 14, 2095-2103 (2008).
5.
Lee,Y.S., Shibata,Y., Malhotra,A., & Dutta,A. A novel class of small RNAs: tRNAderived RNA fragments (tRFs). Genes Dev. 23, 2639-2649 (2009).
6.
Emara,M.M. et al. Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly. J. Biol. Chem. 285, 10959-10968 (2010).
7.
Yamasaki,S., Ivanov,P., Hu,G.F., & Anderson,P. Angiogenin cleaves tRNA and promotes stress-induced translational repression. J. Cell Biol. 185, 35-42 (2009).
8.
Wang,Q. et al. Identification and Functional Characterization of tRNA-derived RNA Fragments (tRFs) in Respiratory Syncytial Virus Infection. Mol. Ther. 21(2), 368-79 (2012).
9.
Falade,A.G. & Ayede,A.I. Epidemiology, aetiology and management of childhood acute community-acquired pneumonia in developing countries--a review. Afr. J. Med. Med. Sci. 40, 293-308 (2011).
t p ri
c us
d e t
an m
p e c
c A
10.
Welliver,R.C. Review of epidemiology and clinical risk factors for severe respiratory syncytial virus (RSV) infection. J Pediatr. 143, S112-S117 (2003).
11.
Zhang,Y. et al. Expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays. J. Virol. 75, 9044-9058 (2001).
12.
Mejias,A. et al. Whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection. PLoS. Med. 10, e1001549 (2013).
13.
Rehmsmeier,M., Steffen,P., Hochsmann,M., & Giegerich,R. Fast and effective prediction of microRNA/target duplexes. RNA. 10, 1507-1517 (2004).
14.
Agnello,V., Abel,G., Elfahal,M., Knight,G.B., & Zhang,Q.X. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc. Natl. Acad. Sci. U. S. A 96, 12766-12771 (1999).
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
15.
Srinivasakumar,N., Ogra,P.L., & Flanagan,T.D. Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay. J. Virol. 65, 4063-4069 (1991).
16.
Reddy,S.S., Connor,T.E., Weeber,E.J., & Rebeck,W. Similarities and differences in structure, expression, and functions of VLDLR and ApoER2. Mol. Neurodegener. 6, 30 (2011).
17.
Lu,B., Ma,C.H., Brazas,R., & Jin,H. The major phosphorylation sites of the respiratory syncytial virus phosphoprotein are dispensable for virus replication in vitro. J. Virol. 76, 1077610784 (2002).
18.
Bermingham,A. & Collins,P.L. The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription. Proc. Natl. Acad. Sci. U. S. A 96, 11259-11264 (1999).
19.
Haasnoot,J. & Berkhout,B. RNAi and cellular miRNAs in infections by mammalian viruses. Methods Mol. Biol. 721, 23-41 (2011).
20.
Franzen,O. et al. The short non-coding transcriptome of the protozoan parasite Trypanosoma cruzi. PLoS. Negl. Trop. Dis. 5, e1283 (2011).
21.
Hsieh,L.C., Lin,S.I., Kuo,H.F., & Chiou,T.J. Abundance of tRNA-derived small RNAs in phosphate-starved Arabidopsis roots. Plant Signal. Behav. 5, (2010).
22.
Hsieh,L.C. et al. Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing. Plant Physiol 151, 2120-2132 (2009).
23.
Peng,H. et al. A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm. Cell Res. 22, 1609-1612 (2012).
t p ri
c us
d e t
an m
p e c
c A
24. Goodarzi,H. et al. Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell 161, 790-802 (2015). 25.
Ren,J. et al. A novel mechanism for the inhibition of interferon regulatory factor-3dependent gene expression by human respiratory syncytial virus NS1 protein. J. Gen. Virol. 92, 2153-2159 (2011).
26.
Langmead,B. & Salzberg,S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359 (2012).
27.
Kent,W.J., Zweig,A.S., Barber,G., Hinrichs,A.S., & Karolchik,D. BigWig and BigBed: enabling browsing of large distributed datasets. Bioinformatics. 26, 2204-2207 (2010).
28.
Roberts,A., Pimentel,H., Trapnell,C., & Pachter,L. Identification of novel transcripts in annotated genomes using RNA-Seq. Bioinformatics. 27, 2325-2329 (2011).
29.
Trapnell,C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562-578 (2012).
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
30.
Deng,J. et al. Human metapneumovirus infection induces significant changes in small noncoding RNA expression in airway epithelial cells. Mol. Ther. Nucleic Acids 3, e163 (2014).
31.
Lee,K. et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA. 17, 1076-1089 (2011).
32.
Ren,J. et al. Human Metapneumovirus M2-2 Protein Inhibits Innate Cellular Signaling by Targeting MAVS. J. Virol. 86, 13049-13061 (2012).
33.
Ren,J. et al. Human metapneumovirus m2-2 protein inhibits innate immune response in monocyte-derived dendritic cells. PLoS. ONE. 9, e91865 (2014).
t p ri
c us
d e t
an m
p e c
c A
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Gene APOER2 NOP56 CALM1 SOX7 FTSJ3 SPATA2 C6orf120 TNFRSF10D PBDC1 EBNA1BP2 FTSJ2 MTF2 PSMA4 DBR1 AMD1 RPL37 RPL23 TCEAL4 C14orf2 SLIRP
Gene Accession # NM_004631 NM_006392 NM_006888 NM_031439 NM_017647 NM_006038 NM_001029863 NM_003840 NM_016500 NM_006824 NM_013393 NM_001164392 NM_002789 NM_016216 NM_001634 NM_000997 NM_000978 NM_001006937 NM_004894 NM_001267863
∆G (free energy) values -42.5 -40.6 -40.1 -39.7 -39.6 -39.0 -38.9 -37.1 -36.8 -36.0 -35.3 -34.2 -33.9 -33.7 -32.6 -32.5 -32.4 -32.3 -31.6 -30.1
t p ri
c us
an m
Table I. List of genes which were downregulated by RSV (log2 fold change<-1) and had increased binding to tRF5-GluCTC (fold change >10) and predicted minimum free energy (∆G) for their interaction with tRF5-GluCTC ≤-30 kcal/mol.
d e t
p e c
c A
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Legends: Figure 1. tRF5-GluCTC controls the expression of APOER2. (A) Workflow to identify tRF5GluCTC-associated mRNAs. (B) Sequence alignment of tRF5-GluCTC with APOER2. The interactive region was identified by both RNAhybrid analysis and the binding assays. (C) The APOER2 mRNAs interacting with biotinylated tRF5-GluCTC was confirmed by RT-PCR, using β-actin as a negative control to demonstrate the binding specificity. (D) The mimic of tRF5-GluCTC decreases APOER2 expression. A549 cells were transfected with 25 nM WT-mimic for tRF5-GluCTC or control mimic
t p ri
(ctrl-mimic). At 15 hr post transfection, total cell lysate was harvested, followed by western blot to measure the expression of APOER2. (E and F). Antisense against tRF5-GluCTC increases APOER2
c us
expression. A549 cells in a 6-well plate were co-transfected with 120 nM antisense oligos, anti-tRF5-
an m
GluCTC or anti-ctrl. After 2 hr post transfection, cells were mock-infected or infected with RSV at MOI of 1, then harvested at 15 hr p.i. to harvest total RNAs or proteins to measure the APOER2’s mRNA by
d e t
qRT-PCR (E) or APOER2 protein by western blot (F) respectively. FC: Fold changes. Figure 2. Targeting elements of tRF5-GluCTC. (A) Construction of the firefly (Pp) luciferase plasmid
p e c
containing the targeted sequence of APOER2, WT or mutants to identify the motif(s) critical for tRF5-
c A
GluCTC’s trans-silencing function. (B) The effect of tRF5-GluCTC on Pp luciferase expression from Pp-WT. A549 cells in a 24-well plate were co-transfected with a Pp- WT and a plasmid expressing renilla Rr luciferase, and 120 nM antisense oligos, anti-tRF5-GluCTC or anti-ctrl. After 2 hr post transfection, cells were mock-infected or infected with RSV at MOI of 1. At 15 hr p.i. cells were lysed to measure the luciferase activity. Pp values were first normalized by Rr values yielding relative Pp/Rr values (y-axis). * denotes P value <0.05, relative to the first white bar. (C-E) Targeting motifs. A549 cells, co-transfected with a Pp luciferase plasmid, Pp-WT, Pp-Mut5 , Pp-MutM, Pp-Mut3, Pp-Mut3-A, or Pp-Mut3-B (0.1 µg/well of 24-well plate), and a Rr luciferase plasmid were infected with RSV at MOI of 1 (C) or treated with 100 nM WT-mimic oligo (D). Mock infection or the ctrl-
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
mimic was used as a control for C and D respectively. At 15 hr p.i. or 30 hr post-transfection, cells were lysed for luciferase assays. Values at y-axis (Pp/Rr) are a representative of three independent experiments and are expressed as mean ± SE. * or ** on the bars denotes P value <0.05 or <0.01 respectively, relative to the black bar (Pp-WT-transfected plus RSV-infected or WT-mimic-treated samples). (E). A549 cells in hexaplicate were co-transfected with a Pp-WT plasmid (0.1 µg/well of 24well plate), an Rr plasmid, and 100 nM mimic (WT or Mut3, please see illustration in A). After 15 hr post transfection, cells were harvested to measure luciferase activities. Values at y-axis (Pp/Rr) are a representative of three independent experiments and are expressed as mean ± SE. **on the second bar
t p ri
(WT-mimic transfected) denotes P value <0.01, relative to the first bar (ctrl-mimic transfected).
c us
Figure 3. Antiviral effect of APOER2. (A-C) The effect of APOER2 silencing on RSV replication. A549 cells were transfected with 100 nM siRNA against APOER2 (si-APOER2) or si-ctrl using
an m
Lipofectamine 2000. At 48 hr post transfection, cells were mock-infected or infected with RSV at MOI of 1 for 15 hr. (A) Total proteins were prepared to confirm the silencing efficiency of si-APOER2. (B)
d e t
Infectious particles were measured by immunostaining and the values expressed as pfu/ml. (C) Genome
p e c
copies were measured by qRT-PCR. (D-F) The effect of APOER2 overexpression on RSV replication. A549 cells in 6-well plate were transfected with a plasmid encoding Flag-tagged APOER2 (2 µg/well)
c A
or a control plasmid for 30 hr, followed by mock infection or RSV infection (MOI of 1) for 15 hr. (D) Confirmation on Flag-APOER2 expression by western blot. (E) Total infectious particles measurement. (F) Genome copies of RSV. * and ** denotes p < 0.05 and < 0.01 respectively, relative to its ctrl plasmid . Figure 4. APOER2 interacts with RSV P protein. (A) APOER2 forms a complex with RSV P protein. A549 cells in 6-well plate were transfected with 2 µg/well plasmids encoding Flag-tagged APOER2 or it control vectors. At 30 hr post transfection, cells were mock infected or infected with RSV at MOI of 1. At 6 hr p.i. total cell lysates were immunoprecipitated with an anti-Flag antibody followed by western blot using an anti-RSV antibody to detect associated RSV protein(s). A small aliquot was also prepared
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
before the IP for equal input assays. (B) Overexpressed RSV P protein restores APOER2-inhibited RSV replication. HEK-293 cells at 60-70 % confluence were transfected with a plasmid encoding V5-tagged RSV P or N (control protein) or their common control vector. After 30 hr cells were mock infected or infected with RSV at MOI of 1 for 15 hr. The viral particles in the supernatant were titrated by immune staining and protein overexpression was confirmed by western blot. (C) A mechanism model for tRF5GluCTC-pormoted RSV replication. RSV-induced tRF5-GluCTC targets APOER2, leading to suppressed APOER2 expression and consequently releasing more RSV P protein to facilitate RSV genome replication.
t p ri
c us
Table I. Common mRNAs identified by methods demonstrated in Fig. 1A and with predicted minimum free energy hybridization (∆G) ≤-30 kcal/mol to interact with tRF5-GluCTC.
an m
Supplementary Table I. mRNAs with enhanced binding to tRF5-GluCTC and decreased expression by
d e t
RSV infection. Cutoff criteria were demonstrated in Fig. 1A.
p e c
Supplementary Figure 1. tRF5-GluCTC inhibits the transcription of APOER2. A549 cells in 6-well
c A
plate were co-transfected with a Pp- WT and 120 nM antisense oligos, anti-tRF5-GluCTC or anti-ctrl. After 2 hr post transfection, cells were mock-infected or infected with RSV at MOI of 1. At 15 hr p.i. cells were lysed to isolate total RNAs. After the treatment of DNase, the transcription of luciferase, controlled by the APOER2-derived target of tRF5-GluCTC, was measured by qRT-PCR samples using paired primers as follow: 5'-GAGGTTCCATCTGCCAGGTATC-3' and 5' – CGGTTTATCATCCCCCTCG-3'. ** denotes P value <0.01, relative to the first white bar.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
Accepted Article Preview: Published ahead of advance online publication Respiratory syncytial virus utilizes a tRNA fragment to suppress antiviral responses through a novel targeting mechanism
t p ri
Junfang Deng, Ryan N Ptashkin, Yu Chen, Zhi Cheng, Guangliang Liu, Thien Phan, Xiaoling Deng, Jiehua Zhou, Inhan Lee, Yong Sun Lee, and Xiaoyong Bao
c us
Cite this article as: Junfang Deng, Ryan N Ptashkin, Yu Chen, Zhi Cheng, Guangliang Liu, Thien Phan, Xiaoling Deng, Jiehua Zhou, Inhan Lee, Yong Sun Lee, and Xiaoyong Bao, Respiratory syncytial virus utilizes a tRNA fragment to suppress antiviral responses through a novel targeting mechanism, Molecular Therapy accepted article preview online 09 July 2015; doi:10.1038/mt.2015.124
d e t
an m
This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
p e c
c A
Received 26 January 2015; accepted 27 June 2015; Accepted article preview online 09 July 2015
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Respiratory syncytial virus utilizes a tRNA fragment to suppress antiviral responses through a novel targeting mechanism Junfang Denga,b#, Ryan N Ptashkinc,d#, Yu Chena, Zhi Chenga, Guangliang Liua,e, Thien Phana , Xiaoling Denga, Jiehua Zhoua, Inhan Leec, Yong Sun Leef, and Xiaoyong Baoa,g * Running Title: RSV regulates host gene expression via a tRF Author Affiliations Department of Pediatricsa, University of Texas Medical Branch, Galveston, TX; Department of
t p ri
Hepatobiliary Surgeryb, The First Affiliated Hospital, College of Medicine, Zhejiang University, China; miRcorec, Ann Arbor, MI; Department of Computational Medicine and Bioinformaticsd, University of
c us
Michigan, MI; Department of Otorhinolaryngologye, Sixth Affiliated Hospital, Sun Yat-Sen University,
an m
China; Department of Biochemistry and Molecular Biologyf and Institute for Translational Scienceg, University of Texas Medical Branch, Galveston, TX.
d e t
#Equal contribution
p e c
*Correspondence should be addressed to: Yong Sun Lee, PhD and Xiaoyong Bao, PhD,
c A
Department of Pediatrics, Division of Clinical and Experimental Immunology & Infectious Diseases, 301 University Boulevard, Galveston, TX 77555-0372 Phone: (409) 772-1777; Fax: (409)772-0460; Email: [email protected] and [email protected] Key Words: RSV, sncRNA, tRFs and APOER2.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Abstract Target identification is highly instructive in defining the biological roles of microRNAs. However, little is known about other small non-coding RNAs; for example, tRNA-derived RNA Fragments (tRFs). Some tRFs exhibit a gene-silencing mechanism distinctly different from that of typical microRNAs. We recently demonstrated that a respiratory syncytial virus (RSV)-induced tRF, called tRF5-GluCTC, promotes RSV replication. RSV is the single most important cause of lower respiratory tract infection in children. By using biochemical screening and bioinformatics analyses, we have identified apolipoprotein E receptor 2 (APOER2) as a target of tRF5-GluCTC. The 3′-portion of
t p ri
tRF5-GluCTC recognizes a target site in the 3′-untranslated region of APOER2 and suppresses its
c us
expression. We have also discovered that APOER2 is an anti-RSV protein whose suppression by tRF5GluCTC promotes RSV replication. Our report represents the first identification of a natural target of a
an m
tRF and illustrates how a virus utilizes a host tRF to control a host gene to favor its replication.
d e t
p e c
c A
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Introduction In addition to microRNAs (miRNAs) many new types of small non-coding RNAs (sncRNAs) are continuously emerging, whose functions and underlying mechanisms are largely unknown 1. tRNAderived RNA Fragments (tRFs) are a recently identified family of sncRNAs. They are present in diverse eukaryotes 2-5, often cleaved from a specific subset of tRNAs (transferRNAs) by specific nucleases under certain biological conditions 4-8. So, tRF production is under tight control. Although some tRFs regulate gene expression, the regulatory mechanisms are largely unexplored.
t p ri
Respiratory syncytial virus (RSV) is the most common cause of life-threatening lower respiratory
c us
tract infection in children, responsible for several hundred thousand deaths annually 9. However, therapeutic strategies and vaccines for RSV are unavailable 10, largely due to the lack of comprehensive
an m
understanding of host-RSV interaction. We have recently discovered that the most dominant sncRNAs 8
induced by RSV are tRFs . Moreover, a specific tRF derived from the 5'-end of tRNA-GluCTC (tRF5-
d e t
GluCTC) demonstrates a trans-silencing activity and promotes viral replication. Herein, we have
p e c
elucidated a molecular mechanism(s) underlying tRF5-GluCTC’s functions by identifying its cellular targets. Because tRFs have distinct trans-silencing mechanism(s) from miRNAs 8, instead of simply
c A
using algorithms for miRNA target prediction we took an unbiased approach combining biochemical screening, analysis of gene expression data, and in silico prediction to identify tRF targets. As a result, we found apolipoprotein E receptor-2 (APOER2) as an endogenous target of tRF5-GluCTC and also a new anti-RSV protein. This is a novel example for a virus controlling host genes through the induction of tRNA cleavage and the first identification of an endogenous target of a tRF.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Results Identification of direct targets of tRF5-GluCTC. According to our recent paper, tRF5-GluCTC is localized exclusively in the cellular cytoplasm and the cis-acting element for its trans-silencing activity lies within the transcribed region of a luciferase sensor gene 8. Therefore, it is reasonable to posit that tRF5-GluCTC’s gene trans-silencing activity is mediated by its interaction with the target mRNA. Therefore, we sought to identify RNAs physically associated with tRF5-GluCTC as the first step for target identification. A synthetic biotinylated tRF5-GluCTC oligonuleotide (tRF5-GluCTC mimic) was transfected into A549 human lung epithelial cells as a bait to capture associated RNAs. The tRF-
t p ri
containing complex was isolated from a cell lysate by pull down with streptavidin beads and the
c us
associated RNAs therein were identified by HIgh-Throughput Sequencing of RNAs Associated with Biotinylated tRF (HITS-ABt).
an m
In prioritizing HITS-ABt-captured targets, we took advantage of published mRNA array data upon RSV infection 11, 12. Given the gene trans-silencing activity of tRF5-GluCTC8, tRF5-GluCTC’s targets
d e t
were expected to be suppressed by RSV. Promising target candidates were the 44 genes identified not
p e c
only associated with tRF5-GluCTC but also inhibited by RSV infection (Fig. 1A and Supplementary
c A
Table I).
A genuine direct target of tRF5-GluCTC was presupposed to harbor a reverse complementary sequence to interact with tRF5-GluCTC; therefore, we searched by the RNAhybrid tool for such sequences in those 44 genes 13. This search was done by scanning the whole transcripts, because tRFs might recognize a target site in the untranslated regions (UTRs) as well as the coding sequences (CDS). Twenty genes, with a minimum free energy hybridization (∆G) value less than -30 kcal/mol or lower were selected (Table I). Among those 20 genes, we chose APOER2 as a primary candidate because it has a target site in its 3'-UTR (Fig. 1B) with a ∆G value (-42.5 kcal/mol) superior to the other genes. Reverse transcription (RT)-PCR revealed that APOER2 mRNA was associated with biotinylated tRF5-GluCTC mimic.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
However, β-actin was not, suggesting a binding specificity of APOER2 to tRF5-GluCTC mimic (Fig. 1C). The input lanes in Fig. 1C showed that APOER2 mRNA began to decrease at 6 hr posttransfection, but was enriched in the complex, ascertaining the association was real. APOER2 protein was also decreased by the tRF5-GluCTC mimic (Fig. 1D). To corroborate the above data we investigated APOER2 expression in the context of RSV infection. Upon infection, both mRNA and protein levels of APOER2 were significantly decreased. This was reversed by an anti-tRF5-GluCTC oligonucleotide (oligo), demonstrating that the suppression is caused specifically by tRF5-GluCTC (Fig. 1E-F). Since the tRF5-GluCTC is in the cytoplasmic compartment, its effect on APOER2 mRNA should
t p ri
be at a post-transcriptional stage. Therefore, the mRNA decrease of APOER2 was most likely due to
c us
mRNA destabilization.
The regulation of APOER2 by tRF5-GluCTC. As depicted in Fig. 1B, the base pairing between
an m
tRF5-GluCTC and APOER2’s 3'-UTR is imperfectly complementary. However, their overall interaction has a ∆G value of -42.5 kcal/mol, much lower than ∆G (~-30 kcal/mol) for common miRNA-target
d e t
interactions 13. We reasoned that tRF5-GluCTC targets APOER2 via this site and tested the hypothesis
p e c
by a luciferase report system that we recently used to demonstrate tRF’s trans-silencing function 8. In brief, the target site of APOER2 was inserted into the 3'-UTR of the firefly (“Pp”) luciferase gene
c A
(designated as “Pp-APOER2-WT” and simplified as “Pp-WT” in Fig. 2A). Cells were then cotransfected with Pp-WT together with a plasmid expressing renilla luciferase (“Rr”) for normalization. The relative luciferase activity (Pp/Rr values in y-axis) was compared among treatments. Compared to mock-infection, RSV infection significantly decreased the relative luciferase activity of Pp-WT (left two bars in Fig. 2B). This decrease was reversed by the co-transfection of “anti-tRF5-GluCTC” (right two bars in Fig. 2B). Our luciferase data demonstrated that the target site alone was sufficient for the suppression by tRF5-GluCTC.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
In addition to decreased luciferase expression, real-time PCR (qRT-PCR) indicated that the Pp mRNA level of Pp-WT was decreased by tRF5-GluCTC (the Supplementary Fig. 1), which was consistent with the decrease of endogenous APOER2 mRNA upon RSV infection (Fig. 1E). These data further supported that tRF5-GluCTC facilitated mRNA destabilization. We further scrutinized the sequence requirement by mutagenesis experiments. Point mutations at 36 bases were introduced in the 3'-, middle- (M-), or 5'-region of the target site to abrogate potential regulation by tRF5-GluCTC (illustrated in Fig. 2A). It should be clarified that the Pp-APOER2-Mut series refers to the regions in tRF5-GluCTC; for example, Pp-Mut3 actually has a mutation at the 5'-
t p ri
portion at the target site and so its interaction to the tRF 3'-portion is abrogated (Fig. 2A). Compared to
c us
Pp-WT, Pp-Mut3 was completely unresponsive to RSV infection (Fig. 2C). To further investigate which nucleotides (nts) are important for the 3'-end-mediated APOER2 suppression, we also constructed Pp
an m
plasmids containing partial mutations at the 5'-portion of the target site (illustrated as Pp-Mut3-A and Pp-Mut3-B in Fig. 2A) and found that these nts were critical for the gene regulatory function of tRF5-
d e t
GluCTC (Fig.2C). In contrast, Pp-Mut5 responded very moderately and Pp-MutM barely responded
p e c
upon RSV infection (Fig. 2C). Overall, our results demonstrates that the 3'-end of tRF5-GluCTC plays a dominant role in suppressing APOER2 and the 5'-end also contributes to the AOPER2 suppression. The
c A
same results were obtained when we compared luciferase activities of Pp-WT and Pp-mutants in response to tRF5-GluCTC WT-mimic. Compared to Pp-WT, Pp-Mut3 failed to response to WT-mimic, while Pp-MutM and -Mut5 had comparable and moderately reduced responses respectively (Fig. 2D). In addition, a mimic harboring mutations in the 3'-portion (Mut3-mimic) failed to suppress the luciferase activity of Pp-WT (Fig. 2E). All these data unequivocally support that the 3'-portion of tRF5-GluCTC is the most critical motif for targeting. This is an intriguing result, given that the base pairing in the 3'portion is poorer than those in the 5'- and M-portions (see Fig. 2A). Compared to eight or nine consecutive canonical base pairings within the 10 nt block in the 5'- or M- portion, there are only four
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
canonical and one wobble G:U base pairings at the 3'-end. Overall, we identified APOER2 as a genuine direct target of tRF5-GluCTC. Antiviral function of APOER2. The next question was the functional significance of the tRF5GluCTC-APOER2 axis in RSV infection. Since tRF5-GluCTC promoted RSV replication, its target APOER2 was expected to be antiviral. Therefore we examined the effect of APOER2 suppression upon RSV replication. Compared to control siRNA (si-ctrl), siRNA against APOER2 (si-APOER2) efficiently decreased APOER2 protein (Fig. 3A), leading to enhanced production of RSV particles and genome copies (Figs. 3B and C). The antiviral role of APOER2 was also investigated in ectopic expression. It
t p ri
should be noted that the exogenous APOER2 lacked its 3'-UTR and therefore was unresponsive to RSV-
c us
induced tRF5-GluCTC (Fig. 3D). APOER2 overexpression significantly decreased RSV replication and viral genome copies (Figs. 3E and F) in a dose-dependent manner (data not shown), confirming its antiRSV function.
an m
Antiviral mechanism of APOER2. APOER2 is a low-density lipoprotein receptor and these
d e t
receptors promote the replication of some Flaviviruses via mediating the endocytosis of viruses 14. The
p e c
role of APOER2 in Paramyxoviruses, including RSV, has not been previously reported. However, our data proved its anti-RSV role (Fig. 3D), suggesting that its impact on viral replication is virus specific.
c A
Since previous studies on RSV entry suggested that RSV, like most other Paramyxoviruses, fuses its membrane directly with the plasma membrane of target cells 15, it is less likely for APOER2 to control RSV replication via receptor-mediated endocytosis. On the other hand, it is well-known that all lowdensity lipoprotein receptor family proteins contain a cytoplasmic tail with at least one motif important for binding intracellular proteins 16. As shown in Fig. 3, APOER2 affected the viral genome synthesis, a process controlled by viral RNA-dependent RNA polymerases (RDRP), which is a cytoplasmic enzyme composed of multiple subunits including phosphoprotein P and large protein L 17, 18. Therefore, it is possible that APOER2 inhibits RSV replication via interacting with those RSV protein(s). As shown by the immunoprecipitation assay (Fig. 4A), RSV P protein was indeed co-precipitated with APOER2,
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
demonstrating their physical interaction, which may lead to sequestration of P protein away from the formation of the functional RDRP complex and consequently to suppression of RSV replication. In support of this scenario, supplement of exogenous P protein offset the inhibitory effect of APOER2 on RSV replication (Fig. 4B). Overall, we have shown here a novel regulatory network between RSV and host genes. In our model (Summarized in Fig. 4C), RSV infection induces tRNA cleavage to produce tRF5-GluCTC which targets APOER2 to make more P proteins available for RSV replication.
t p ri
Discussion
c us
In our previous work, we found that tRF5-GluCTC is induced by RSV and promotes RSV replication. Here, we signally substantiated this by identifying its natural cellular target genes. Because a
an m
target prediction algorithm for tRFs is not available, we used experimental and computational tools and identified several candidate targets. Among them, we selected APOER2 for experimental validation and
d e t
found that tRF5-GluCTC suppresses APOER2 mRNA using its 3′-portion to recognize a target site in
p e c
the APOER2 3′-UTR.
Our target identification here is an important initial process in understanding the basic biological
c A
role of tRFs. The knowledge regarding the effects of tRF5-GluCTC and its target(s) on RSV replication will provide insights critical for the development of new therapeutic strategies to control RSV replication. It should be highlighted that our scheme was prudently designed in a logical and unbiased manner. We first clarified that tRF5-GluCTC is different from miRNAs in target recognition. It is known that the 5′-end of miRNAs is generally important for its gene silencing function 19. However, in the case of tRFs, we found that the 3′-portion of tRF5-GluCTC is critical for gene targeting. Then we combined a biochemical approach (HITS-ABt), global expression data from mRNA array, and in silico prediction of RNA-RNA interaction for target identification. Our search turned out to be influential, because all our data proved that the best candidate, APOER2, is a real direct target. We plan to test more candidates as
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
well as to apply the same strategy to identify targets of other tRFs besides tRF5-GluCTC. As the list of genuine target genes increases, we will be able to decipher a target recognition rule more accurately. Once established, the rule will greatly facilitate the studies on tRFs, like the miRNA studies of the last decade. We have also discovered that APOER2 is an anti-RSV protein. According to our experimental data, the antiviral ability of APOER2 was surprisingly strong; it seemed as if the decrease of APOER2 by tRF5-GluCTC accounted for the entire effect of tRF5-GluCTC on viral replication. However, this does not necessarily mean that APOER2 is the sole functional target for the tRF5-GluCTC effect because the
t p ri
degree of suppression of APOER2 by siRNA is not necessarily equivalent to that of APOER2 decrease
c us
by tRF5-GluCTC in the context of RSV infection. Overall, our RNA pull-down assay demonstrated several other potential candidates of tRF5-GluCTC. We will delineate the contribution of other targets to RSV replication in future studies.
an m
The tRF study is at a beginning stage and the whole tRF-target network is sketchy at present. We
d e t
plan to expand this network by adding more tRFs and more targets. Although our study on tRFs is
p e c
currently limited to infectious diseases, strategies on tRFs’ function characterization and target identification should be useful in other biological settings given the fact tRFs are found in events
c A
associated with cell proliferation, cellular stress responses and development 5, 8, 20-24. In addition, our results on antiviral target identification and the targeting mechanism will provide new insights important for the design of preventive and therapeutic strategies involving RSV replication. Materials and Methods Cell lines, virus and antibody. HEp-2, HEK-293 and A549, human alveolar type II-like epithelial cells (all from ATCC, Manassas, VA) were maintained as we previously described 8, 26. RSV A2 strain was grown in HEp-2 cells and purified by sucrose gradient as described 8, 26. Viral titer was determined by immunostaining in HEp-2 cells using polyclonal biotin-conjugated goat anti-RSV antibody (Ad
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Direct, Barberton, OH) and streptavidin peroxidase polymer (Sigma-Aldrich, St Louis, MO) sequentially, as described 8, 25. RNA pull-down. Biotinylated tRF5-GluCTC mimic is a synthetic oligoribonucleotide containing a biotin moiety at the 5'-end 8. 100 nM biotin-tRF5-GluCTC or its control (Sigma-Aldrich) were transfected into uninfected A549 cells in 10-cm dishes using Lipofectamine 2000 (Life Technologies, Grand Island, NY) according to the manufacturer’s instruction. Mimics at 100 nM did not induce a significant amount of antiviral mediator, such as IFN-β, or cellular toxicity as determined by lactate dehydrogenase release (data not shown). At 6 hr post transfection, cells were treated with UV
t p ri
crosslinking (UV Stratalinker 1800, Stratagene/Agilent, Santa Clara, CA) and then harvested using cell
c us
lysis buffer (Roche, Cat. No. 11719394001, Indianapolis, IN) supplemented with RNase inhibitors. Streptavidin beads (Pierce, Rockford, IL) were added to pull down biotinylated oligos. The beads were
an m
washed with detergent-free lysis buffer twice and then treated with protease K (New England BioLabs, Ipswich, MA). Associated RNAs in pull-downed biotinylated tRF5-GluCTC mimic were extracted by
d e t
TRIzol® (Life Technologies) for RNA sequencing.
p e c
Library construction and RNA sequencing. The library construction and RNA sequencing were performed by the Next Generation Sequencing Core at the University of Texas Medical Branch,
c A
Galveston, TX. In brief, ribosomal RNAs (rRNAs) were removed from 1 ug of total RNA using RiboZero biotinylated, target-specific oligos (Epicentre, Madison, WI). Poly-A+ RNA was removed using poly (dT)-magnetic beads and the bound RNA containing predominantly adenylated mRNAs were fragmented by incubation at 94°C for 8 min in 19.5 ul of fragmentation buffer (Cat. No.15016648, Illumina, San Diego, CA). First strand synthesis was performed using reverse transcriptase (Superscript II, Life Technologies) and random primers. Second strand synthesis using DNA polymerase I and RNAse H was performed using a deoxyribonucleotide mixture that had dUTP substituted for dTTP. End polishing, addition of 5' phosphates and 3' adenylation and adapter ligation were performed as prescribed by the Illumina TruSeq Stranded protocol. Samples were tracked using “index tags”
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
incorporated into the adapters. Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Quantitation of library DNA templates was performed using qPCR and a known-size reference standard. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Template input was adjusted to obtain a cluster density of 700-850 K/mm2. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000 using protocols defined by the manufacturer.
t p ri
Genome alignments for tRF5-GluCTC-bound RNAs. The raw paired-end sequence reads for
c us
RNAs bound to biotin-labeled tRF5-GluCTC or control mimic were first aligned to a custom database of rRNA sequences. Those read pairs that aligned concordantly to the rRNA database were excluded from
an m
the proceeding analysis. The remaining read pairs were aligned to human reference (hg19). All alignments were performed with Bowtie2 using default parameters26. Read pairs that mapped
d e t
concordantly to human reference were converted to BED file format. These reads were further
p e c
characterized by searching for overlap to databases for the following genomic regions downloaded from UCSC table browser: coding sequences (CDS), 5UTR, 3UTR, introns, and intergenic. Comparisons
c A
were made to the above databases via the intersectBed function of BEDTools 27. We used a parameter of minimum overlap of 0.5 as a fraction of the first bed file. The resulting alignment files for mRNAs were then processed with Cuffdiff to determine gene and transcript level expression and test for differential expression 28, 29. Isoform-level abundances were estimated using a maximization likelihood function (Cufflinks) and upper quartile normalized to account for differences in library sizes29. Gene counts were calculated as the summed counts of transcripts sharing each gene identification (ID). We further compared mRNAs which have enhanced binding to tRF5-GluCTC (tRF5-GluCTC vs control oligo, with fold changes in binding >10 and P<0.05) to mRNAs shown to be decreased from previous microarray data. The overlap of mRNAs from these two data sets were then identified
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
(Supplementary Table I). The tRF5-GluCTC sequence was also queried against whole gene sequences of common genes using RNAhybrid. Genes with a hybridization energy value (∆G value) less than -30 kcal/mole were listed in Table I. Northern blot. Northern hybridization for sncRNAs was performed as we previously described 8, 30. Briefly, RNA was separated in 15% denaturing polyacrylamide gel with 7M urea and then transferred to a positively charged nylon membrane (Amersham Biosciences, Piscataway, NJ). The membrane was hybridized with 32P-labled probes in ULTRAhyb®-Oligo solution (Life Technologies), followed by washing according to the manufacturer’s instruction.
t p ri
Western blot. The total cellular extracts of uninfected and infected cells were prepared using RIPA
c us
buffer (New England BioLabs) supplied with protease inhibitor cocktail (Roche). After protein quantification, the lysates were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride
an m
membranes. Membranes were blocked with 5% milk in TBS-Tween 20 and incubated with the proper primary antibodies according to manufacturer's instruction. Anti-APOER2 and β-actin antibodies were
d e t
obtained from Abcam (Cambridge, MA) and Sigma-Aldrich respectively.
p e c
Construction of luciferase sensor plasmids and luciferase assays. The sensor plasmid “Pp-
c A
APOER2-WT” (simplified as “Pp-WT”) was constructed by inserting a tRF5-GluCTC-targeting sequence complementary to APOER2’s three prime untranslated region (3'-UTR) into EcoRI/XhoI sites of pcDNA3.1-Zeo(+)-Pp as described 31 (also illustrated in Figs. 1B and 2A). Paired primers used for insertion were: 5'-AATTGTTCTGCCACTTACTCCCACTAGACAACCAGGGA-3' and 5'- TCGATCCCTGGTTGTCTAGTGGGAGTAAGTGGCAGAAC-3' (bold letters represent extra nts to generate EcoRI/XhoI overhangs). Five mutant plasmids were constructed in the same manner (their mutated sequences shown in Fig. 2A).
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
To investigate the effect of RSV-induced tRF5-GluCTC on APOER2 mRNA expression A549 cells were co-transfected with Pp-WT sensor plasmids (firefly plasmids), pRL-CMV plasmids expressing Rr (renilla luciferase), and anti-tRF5-GluCTC oligo or its control (anti-ctrl), using Lipofetamine 2000 according to the manufacturer’s instruction (Invitrogen, Grand Island, NY). Synthetic anti-tRF5GluCTC oligo used in this study contained a backbone phosphorothioate and had 5 nts on each end substituted with 2'-O-methyl ribonucleotides which have been shown to be effective in suppressing RSV-induced tRF5-GluCTC 8. After 2 hr of transfection, the cells were infected with mock or RSV. At 15 hr post infection (p.i.) cells were lysed for luciferase assays using a dual-luciferase kit (Promega,
t p ri
Madison, WI). Data processing and normalization were described in Fig. 2 legend.
c us
Pp-WT sensor plasmids were mutated as shown in Fig. 2A to identify the targeting specificity of tRF5-GluCTC. A549 cells were co-transfected with each individual mutant and Rr expressing plasmids,
an m
followed by mock or RSV infection with a multiplicity of infection or MOI of 1. Cells were lysed for luciferase assays at 15 hr p.i. (Fig. 2C). Similarly, cells transfected with Pp-WT or its mutant were co-
d e t
transfected with WT-mimic for tRF5-GluCTC or its control (ctrl-mimic) to confirm the targeting
p e c
specificity (Fig. 2D). Specificity was further confirmed by comparing the luciferase activity of Pp-WT in response to WT-mimic and a mimic containing mutations at the 3'-end of tRF5-GluCTC (Mut3-
c A
mimic). In brief, A549 cells were co-transfected with Pp-WT sensor plasmids, Rr expressing plasmids, and WT- or Mut3-mimic for 15 hr, followed by luciferase assays (Fig. 2E). All other descriptions, such as the concentrations of oligos and plasmids were described in the legend of Fig. 2. RNA interference. siRNAs were purchased from Sigma-Aldrich. 100 nM of siRNA was transfected into A549 cells, by using Lpofectamine 2000. After 48 hr, A549 cells were mock- or RSVinfected for 6 hr at a MOI of 1. Quantitative real-time PCR (qRT-PCR). Total cellular RNAs were extracted using TRIzol® reagents. qRT-PCR for viral replication was performed by using SYBR as we previously described 8, 25.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Co-immunoprecipitation (Co-IP). Logarithmically growing A549 cells in 6-well plates were cotransfected with 2 µg of pcDNA3 containing Flag-tagged APOER2 or empty vector. Cells were then mock infected or infected with RSV at MOI of 1 for 6 hr, followed by cell lysis and immunoprecipitation (IP kit from Roche, Cat. no. 11719386001), similarly as previously described 32, 33. In brief, 6×106 cells were lysed using 1.5 ml of lysis buffer, followed by a preclearing. Precleared samples were exposed to 5 µg of antibody against Flag for 1 hr at 4°C. 50 µl of the protein A/G-agarose were added to the samples and incubated overnight at 4°C. The IP complexes were then recovered by centrifugation and washed three times using buffers provided by the kit. The IP complexes were eluted
t p ri
from the beads and subjected to SDS-PAGE followed by Western blot using an anti-RSV antibody. The
c us
membrane was stripped and reprobed with an anti-Flag antibody to ensure the proper IP process. Statistical Analysis. Statistical significance for sequencing data was analyzed using analysis of
an m
variance (ANOVA) or student’s t-test. P value of <0.05 was considered significant. Mean ± standard error (SE) is shown.
d e t
p e c
Acknowledgements
c A
The authors concur there are no conflicts of interest associated in this published work. This work was supported by grants from the National Institutes of Health-National Institute of Allergy and Infectious Diseases 1 R56 AI107033-01A1, the American Thoracic Society Unrestricted Research Grants, Institute for Translational Science Novel Method Grant (UTMB) to X.B. We also thank Betty H. Johnson for her assistance with manuscript editing.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Reference:
1.
Esteller,M. Non-coding RNAs in human disease. Nat. Rev. Genet. 12, 861-874 (2011).
2.
Lee,S.R. & Collins,K. Starvation-induced cleavage of the tRNA anticodon loop in Tetrahymena thermophila. J. Biol. Chem. 280, 42744-42749 (2005).
3.
Haiser,H.J., Karginov,F.V., Hannon,G.J., & Elliot,M.A. Developmentally regulated cleavage of tRNAs in the bacterium Streptomyces coelicolor. Nucleic Acids Res. 36, 732-741 (2008).
4.
Thompson,D.M., Lu,C., Green,P.J., & Parker,R. tRNA cleavage is a conserved response to oxidative stress in eukaryotes. RNA. 14, 2095-2103 (2008).
5.
Lee,Y.S., Shibata,Y., Malhotra,A., & Dutta,A. A novel class of small RNAs: tRNAderived RNA fragments (tRFs). Genes Dev. 23, 2639-2649 (2009).
6.
Emara,M.M. et al. Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly. J. Biol. Chem. 285, 10959-10968 (2010).
7.
Yamasaki,S., Ivanov,P., Hu,G.F., & Anderson,P. Angiogenin cleaves tRNA and promotes stress-induced translational repression. J. Cell Biol. 185, 35-42 (2009).
8.
Wang,Q. et al. Identification and Functional Characterization of tRNA-derived RNA Fragments (tRFs) in Respiratory Syncytial Virus Infection. Mol. Ther. 21(2), 368-79 (2012).
9.
Falade,A.G. & Ayede,A.I. Epidemiology, aetiology and management of childhood acute community-acquired pneumonia in developing countries--a review. Afr. J. Med. Med. Sci. 40, 293-308 (2011).
t p ri
c us
d e t
an m
p e c
c A
10.
Welliver,R.C. Review of epidemiology and clinical risk factors for severe respiratory syncytial virus (RSV) infection. J Pediatr. 143, S112-S117 (2003).
11.
Zhang,Y. et al. Expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays. J. Virol. 75, 9044-9058 (2001).
12.
Mejias,A. et al. Whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection. PLoS. Med. 10, e1001549 (2013).
13.
Rehmsmeier,M., Steffen,P., Hochsmann,M., & Giegerich,R. Fast and effective prediction of microRNA/target duplexes. RNA. 10, 1507-1517 (2004).
14.
Agnello,V., Abel,G., Elfahal,M., Knight,G.B., & Zhang,Q.X. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc. Natl. Acad. Sci. U. S. A 96, 12766-12771 (1999).
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
15.
Srinivasakumar,N., Ogra,P.L., & Flanagan,T.D. Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay. J. Virol. 65, 4063-4069 (1991).
16.
Reddy,S.S., Connor,T.E., Weeber,E.J., & Rebeck,W. Similarities and differences in structure, expression, and functions of VLDLR and ApoER2. Mol. Neurodegener. 6, 30 (2011).
17.
Lu,B., Ma,C.H., Brazas,R., & Jin,H. The major phosphorylation sites of the respiratory syncytial virus phosphoprotein are dispensable for virus replication in vitro. J. Virol. 76, 1077610784 (2002).
18.
Bermingham,A. & Collins,P.L. The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription. Proc. Natl. Acad. Sci. U. S. A 96, 11259-11264 (1999).
19.
Haasnoot,J. & Berkhout,B. RNAi and cellular miRNAs in infections by mammalian viruses. Methods Mol. Biol. 721, 23-41 (2011).
20.
Franzen,O. et al. The short non-coding transcriptome of the protozoan parasite Trypanosoma cruzi. PLoS. Negl. Trop. Dis. 5, e1283 (2011).
21.
Hsieh,L.C., Lin,S.I., Kuo,H.F., & Chiou,T.J. Abundance of tRNA-derived small RNAs in phosphate-starved Arabidopsis roots. Plant Signal. Behav. 5, (2010).
22.
Hsieh,L.C. et al. Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing. Plant Physiol 151, 2120-2132 (2009).
23.
Peng,H. et al. A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm. Cell Res. 22, 1609-1612 (2012).
t p ri
c us
d e t
an m
p e c
c A
24. Goodarzi,H. et al. Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell 161, 790-802 (2015). 25.
Ren,J. et al. A novel mechanism for the inhibition of interferon regulatory factor-3dependent gene expression by human respiratory syncytial virus NS1 protein. J. Gen. Virol. 92, 2153-2159 (2011).
26.
Langmead,B. & Salzberg,S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359 (2012).
27.
Kent,W.J., Zweig,A.S., Barber,G., Hinrichs,A.S., & Karolchik,D. BigWig and BigBed: enabling browsing of large distributed datasets. Bioinformatics. 26, 2204-2207 (2010).
28.
Roberts,A., Pimentel,H., Trapnell,C., & Pachter,L. Identification of novel transcripts in annotated genomes using RNA-Seq. Bioinformatics. 27, 2325-2329 (2011).
29.
Trapnell,C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562-578 (2012).
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
30.
Deng,J. et al. Human metapneumovirus infection induces significant changes in small noncoding RNA expression in airway epithelial cells. Mol. Ther. Nucleic Acids 3, e163 (2014).
31.
Lee,K. et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA. 17, 1076-1089 (2011).
32.
Ren,J. et al. Human Metapneumovirus M2-2 Protein Inhibits Innate Cellular Signaling by Targeting MAVS. J. Virol. 86, 13049-13061 (2012).
33.
Ren,J. et al. Human metapneumovirus m2-2 protein inhibits innate immune response in monocyte-derived dendritic cells. PLoS. ONE. 9, e91865 (2014).
t p ri
c us
d e t
an m
p e c
c A
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Gene APOER2 NOP56 CALM1 SOX7 FTSJ3 SPATA2 C6orf120 TNFRSF10D PBDC1 EBNA1BP2 FTSJ2 MTF2 PSMA4 DBR1 AMD1 RPL37 RPL23 TCEAL4 C14orf2 SLIRP
Gene Accession # NM_004631 NM_006392 NM_006888 NM_031439 NM_017647 NM_006038 NM_001029863 NM_003840 NM_016500 NM_006824 NM_013393 NM_001164392 NM_002789 NM_016216 NM_001634 NM_000997 NM_000978 NM_001006937 NM_004894 NM_001267863
∆G (free energy) values -42.5 -40.6 -40.1 -39.7 -39.6 -39.0 -38.9 -37.1 -36.8 -36.0 -35.3 -34.2 -33.9 -33.7 -32.6 -32.5 -32.4 -32.3 -31.6 -30.1
t p ri
c us
an m
Table I. List of genes which were downregulated by RSV (log2 fold change<-1) and had increased binding to tRF5-GluCTC (fold change >10) and predicted minimum free energy (∆G) for their interaction with tRF5-GluCTC ≤-30 kcal/mol.
d e t
p e c
c A
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
Legends: Figure 1. tRF5-GluCTC controls the expression of APOER2. (A) Workflow to identify tRF5GluCTC-associated mRNAs. (B) Sequence alignment of tRF5-GluCTC with APOER2. The interactive region was identified by both RNAhybrid analysis and the binding assays. (C) The APOER2 mRNAs interacting with biotinylated tRF5-GluCTC was confirmed by RT-PCR, using β-actin as a negative control to demonstrate the binding specificity. (D) The mimic of tRF5-GluCTC decreases APOER2 expression. A549 cells were transfected with 25 nM WT-mimic for tRF5-GluCTC or control mimic
t p ri
(ctrl-mimic). At 15 hr post transfection, total cell lysate was harvested, followed by western blot to measure the expression of APOER2. (E and F). Antisense against tRF5-GluCTC increases APOER2
c us
expression. A549 cells in a 6-well plate were co-transfected with 120 nM antisense oligos, anti-tRF5-
an m
GluCTC or anti-ctrl. After 2 hr post transfection, cells were mock-infected or infected with RSV at MOI of 1, then harvested at 15 hr p.i. to harvest total RNAs or proteins to measure the APOER2’s mRNA by
d e t
qRT-PCR (E) or APOER2 protein by western blot (F) respectively. FC: Fold changes. Figure 2. Targeting elements of tRF5-GluCTC. (A) Construction of the firefly (Pp) luciferase plasmid
p e c
containing the targeted sequence of APOER2, WT or mutants to identify the motif(s) critical for tRF5-
c A
GluCTC’s trans-silencing function. (B) The effect of tRF5-GluCTC on Pp luciferase expression from Pp-WT. A549 cells in a 24-well plate were co-transfected with a Pp- WT and a plasmid expressing renilla Rr luciferase, and 120 nM antisense oligos, anti-tRF5-GluCTC or anti-ctrl. After 2 hr post transfection, cells were mock-infected or infected with RSV at MOI of 1. At 15 hr p.i. cells were lysed to measure the luciferase activity. Pp values were first normalized by Rr values yielding relative Pp/Rr values (y-axis). * denotes P value <0.05, relative to the first white bar. (C-E) Targeting motifs. A549 cells, co-transfected with a Pp luciferase plasmid, Pp-WT, Pp-Mut5 , Pp-MutM, Pp-Mut3, Pp-Mut3-A, or Pp-Mut3-B (0.1 µg/well of 24-well plate), and a Rr luciferase plasmid were infected with RSV at MOI of 1 (C) or treated with 100 nM WT-mimic oligo (D). Mock infection or the ctrl-
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
mimic was used as a control for C and D respectively. At 15 hr p.i. or 30 hr post-transfection, cells were lysed for luciferase assays. Values at y-axis (Pp/Rr) are a representative of three independent experiments and are expressed as mean ± SE. * or ** on the bars denotes P value <0.05 or <0.01 respectively, relative to the black bar (Pp-WT-transfected plus RSV-infected or WT-mimic-treated samples). (E). A549 cells in hexaplicate were co-transfected with a Pp-WT plasmid (0.1 µg/well of 24well plate), an Rr plasmid, and 100 nM mimic (WT or Mut3, please see illustration in A). After 15 hr post transfection, cells were harvested to measure luciferase activities. Values at y-axis (Pp/Rr) are a representative of three independent experiments and are expressed as mean ± SE. **on the second bar
t p ri
(WT-mimic transfected) denotes P value <0.01, relative to the first bar (ctrl-mimic transfected).
c us
Figure 3. Antiviral effect of APOER2. (A-C) The effect of APOER2 silencing on RSV replication. A549 cells were transfected with 100 nM siRNA against APOER2 (si-APOER2) or si-ctrl using
an m
Lipofectamine 2000. At 48 hr post transfection, cells were mock-infected or infected with RSV at MOI of 1 for 15 hr. (A) Total proteins were prepared to confirm the silencing efficiency of si-APOER2. (B)
d e t
Infectious particles were measured by immunostaining and the values expressed as pfu/ml. (C) Genome
p e c
copies were measured by qRT-PCR. (D-F) The effect of APOER2 overexpression on RSV replication. A549 cells in 6-well plate were transfected with a plasmid encoding Flag-tagged APOER2 (2 µg/well)
c A
or a control plasmid for 30 hr, followed by mock infection or RSV infection (MOI of 1) for 15 hr. (D) Confirmation on Flag-APOER2 expression by western blot. (E) Total infectious particles measurement. (F) Genome copies of RSV. * and ** denotes p < 0.05 and < 0.01 respectively, relative to its ctrl plasmid . Figure 4. APOER2 interacts with RSV P protein. (A) APOER2 forms a complex with RSV P protein. A549 cells in 6-well plate were transfected with 2 µg/well plasmids encoding Flag-tagged APOER2 or it control vectors. At 30 hr post transfection, cells were mock infected or infected with RSV at MOI of 1. At 6 hr p.i. total cell lysates were immunoprecipitated with an anti-Flag antibody followed by western blot using an anti-RSV antibody to detect associated RSV protein(s). A small aliquot was also prepared
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
before the IP for equal input assays. (B) Overexpressed RSV P protein restores APOER2-inhibited RSV replication. HEK-293 cells at 60-70 % confluence were transfected with a plasmid encoding V5-tagged RSV P or N (control protein) or their common control vector. After 30 hr cells were mock infected or infected with RSV at MOI of 1 for 15 hr. The viral particles in the supernatant were titrated by immune staining and protein overexpression was confirmed by western blot. (C) A mechanism model for tRF5GluCTC-pormoted RSV replication. RSV-induced tRF5-GluCTC targets APOER2, leading to suppressed APOER2 expression and consequently releasing more RSV P protein to facilitate RSV genome replication.
t p ri
c us
Table I. Common mRNAs identified by methods demonstrated in Fig. 1A and with predicted minimum free energy hybridization (∆G) ≤-30 kcal/mol to interact with tRF5-GluCTC.
an m
Supplementary Table I. mRNAs with enhanced binding to tRF5-GluCTC and decreased expression by
d e t
RSV infection. Cutoff criteria were demonstrated in Fig. 1A.
p e c
Supplementary Figure 1. tRF5-GluCTC inhibits the transcription of APOER2. A549 cells in 6-well
c A
plate were co-transfected with a Pp- WT and 120 nM antisense oligos, anti-tRF5-GluCTC or anti-ctrl. After 2 hr post transfection, cells were mock-infected or infected with RSV at MOI of 1. At 15 hr p.i. cells were lysed to isolate total RNAs. After the treatment of DNase, the transcription of luciferase, controlled by the APOER2-derived target of tRF5-GluCTC, was measured by qRT-PCR samples using paired primers as follow: 5'-GAGGTTCCATCTGCCAGGTATC-3' and 5' – CGGTTTATCATCCCCCTCG-3'. ** denotes P value <0.01, relative to the first white bar.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
ACCEPTED ARTICLE PREVIEW
© 2015 The American Society of Gene & Cell Therapy. All rights reserved