Listeria monocytogenes secretes a small RBP that we named Zea. We show that Zea binds a subset of L. monocytogenes RNAs, causing their accumulation in the extracellular medium. Furthermore, during L. monocytogenes infection, Zea binds RIG-I, the non-self-RNA innate immunity sensor, potentiating interferon-β production. Mouse infection studies reveal that Zea affects L. monocytogenes virulence. Together, our results unveil that bacterial RNAs can be present extracellularly in association with RBPs, acting as “social RNAs” to trigger a host response during infection.RNA-binding proteins (RBPs) perform key cellular activities by controlling the function of bound RNAs. The widely held assumption that RBPs are strictly intracellular has been challenged by the discovery of secreted RBPs. However, extracellular RBPs have been described in eukaryotes, while secreted bacterial RBPs have not been reported. Here, we show that the bacterial pathogen   •L. monocytogenes secretes an RNA-binding protein, Zea•L. monocytogenes RNA, resulting in extracellular RNA accumulationZea binds and protects •During infection, Zea binds RIG-I and modulates RIG-I-dependent IFN response•L. monocytogenes virulence in miceZea plays a role in  L. monocytogenes secretes an RNA-binding protein, Zea, which associates extracellularly with L. monocytogenes RNA. During infection, Zea binds RIG-I and modulates the type I IFN response. These results reveal that RNA can traffic between organisms via RBPs.Extracellular RBPs have only been described in eukaryotes. Pagliuso et al. report that  RNA-binding proteins (RBPs) are found in all living organisms. By binding RNAs, RBPs assemble in ribonucleoprotein complexes that dictate the fate and the function of virtually every cellular RNA molecule. In bacteria, RBPs interact with their cognate RNAs via classical RNA-binding domains (RBDs), structurally well-defined signatures that recognize specific RNA sequences and/or motifs  response. We further show that Lmo2686 affects L. monocytogenes virulence in vivo. Based on these findings, we propose to rename this protein Zea—as Zea, also known as Hecate, is an ancient Greek goddess who protected and guided the travelers. The presence of Zea orthologs in other bacterial species revealed that secretion of RBPs is a conserved phenomenon in prokaryotes.In this study, we report the identification of a secreted bacterial RBP, the lmo2686/zea open-reading frame is 534 bp long  data have revealed a transcriptional start site upstream of the start codon of zea A (Wurtzezea . zea appd at 4°C .zea gene encodes a protein of 177 amino acids (aa) (The ids (aa) B. Analysids (aa) B. Of notids (aa) . We coulL. monocytogenes cytosol and in the culture medium. Immunoblot analysis revealed that Zea could be recovered from the culture medium, indicating secretion of the protein (zea-deleted strain (Δzea) did not show any immunoreactive band, thus confirming the specificity of our antibodies. The secretion of Zea was also confirmed by engineering a L. monocytogenes strain carrying a chromosomally integrated copy of the C-terminally FLAG-tagged zea gene under the control of a constitutive promoter (zeaFLAG)  D. QuantizeaFLAG) D.4K15). Zea is a toroid-shaped homohexamer in which every monomer essentially contacts the neighboring molecule via a beta sheet-hairpin-beta sheet unit  by Minasov and colleagues  have the intrinsic capability to bind RNA . InteresL. monocytogenes cytosol and culture medium  co-immunoprecipitation of hemagglutinin (HA)- and FLAG-tagged versions of Zea G, (2) sie medium H, and . Zea was immunoprecipitated from L. monocytogenes cytosol and culture medium, and the Zea-bound RNAs were subsequently extracted and sequenced. As a control, we performed a mock IP using an unrelated antibody of the same isotype. Remarkably, RIP-seq analysis revealed the presence of L. monocytogenes RNAs almost exclusively in the culture medium compared with the control samples (2 fold change (log2FC) >1.5, corresponding to an almost 3-fold increase, was used for the identification of Zea-associated RNAs. Importantly, the enrichment of specific RNAs in the Zea IP was uncorrelated to their expression levels (We then examined whether Zea could bind RNA. We performed RNA immunoprecipitation (IP) of cytosolic extract and culture supernatant followed by high-throughput sequencing (RIP-seq) A. Given  samples A, indican levels B  by using recombinant HisZea and s to RNA A and 3B.mall RNA C and 3D.in vitro-transcribed biotinylated RNA and recombinant HisZea. Here, in addition to rli143 and rli92, we tested two other small RNAs (rli18 and rli1), which also displayed specific binding to Zea in the RIP-seq dataset (rli80) that was not specifically bound by Zea. Using this alternative approach, we confirmed binding above background level for rli143  in the culture medium. EF-Tu was undetectable in the culture medium, indicating that bacterial lysis under our experimental conditions was negligible , Δzea, and zea+. For this purpose, L. monocytogenes was grown in minimal medium (MM), because rich medium (BHI) contains RNA. qPCR analysis on RNA extracted from the culture medium revealed that overexpression of Zea increased the amount of the extracellularly detected phage RNA  and performed sequencing. Differential gene expression analysis revealed that, besides the overexpressed Zea RNA, 36 endogenous transcripts were significantly more abundant in the medium of the zea+ strain (−5). Of note, among these 12 enriched secreted RNAs, 8 RNAs proceeded from the A118 phage. qPCR analysis of intrabacterial phage RNA from the WT and zea+ strains revealed similar amounts of the majority of the phage genes tested, indicating that Zea does not affect the expression of phage genes  with L. monocytogenes WT and pulled down the Strep-tagged proteins. Co-purified RNA molecules from three independent replicates were sequenced and mapped to the L. monocytogenes genome. We found 15 RNAs specifically enriched in the RIG-I pull-down, and 9 of them (60%) belonged to the phage A118 locus  family  can sense non-self RNA in the cytoplasm, but only RIG-I and MDA5 can trigger the type I IFN signaling cascade . A recen18 locus . We did 18 locus , in agrenfection . These dL. monocytogenes WT versus cells infected with zea+. qPCR analysis revealed that overexpression of Zea increased the amount of IFN-β while IFN-γ was undetectable  . Transfe did not F. This s did not G. We con did not .Figure 7L. monocytogenes secretes Zea, a small RBP, which associates extracellularly with a subset of L. monocytogenes RNAs. We found that the overexpression of Zea correlates with an increased amount of its RNA ligands in the culture medium. This might be due to two non-mutually exclusive phenomena: (1) increased secretion of Zea-RNA complexes and (2) increased stabilization of Zea RNA targets following Zea accumulation in the extracellular medium. In the latter case, the pathway regulating RNA secretion would remain to be identified. The presence of Zea orthologs in other bacterial species indicates that the secretion of RBPs is a conserved phenomenon in prokaryotes. Our results suggest that additional RBPs remain to be identified in bacteria; however, their identification might be difficult, as bacterial RBPs are likely devoid of classical RBDs  RNAs from an invading bacterium. These data reinforce the concept that RIG-I has primarily evolved to sense viral RNAs and highlight that phage RNAs from a bacterial pathogen contribute to RIG-I activation.Our work reveals that the secretion of an RBP during bacterial infection regulates the induction of the INF response. We provide evidence that during L. monocytogenes virulence in vivo as its deletion results in an increased bacteria burden in the organs of infected mice. Strikingly, and in agreement with our results, Zea is absent in the hypervirulent strains of L. monocytogenes (lineage I), but it is well conserved in the strains from lineage II, which include a smaller number of clinical isolates. Given the complexity of the innate immune response to L. monocytogenes infection .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Pascale Cossart  medium (GIBCO) with shaking at 200 rpm at 37°C. E. coli cells were grown in LB broth. When required, antibiotics were added . LoVo cells were maintained in Ham’s F-12K medium (GIBCO) supplemented with 20% fetal calf serum and Glutamax (GIBCO). Strep-tagged RIG-I, MDA5, LGP2 and mCherry cell lines  were fused via splicing by overlap extension PCR and cloned with appropriate restriction sites into the integrative suicide vector pMAD as previously described  were purchased by Charles River, Inc. All animal experiments were carried out in strict accordance with the French national and European laws and conformed to the Council Directive on the approximation of laws, regulations, and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes (86/609/Eec). Experiments that relied on laboratory animals were performed in strict accordance with the Institut Pasteur’s regulations for animal care and use protocol, which was approved by the Animal Experiment Committee of the Institut Pasteur .L. monocytogenes of both full-length ZeaFlag, full-length untagged Zea and Lmo2595, the entire ORFs (with or without a Flag tag at the C terminus for Zea) were synthesized as a gBlocks (Integrated DNA Technologies) and subcloned into the integrative plasmid pAD, downsteam of the Phyper promoter (pHyper promoter was synthesized as a gBlock (Integrated DNA Technologies) and cloned in the pP1 plasmid with the appropriate restriction enzymes. To create a plasmid for the overexpression of HisZea in E. coli , the zea ORF was amplified by PCR from L. monocytogenes genomic DNA and cloned with the appropriate restriction enzymes into the pET28a plasmid, downstream of the polyhistidine tag. To create a plasmid for the overexpression of ZeaFlag in mammalian cells, the cDNA encoding the predicted mature form of Zea was codon-optimized for human expression and synthesized (GeneCust) with a 2xFlag tag at the N terminus. The resulting construct was then subcloned into pcDNA3.1(+) using the appropriate restriction sites. Modified pCineo plasmid carrying GW cassette (pCineoGW) and the Cherry coding sequence was provided by Dr.Yves Jacob (Institut Pasteur). pEXPR-IBA105-RIG-I and pEXPR-IBA105-mCherry plasmids for the overexpression of Strep-RIG-I and Strep-mCherry, respectively, were previously described . Two rabbits were injected with each antigen supplemented with Freund’s adjuvant . The total IgG fractions were affinity-purified via a resin column containing the antigenic peptide. The affinity-purified antibodies were dialyzed against PBS and 50% glycerol and stored at −20°C. A mix of the three antibodies  was used for immunoblotting. The specificity of the anti-Zea antibodies in immunoblotting and immunoprecipitation was verified by comparing bacterial extract and culture medium prepared from the L. monocytogenes was grown to exponential phase (OD600 = 1). Bacteria were harvested by centrifugation  and proteins in the culture medium fraction were precipitated by addition of 40% ammonium sulfate and incubated at 4°C . Protein were recovered by centrifugation  and resuspended in water. Samples were dialyzed against water , concentrated using an Amicon centrifugal filter units  and resuspended in LDS-PAGE sample loading buffer . The bacterial pellet was washed twice in PBS and resuspended in lysis buffer . Bacteria were transferred to 2 mL lysing matrix tubes  and mechanically lysed by bead beating in a FastPrep apparatus . Subsequently, tubes were centrifuged  to remove cellular debris. To quantify the partition of Zea between bacterial cytosol and culture medium, equal volumes of culture supernatant and bacterial cytosol were analyzed by gradient SDS-PAGE and subjected to immunoblotting via wet transfer onto 0.45 μm nitrocellulose membrane (Millipore). EF-Tu and InlC proteins were used as marker of intracellular and extracellular fractions . The protein pellets were washed twice with ice-cold acetone and dried at 95°C for 5 min. Proteins were resuspended in NuPage LDS sample buffer and an equal percentage of bacterial cytosol and culture medium were subjected to immunoblotting as above.For detection of endogenous Zea in culture medium, ractions . Detectiescribed . BrieflyE. coli C43 bacteria which were grown at 37°C in Terrific broth (TB) (Thermo Fisher Scientific) supplemented with 50 μg/mL kanamycin. Expression was induced by the addition of IPTG to a final concentration of 1 mM at OD600nm = 0.7 AU. Cultures were incubated overnight, and cells were harvested by centrifugation . The bacterial pellet was resuspended in Buffer A . All subsequent steps were performed at 4°C. Cell lysis was carried out by passing the samples three times through a pre-cooled microfluidizer operating at 17,000 psi. The soluble fraction was then obtained by centrifugation at 39,000 × g for 45 min at 4°C. Subsequently, the supernatant was loaded onto a pre-equilibrated Ni-NTA column (QIAGEN) at 0.5 mL/min with a peristaltic pump at 4°C. The washing and elution steps were performed on an AKTA system using steps of 35% and 100% Buffer B . The fractions containing Zea were pooled, concentrated with an Amicon centrifugal filter unit , and further purified by size-exclusion chromatography on a Hi Load S200 10/300 column  pre-equilibrated in Buffer C . Peak fractions were pooled, concentrated to 10 mg/mL, and subsequently dialyzed against Buffer D . After dialysis, protein concentration was assessed again and the sample was flash-frozen in liquid nitrogen. During purification, the purity and homogeneity of the sample were monitored by SDS-PAGE.pET28a-HisZea (described above) was used to transform L. monocytogenes was extracted as previously described (600nm = 0.8-1.0 for growth cultures in BHI or OD600nm = 0.4 for growth cultures in MM) or stationary phase  were pelleted by centrifugation . Pellets were resuspended in 1 mL TRIzol Reagent (Ambion), transferred to 2 mL Lysing Matrix tubes and mechanically lysed by bead beating in a FastPrep apparatus . Subsequently tubes were centrifuged  in a tabletop centrifuge and lysates were transferred to a 2 mL Eppendorf tube. RNA isolation proceeded according to the manufacturer’s instructions. Briefly, 200 μL of chloroform (Sigma) were added to the lysate, shaken and incubated for 10 min at room temperature, followed by centrifugation . The upper aqueous phase was removed and transferred to a new 1.5 mL Eppendorf tube and RNA was precipitated by the addition of 500 μL isopropanol and incubation at room temperature for 5– 10 min. RNA pellets  were washed twice with 75% ethanol and resuspended in 50 μL of nuclease-free water (Ambion).Total RNA from escribed . BrieflyL. monocytogenes was grown until exponential phase (OD600nm = 0.4). Medium was recovered by centrifugation , filtered (0.22 μm) and concentrated 10 times using an Amicon centrifugal filter unit (3K cut-off). Medium was then brought back to the initial volume by adding nuclease-free water and concentrated again. This desalting process was repeated three times to avoid co-precipitation of salts during the subsequent RNA isolation. RNA was then extracted twice with acid phenol/chloroform, precipitated with ethanol/0.3 M sodium acetate and resuspended in nuclease-free water.To extract total secreted RNA from MM, g, at 4°C), the upper aqueous phase was transferred to a new Eppendorf tube and RNA was precipitated by the addition of an equal volume of isopropanol and incubation at room temperature for 10 min. RNA pellet was washed twice with 70% ethanol and resuspended in 50 μL of nuclease-free water.RNA extraction from LoVo cell monolayers in 6-well plates was performed by using TRIzol Reagent. Briefly, cells were washed once with ice-cold PBS and directly lysed in the well by adding 1 mL of TRIzol and gentle pipetting. Samples were vortexed thoroughly for 30 s before the addition of 200 μL chloroform and then incubated 3 min at room temperature. After centrifugation  and resuspended in nuclease-free water. Purified cDNA (200 ng) was transcribed in vitro by using the MAXIscript T7 in vitro transcription kit (Invitrogen) according to the manufacturer’s recommendation. The quality of the in vitro-transcribed RNA was verified by SYBR Gold (Life Technologies) staining after running on 6% Novex TBE-Urea gel (Thermo Fisher Scientific) or by the Bioanalyser RNA nano kit (Agilent). The p2RZ vector expressing a part of Cherry protein transcript was described elsewhere  was included in the reaction mixture.cDNA templates of the lsewhere  and lineIn vitro formation of HisZea - RNA complexes was assessed by electrophoretic mobility gel shift assay (EMSA). For in vitro RNA synthesis, 1 μg of cDNA template carrying a T7 promoter was amplified by PCR and in vitro-transcribed using the MAXIscript T7 in vitro transcription kit according to the manufacturer’s instructions. The quality of the in vitro-transcribed RNA was verified as described above. RNA was purified and concentrated using “RNA clean & concentrator” (Zymo research) before dephosphorylation and 5′ end labeling as previously described  and discarded. The HisZeap-containing supernatant was used in the subsequent steps. This preclearing step was performed in order to remove the Zea fraction which aggregated non-specifically onto the beads. Biotinylated RNA (500 nM) was added to the HisZea-containing supernatant and incubated for 30 min, 4°C with shaking. Then, 50 μL of equilibrated streptavidin magnetic beads were added for a further incubation . The beads were then washed four times in binding buffer and bound HisZea was recovered by addition of NuPage LDS sample buffer.HisZea (2.5 μg) was incubated with 50 μL of equilibrated streptavidin magnetic beads (BioLabs) in 250 μL of binding buffer  for 45 min at 4°C with shaking. Beads were recovered by centrifugation  and incubated at 25°C for 30 min. Then, 0.0033U of RNaseI (Ambion) were added before incubation for either 1 or 3 min at 37°C. Reactions were stopped by addition of NuPage LDS sample buffer and samples were loaded on 8% Novex TBE-Urea gels (Thermo Fisher Scientific). Signals were detected by autoradiography (at least one-h exposure at −80°C in presence of an intensifying screen).Equimolar concentrations of HisZea or GST (1 μM) were mixed with L. monocytogenes overnight cultures expressing either ZeaFlag alone, or both ZeaFlag and ZeaHA were centrifuged  to collect bacteria and culture medium. The recovered medium was filtered by using Millex-GP 0.22 μm filters (Millipore), supplemented with 0.2% of Triton X-100, before adding 20 μL of M2 Flag magnetic beads (Sigma). Samples were shaken for 2 h at 4°C and then washed four times with lysis buffer . The immunoprecipitated material was finally eluted using 100 μg/mL of 3xFlag peptide (Sigma) according to the manufacturer’s instructions. Bacterial pellet was washed twice in ice-cold PBS, resuspended in lysis buffer and lysed in a FastPrep apparatus . The samples were then clarified by centrifugation  and protein concentration determined by Bradford assay. The same percentage of bacterial cytosol compared to the culture medium was used to immunoprecipitate ZeaFlag, under the same condition used for the culture medium. Equal amounts of eluted proteins were subjected to immunoblotting via wet transfer onto a 0.45 μm nitrocellulose membrane.To assess the interaction between ZeaFlag and ZeaHA, 25 mL of L. monocytogenes wt and zeaFlag overnight cultures were recovered by centrifugation  and filtered by using Millex-GP 0.22-μm filters. Filtered culture medium was supplemented with 0.2% Igepal. Then, 25 μL of M2 Flag magnetic beads were added and samples were incubated overnight at 4°C with shaking. Magnetic beads were recovered and washed four times with washing buffer  and left on ice while preparing the cell lysate. HEK293 cells stably transfected with Strep-RIG-I were lysed in lysis buffer , sonicated twice for 15 s at 20% amplitude and incubated on ice for 30 min. The cell lysate was cleared by centrifugation  with the supernatant assayed for protein concentration with Bradford assay and used fresh. At least 1 mg of cell lysate was added to the Zea-containing washed Flag magnetic beads (prepared above) and incubated overnight at 4°C with shaking. Beads were washed four times in washing buffer and twice in washing buffer without Igepal. For RNaseA treatment, 100 μg/mL of RNaseA (Roche) in lysis buffer without Igepal were added to the beads  followed by two further washes in the same buffer. Zea was eluted from the magnetic beads with 3xFlag peptide at 100 μg/mL, according to the manufacturer’s recommendations, in a total volume of 50 μL. Samples were then subjected to immunoblotting via wet transfer onto a 0.45-μm nitrocellulose membrane.To assess the interaction between ZeaFlag and Strep-RIG-I, 25 mL of culture medium from 2 dishes were transiently co-transfected with 7 μg of each DNA (ZeaFlag and Strep-RIG-I) using 24 μL of Lipofectamine LTX (Thermo Fisher Scientific). 24 h after transfection, the cells were washed twice with PBS and lysed using 1 mL lysis buffer per dish . The lysate was sonicated for 15 s, at 20% amplitude and incubated on ice for 30 min with shaking. The lysate was then clarified  and assayed for protein concentration (Bradford). 0.5 mg of total lysate was incubated with 15 μL of M2 Flag magnetic beads . Beads were recovered and washed three times in lysis buffer before treatment with RNase A and elution with the 3xFlag peptide (both performed as above). Samples were then subjected to immunoblotting via wet transfer onto a 0.45-μm nitrocellulose membrane.For ZeaFlag immunoprecipitation from mammalian cells, LoVo cells in 10-cmg, 30 min, 4°C). The bacterial pellet was washed twice with ice-cold PBS and mechanically lysed in 1 mL of lysis buffer  in 2 mL Lysing Matrix tubes by bead beating in a FastPrep apparatus . Bacterial lysate was clarified by centrifugation  and protein concentration was determined by Bradford assay. The culture medium was filtered by using Millex-GP 0.22-μM filters. 5 μL of anti-Hfq anti-serum were added to an equal percentage of bacterial cytosol and culture medium and incubated overnight at 4°C under shaking condition. Then, 50 μL of protein A Sepharose beads  were added for a further h of incubation . The immune complexes were collected by centrifugation . After three washes with lysis buffer, the bound protein was eluted from the protein A Sepharose beads by boiling (10 min) in 50 μL LDS sample buffer.For Hfq immunoprecipitation, bacteria were grown in 20 mL of BHI until stationary phase and pelleted , 20 μL of pre-equilibrated M2 Flag magnetic beads were added to equal percentage of cytosolic and nuclear fractions. Samples were incubated overnight at 4°C with shaking. After three washes in lysis buffer , immune complexes were retrieved by adding 100 mg/mL of 3xFlag peptide. Samples were then subjected to immunoblotting via wet transfer onto a 0.45-μM nitrocellulose membrane.Immunofluorescence was performed as previously described . Brieflyg, 4°C). The supernatant was isolated as the cytosolic fraction and recentrifuged as before to eliminate cell nuclear debris. The pellet was washed in buffer B  glycerol) and centrifuged 5 min at 500 × g, 4°C. Buffer A, B and SR were supplemented with 0.15 mM spermidine, 0.15 mM spermine, 1 mM DTT and protease inhibitor. The washed pellet was resuspended in sucrose buffer , followed by the addition of a high-salt buffer  to obtain a final salt concentration of 250 mM. Samples were incubated for 25 min and centrifuged for 10 min at 13,000 × g, 4°C. The supernatant was isolated as the nuclear soluble fraction from the pellet which represents chromatin and nuclear insoluble material. The pellet was resuspended in sucrose buffer supplemented with 0.0025 U/ μL) and 1 mM CaCl2 and was incubated at 37°C for 10 min. 4mM EDTA was added and samples were sonicated using the Bioruptor (Diagenode) for 7.5 min (15 s on and 1 min off) and centrifuged for 15 min at 13,000 × g, 4°C. The supernatants represent a soluble chromatin fraction.Fractionation of cultured cell lines was performed as previously described . Cell weL. monocytogenes was grown in 20 mL of BHI until stationary phase. Bacteria were pelleted  and then stained with LIVE/DEAD BacLight (Molecular Probes), following the manufacturer’s recommendation. Bacterial suspension (20 μL) was then deposited on a glass coverslip and immediately imaged by using a Zeiss AxioObserver.Z1 inverted fluorescence microscope equipped with an Evolve EM-CCD camera (Photometrics). Images were acquired with a 100 × N.A. 1.4 oil objective using MetaMorph.Δzea+zea+L. monocytogenes stationary phase (OD600nm = 3.5) culture (a zea-deletion strain in which one copy of the zea gene was integrated in the L. monocytogenes genome under the control of a constitutive promoter) was centrifuged  to recover the culture medium and the bacterial pellet. 10 mL culture medium were filtered (0.22-μm) and concentrated to 1 mL by using Amicon centrifugal filter unit (3K cut-off). Concentrated medium was then supplemented with 0,05% of Triton X-100, centrifuged again  and left on ice while preparing bacterial cytosolic extracts. The bacterial pellet was washed thrice in ice-cold PBS and lysed by mechanical shaking in a FastPrep apparatus (described above) in 1 mL of lysis buffer  supplemented with protease inhibitors mixture. Bacterial cytosol was recovered by centrifugation  and protein concentration determine by Bradford assay. One-milliliter of bacterial cytosol and concentrated culture medium  were individually loaded on a Superose 6 10/300 GL column pre-equilibrated with lysis buffer without Triton X-100. About 161 fractions of 220 μL were collected, and one out of every seven fractions was concentrated by acetone precipitation and the presence of Zea and EF-Tu analyzed by immunoblotting after wet transfer onto a nitrocellulose membrane. Fractions containing the complexes (A or B) were then pooled and processed for immunoprecipitation assays. Briefly, each sample was incubated overnight at 4°C with shacking, with a mix of 30 μg of anti-Zea antibodies (10 μg of each antibody) or 30 μg of normal rabbit IgG . Then, 50 μL of Protein A Sepharose were added for further 2 h to recover immunocomplexes. The beads were washed four times with lysis buffer and treated with Turbo DNase (Ambion) for 10 min at 37°C in 200 μL 1X Turbo DNase buffer. Samples were vortexed for 30 s after the addition of 200 μL of acid phenol (Ambion). Then, 50 μL chloroform (Sigma) were added and samples were centrifuged for 5 min at 10 000 × g at 4°C. The aqueous upper phase was recovered and transferred to a new Eppendorf tube. RNA was precipitated by adding the same volume of isopropanol and 0.3 M sodium acetate (Ambion) and 1 μL of glycogen (Invitrogen). Samples were centrifuged , and RNA was washed once with 70% ethanol before being resuspended in 25 μL of nuclease-free water. RNA was analyzed with the Bioanalyser RNA pico kit (Agilent). Purified RNA was fragmented with the “RNA fragmentation reagents” (Thermofisher), purified by ethanol precipitation and quality-controlled with the Bioanalyser, as described above. Directional RNA-seq libraries were prepared with 30 ng of purified RNA for each sample by using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer’s instructions. When required, the RNA samples were spiked-in with a synthetic in vitro-transcribed AdML splicing reporter (50 mL reporter  in orderL. monocytogenes EGD-e genome (NC_003210) and a list of 3160 transcripts  were downloaded from the Listeriomics database (samtools view -b –q 1 parameters) (https://github.com/becavin-lab/RIPSeq-Listeria). Data were normalized with the TMM (2(Fold changes) were calculated by subtraction of log2(TMM) normalized expression values.The database . After tdatabase . They wedatabase . Mappingameters) , and savameters) . The quaameters) . The numameters) . Statistameters)  and in-h the TMM  (edgeR pL. monocytogenes strains (wt and Δzea+zea+) were grown to exponential phase (OD600nm = 0.4) in 14 mL of MM under microaerophilic conditions using Oxoid AnaeroGen 2.5L gas packs (Thermo Fisher) at 25°C. Under this condition zea appeared slightly upregulated compared to standard growth conditions   and filtered (0.22 μm). The bacterial pellet was stored at −80°C for subsequent RNA extraction. 10 mL of the filtered culture medium were desalted and the RNA was extracted as described above. The quality of the RNA was checked by using the Bioanalyser RNA nano kit. The amount of recovered RNA was similar in all the samples. Total secreted RNA (5 μg) was ribodepleted by using the Ribo Zero rRNA removal kit (Illumina) following the manufacturer’s instructions. Ribodepletion was controlled by the Bioanalyser RNA pico kit. Directional RNA-seq libraries were prepared with 100 ng of purified RNA for each sample by using NEBNext Multiplex Small RNA Library Prep Set for Illumina according to the manufacturer’s instructions. Libraries were sequenced on an Illumina NextSeq500 platform (SR75).To extract total secreted RNA from the culture medium,  medium) . A paral-l 45) (samtools view -b –q 1 parameters)   . They we-l 45) . Mappingameters) , and savameters) . The quaameters) . The numameters) . Differeameters)  and DESeameters)  packagesL. monocytogenes bacterial cultures (Δzea+zea+ strain) were processed essentially as described for the RIP-seq experiment unless otherwise stated. In summary, L. monocytogenes was grown until the stationary phase (OD600nm = 3.5) and, for every sample, 50 mL of bacterial culture were processed as follows. Bacteria were pelleted at 2862 × g, 20 min, 4°C and culture supernatant was filtered and processed (5 mL) for total RNA purification , by performing two sequential phenol/chloroform extractions followed by ethanol/sodium acetate precipitation. The RNA pellet was washed once with ethanol 70% and resuspended in 20 μL nuclease-free water. Purified RNA was then treated with Turbo DNase and purified again, as described above. The remaining medium  was processed for Zea immunoprecipitation. Briefly, 20 μg of a mix of Zea antibodies (6.6 μg of each antibody) were coupled to 100 μL of Protein A Dynabeads (Invitrogen) for 2 h in 500 μL of PBS . Beads were then washed twice with PBS and once with lysis buffer . Culture medium was supplemented with 0.05% Triton X-100 before the addition of the anti-Zea antibody-coupled beads. Samples were then incubated overnight . Beads were washed four times with lysis buffer, treated with Turbo DNase and processed for RNA extraction. Purified RNA was stored at −80°C until use. The bacterial pellet was washed thrice in ice-cold PBS and then mechanically lysed by using FastPrep apparatus in 1 mL of lysis buffer supplemented with protease inhibitors mixture and RNasin (Promega) at 12.5 U/μL. Bacterial lysate was clarified by two sequential centrifugations and the final volume was carefully measured. A volume corresponding to 10% of the total sample (input) was treated with DNase and processed for RNA isolation by phenol/chloroform extraction and ethanol/sodium acetate precipitation. Purified RNA was resuspended in nuclease-free water and stored at −80°C until use. The remaining bacterial cytosol was incubated with an anti-Zea antibody coupled to Protein A as described above . Beads were washed four times with lysis buffer, treated with Turbo DNase and processed for RNA extraction. For qPCR analysis, 100 ng of purified RNA were subjected to reverse transcription in 20 μL final volume using the Reverse Transcription Kit (QIAGEN) according to the manufacturer’s instructions. Reactions were then diluted by adding 180 μL of nuclease-free water. qPCR was assayed in 10 μL reactions with Brillant III Ultra Fast SYBR-Green qPCR Master Mix (Agilent). Reactions were carried out in a Stratagene MX3005p system with the following thermal profile: 5 min at 95°C, 37 cycles of 10 s at 95°C and 12 s at 60°C. Results were analyzed with an MxPro software, as described earlier , bacterial strains were grown in MM until exponential phase (OD600nm = 0.4). L. monocytogenes wt, Δzea and Δzea+zea+ strains were used for the phage and lma-monocin quantification; L. monocytogenes wt, Δzea, zea+ and lmo2595+L. monocytogenes strains were used for the quantification of rli143; L. innocua wt, zea-pAD and lmo2595-pAD strains were employed for the quantification of rli143. The bacterial OD was measured, and the cultures were recovered when OD was equal for all the strains. MM was collected by centrifugation, filtered and processed for RNA extraction (as described above). Purified RNA (5-10 μg) was subjected to DNase treatment using the DNase treatment and removal kit (Ambion). Treated RNA (500 ng) was mixed with an equal amount of CleanCap™ OVA mRNA (TriLink) which serves as an internal control for normalization, and processed for reversed transcription and qPCR, as above. Gene expression levels were normalized to the OVA mRNA, and the fold change was calculated using the ΔΔCT method.For qPCR of Listeria genes from total (intracellular) L. monocytogenes RNA, the RNA was extracted, as described in the RNA extraction section and treated, as described above, except that the OVA mRNA was not included in the reverse transcription reaction. Gene expression levels were normalized to the rpob gene, and the fold change was calculated using the ΔΔCT method.For qPCR of For qPCR of Hfq-associated RNAs, RNA was extracted from immunoprecipitated Hfq, by using the protocol described for the RIP-seq of Zea. DNase-treated RNA (120 ng) was subjected to reverse transcription. Gene expression levels were normalized to the input fractions, and the fold change was calculated using the ΔΔCT method.For qPCR of IFNβ, IFNγ and IL-8, mammalian RNA was extracted, as described in the RNA extraction section. Purified RNA (5-10 μg) was subjected to DNase treatment, and 1 μg processed for reverse transcription, as described above. Gene expression levels were normalized to the actin mRNA and to the uninfected samples, and the fold change was calculated using the ΔΔCT method.2 tissue culture dishes per cell line were pretreated with 0.1 mg/mL poly-L-Lysine-hydrobromide (Sigma), rinsed with distilled water and dried for 1 h before plating the cells. Cells (30-40x106) were plated per dish in 20 mL of DMEM medium for 24 h before infection. Overnight L. monocytogenes EGD-e cultures in BHI were diluted 1/20 in fresh BHI the day of infection and grown up to OD600nm = 1. Each plate was infected with an MOI of 50 for 1 h before replacing the media with complete DMEM containing 10 μg/mL of gentamicin to kill extracellular bacteria. After an additional 3 h , plates were rinsed twice with ice-cold PBS, crosslinked at 400 mJ/cm2 in 10 mL of ice-cold PBS/plate and cells were then scraped, pelleted and resuspended in 8 mL of MOPS lysis buffer . Cell lysates were incubated on ice for 20 min with gentle mixing every 5 min and then clarified by centrifugation at 16000 × g for 15 min at 4°C. Streptactin Sepharose beads  were washed in MOPS washing buffer . Each sample was treated with proteinase K (Roche) in v/v of 2X proteinase K buffer  for 20 min at 4°C that has been preincubated 20 min at 37°C to remove RNase contamination. RNA purification was performed using TRI Reagent LS (Sigma). RNA was dissolved in 50 μL of DNase-free and RNase-free ultrapure water. Extracted RNAs were analyzed using Nanovue  and Bioanalyser RNA nano kit (Agilent) before being processed for next-generatio sequencing .Four 15-cmsamtools view -b –q 1 parameters)  (https://github.com/becavin-lab/RIPSeq-Listeria).Due to the high number of eukaryotic RNAs in the datasets and presence of insertions and deletions, the reads were trimmed (AlienTrimmer 0.4.0) , and mapameters) , and savameters) . The quaameters) . The numameters) . Differeameters)  and Edgeameters)    were seerry RNAs  using Lirry RNAs ) were usL. monocytogenes was thawed from glycerol stocks stored at −80°C and diluted in phosphate-buffered saline (PBS) before injection. A sublethal dose (104L. monocytogenes) was injected into the lateral vein of the tail of each mouse. The number of bacteria in the inoculum was confirmed by plating serial dilutions of the bacterial suspension onto BHI agar plates. For determination of bacterial loads, livers and spleens were recovered and disrupted in PBS at the indicated time points post-infection. Serial dilutions of organ homogenates were plated onto BHI agar plates, and colony forming units (CFUs) were counted after growth at 37°C for 48 h.All data are expressed as mean and standard error of the mean. Student’s t test or ANOVA were used for statistical analysis. Differences in means were considered statistically significant at p < 0.05. Sample number (n) indicates the number of independent biological samples in each experiment, for each set of experiments this information is provided in the figure legends.E-MTAB-7665]. All scripts used for the analysis have been deposited on the Institut Pasteur GitLab: https://github.com/becavin-lab/RIPSeq-Listeria.The accession number for RIP-Seq, RNA-seq and RLRs purification and sequencing data reported in this paper is [Array Express at EMBL-EBI]: [


Emerging evidence implicates a wide range of post-transcriptional RNA modifications that play crucial roles in fundamental biological processes including regulating gene expression. Collectively, they are known as epitranscriptomics. Recent studies implicate 3′ RNA uridylation, the non-templated addition of uridine(s) to the terminal end of RNA, as a key player in epitranscriptomics. In this review, we describe the functional roles and significance of 3′ terminal RNA uridylation that has diverse functions in regulating both mRNAs and non-coding RNAs. In mammals, three Terminal Uridylyl Transferases (TUTases) are primarily responsible for 3′ RNA uridylation. These enzymes are also referred to as polyU polymerases. TUTase 1 (TUT1) is implicated in U6 snRNA maturation via uridylation. The TUTases TUT4 and/or TUT7 are the predominant mediators of all other cellular uridylation. Terminal uridylation promotes turnover for many polyadenylated mRNAs, replication-dependent histone mRNAs that lack polyA-tails, and aberrant structured noncoding RNAs. In addition, uridylation regulates biogenesis of a subset of microRNAs and generates isomiRs, sequent variant microRNAs that have altered function in specific cases. For example, the RNA binding protein and proto-oncogene LIN28A and TUT4 work together to polyuridylate pre-let-7, thereby blocking biogenesis and function of the tumor suppressor let-7 microRNA family. In contrast, monouridylation of Group II pre-miRNAs creates an optimal 3′ overhang that promotes recognition and subsequent cleavage by the Dicer-TRBP complex that then yields the mature microRNA. Also, uridylation may play a role in non-canonical microRNA biogenesis. The overall significance of 3′ RNA uridylation is discussed with an emphasis on mammalian development, gene regulation, and disease, including cancer and Perlman syndrome. We also introduce recent changes to the HUGO-approved gene names for multiple terminal nucleotidyl transferases that affects in part TUTase nomenclature . Epitranscriptomics refer to a diverse set of RNA chemical modifications and post-transcriptional nucleotide additions that play central roles in pre-mRNA splicing, translation, and regulation of gene expression. For example, ribosomal and transfer RNAs undergo extensive chemical modifications that are required for function through stabilization of their RNA secondary structure, while non-templated nucleotide additions are critical for polyadenylated mRNAs and aminoacylation of tRNAs. Recent evidence implicates 3′ RNA uridylation, the addition of non-templated uridine(s) to the RNA end, in several biological processes. This review presents our current understanding of the biochemical and functional roles for 3′ terminal RNA uridylation with a focus on mammalian biology. Over the past decade, 3′ RNA uridylation has emerged to be functionally significant for multiple RNA types such as mRNAs, microRNAs, and structured non-coding RNAs. Terminal Uridylyl Transferase (TUTases) that are also known as polyU polymerases are the enzymes responsible for 3′ RNA uridylation.kcat values of 0.059 and 0.002 s−1  complex, is required for splicing and contains five essential snRNPs . U6 small nuclear RNA (U6 snRNA) has a unique maturation mechanism  that primarily function as negative regulators of their mRNA targets  to the 3′ end of microRNAs is an important post-transcriptional modification that has been shown to impact activity and biogenesis of miRNAs. Specific pre-miRNAs are substrates for monouridylation and/or oligouridylation that can impact microRNA biogenesis positively or negatively, respectively. As the name indicates, monouridylation is the addition of a single non-templated uridine, while oligouridylation and polyuridylation refer to the addition of polyU-tails that can be as long as 30 nucleotides.in vivo. Sequencing of pre-let-7 revealed that roughly 20% of all pre-let-7 microRNAs were monouridylated at their 3′ ends in HeLa cells that lack expression of both LIN28A and LIN28B. In contrast, all other single nucleotide additions to pre-let-7 combined represent only 1%. Analysis of numerous deep sequencing libraries indicated that the majority of mature Group II let-7 microRNAs are likely processed from monouridylated precursors, as evidenced by their let-7-3p reads. In vitro uridylation assays identified three terminal ribonucleotidyl transferases  competent for mono-uridylating pre-let-7a-1 in vitro and each had a preference for pre-let-7a-1 with a 1nt vs. 2nt 3′ overhang. The NTP specificity was determined for these enzymes, revealing that TUT4 and TUT7 utilized UTP, while TENT2 could use UTP, GTP, or ATP. In vivo knockdowns performed singly or in combination revealed that these enzymes function redundantly in HeLa cells where triple knockdowns resulted in the most significant loss of Group II mature let-7 and mono-uridylated pre-let-7. For single knockdown, TUT7 had the most prominent effect. Similar results were observed for miR-105 in the triple knockdowns. It remains unclear if TENT2 is an in vivo TUTase and whether its effects are mediated by monoadenylation rather than monouridylation, given the reported affinity of TENT2 for ATP over UTP  and CACNA1C (Cav 1.2), leading to dysregulation of important gap junction and calcium channel proteins in the heart.Polyuridylation has also been proposed to regulate miR-1 biogenesis in myotonic dystrophy patients , leading to incorporation into the RNA-induced silencing complex (RISC). Although Ago1-4 are individually competent to bind and load microRNAs and siRNAs into RISC, Ago2 is unique in having slicer activity that is responsible for siRNA-mediated (and to a much lesser extent miRNA-directed) cleavage of their target mRNAs.dicer mutants, and maternal-zygotic ago2 mutants at 48 h post fertilization   and guanylation at the end of mRNAs with longer polyAs (>40 nts) at the downstream of the poly(A) tail. Roughly 50 and 80% of polyadenylated transcripts are uridylated at a frequency of >5 and >2%, respectively, with specific transcript as high as 40%. Considering that uridylation targets mRNAs for degradation, the levels of uridylated transcripts at steady state are rather remarkable. Furthermore, they showed that the TUT4 and TUT7 are the responsible TUTases for mRNA uridylation and their knockdown increased the half-life of numerous transcripts. Overall, they discovered more than >600 genes upregulated in HeLa cells upon TUTase loss. Their data supports the idea that uridylated mRNAs are degraded by canonical pathways where DIS3L2 only has a minor role as anticipated given the limited number of Us added to mRNA. The further importance of TUTases in regulation of the maternal transcriptome during oocyte development and in the maternal-to-zygotic transition that occurs shortly after fertilization is discussed in section TUTases in Vertebrate Development and Physiology.During apoptosis, uridylation plays a critical role in the rapid degradation of global mRNAs that relies prominently on DIS3L2  in multiple vertebrate species that include frogs, fish, and mice  in comparison to their non-uridylated counterparts, highlighting that a polyU-tail directs at least in part the activity of this enzyme. In the study of Ustianenko and colleagues, they identified Dis3L2 initially as an oligoU binding protein (Ustianenko et al., Recent work has identified multiple non-coding RNAs as direct targets for Dis3l2-mediated decay where the underlying theme is terminal polyuridylation is the key specificity determinant. Chang and colleagues showed that Dis3l2 knockdown in mouse embryonic stem cells does not affect mature let-7 levels, consistent with polyuridylated pre-let-7 no longer being a Dicer substrate; however, an increase in polyuridylated pre-let-7 levels was observed. They normalized their data to U6 snRNA, a key spliceosomal component that undergoes oligouridylation and trimming. bona fide molecular targets for therapeutic intervention in cancer where their specific inhibition may be well-tolerated in patients (Piskounova et al., Although the last decade has revealed the central importance of uridylation in mammals, much remains to be discovered that has significant implications for basic biology and translational medicine. For example, the LIN28/let-7 pathway is directly implicated in numerous poor prognosis cancers where expression of either LIN28 paralog may confer resistance to ionizing radiation and several frontline chemotherapies such as doxorubicin, 5-fluorouracil, taxanes, and platinum-based drugs (reviewed in Lee et al., Further research is also needed to define the precise molecular mechanisms by which TUT4 promotes the development of poor prognosis breast cancers and high grade gliomas, independently of the LIN28/let-7 pathway. Ongoing research continues to unravel how mutations in DIS3L2 contribute to Perlman syndrome and Wilms tumors. Also, advances in next generation sequencing technology such as nanopore sequencing may enhance our ability to look at terminal RNA uridylation that does not involve cumbersome sequencing library construction that is used for small-RNA-Seq or TAIL-Seq. In terms of more basic biology, uridylation is likely regulated in cell-type and disease-specific manners. Therefore, the complete repertoire of functionally significant uridylation targets remains to be elucidated. Numerous biological questions remain about RNA uridylation. What regulates TUTase expression and function? Are there other tissue specific RNA binding proteins that function like LIN28A to target specific microRNAs or other RNA substrates for uridylation-dependent degradation? How functionally significant are isomiRs generated by uridylation in development, normal physiology, and disease biology? What are the precise roles that uridylation plays in non-canonical microRNA biogenesis? Are there cis- and trans-acting factors that modulate uridylation activity on specific RNA substrates such as mRNAs with short polyA-tails? Does dysregulated uridylation contribute to pathology in other diseases and by what mechanisms? The answers to these questions as well as others should provide critical new insights into how uridylation contributes to post-transcriptional gene regulation in the emerging field of RNA epitranscriptomics.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. MRM and JB contributed equally to this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aphis glycines Matsumura; SBA) is the most economically damaging insect of soybean (Glycine max) in the United States. One previous study demonstrated that avirulent (biotype 1) and virulent (biotype 2) biotypes could co-occur and interact on resistant  and susceptible soybean resulting in induced susceptibility after 11 days of feeding. The main objective of this research was to employ RNA sequencing (RNA-seq) technique to compare the induced susceptibility effect of biotype 2 on susceptible and resistant soybean at day 1 and day 11 .Soybean aphid . Ten RNA datasets are reported with 266,535,654 sequence reads (55.2 GB) obtained from pooled samples derived from the leaves collected at day 1 and day 11 post SBA infestation. A comprehensive understanding of these transcriptome data will enhance our understanding of interactions among soybean and two different biotypes of soybean aphids at the molecular level.We investigated susceptible and  Aphis glycines Matsumura; SBA) is the most economically damaging insect pest of soybean (Glycine max) in the United States (US) [Rag1, biotype 3: virulent to Rag2, biotype 4: virulent to Rag1, Rag2 and Rag1 + Rag2) in the US [Rag1) and susceptible soybean, which is defined as induced susceptibility [Soybean aphid (tes (US) . In the tes (US) . Althougtes (US) . The deptes (US) , 5. Hostn the US –8. Initin the US –8. Howevn the US  demonstrtibility . Furthertibility  , and two SBA populations . A detaLeaf samples collected at day 1 and day 11 from resistant and susceptible cultivars  were used to isolate RNA using PureLink RNA mini kit . Isolated RNA was treated with TURBO™ DNase  to remove any DNA contamination, following the manufacturer’s instructions. The RNA samples from three replicates were pooled in equimolar concentration, and RNA-seq libraries were sequenced on an Illumina NextSeq 500 at 75 cycles. Ten RNA libraries were prepared and sequenced with the sequencing depth ranging from 24,779,816 to 29,72,4913 reads  were obtained from the Phytozome database and aligned using Salmon ver.0.9.1 [Quality control of reads was assessed using FastQC program (version 0.11.3) . The Faser.0.9.1  accesseder.0.9.1  . After trimming, over 99% of the reads were retained as the clean and good quality reads. Upon mapping these reads, we obtained high mapping rate ranging from 90.4 to 92.9%. Among the mapped reads, 85.8% to 91.9% reads were uniquely mapped. After filtering with 0.5 counts per million (CPM) in at least one sample and rlog transformation, a total of 37,468 genes  were retained for transformation (Data file 21). The hierarchical clustering based on 3000 most variable genes, sample distances  indicated that sample clustering followed the time points of sample collection . The correlation between the samples using the top 75% of genes showed in a range of 0.96–1 .https://www.qiagenbioinformatics.com/) and analysis guided by the reference genes could be used to study the differential gene expression for pooled samples with no replications.The quality filtering of downloadable raw fastq files is recommended before use. Kal’s z-test  integrat


Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modification on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ affected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification. Ψ has historically been viewed as an attribute of stable noncoding RNAs including tRNA, rRNA and snRNA2. The development of next-generation sequencing methods for detection and mapping of RNA modifications revealed that Ψs are also abundant in mRNA and long noncoding RNA. The number of Ψs in mRNA was found to be dynamically regulated in response to cellular conditions6. Recent studies have discovered that Ψ participates in the control of various layers of gene expression regulation8. Research on Ψ, whose role ranges from fine-tuning to being functionally essential to the target RNA, is gaining renewed interest11.RNA features a vast collection of chemically and structurally diverse posttranscriptionally modified nucleosides. Pseudouridine (Ψ) was the first such modification discovered2. In most of the structures studied so far, Ψ contributed to the structural stability2. The impact of the Ψ modification depends on the RNA secondary structure context (stems or loops). Early NMR studies revealed that the presence of Ψ in single strand RNA favors the formation of the C3′-endo conformation and promotes base stacking, thus stabilizing RNA structures12. In RNA hairpins Ψ stabilizes base pairing and stacking interactions. In particular, in the anticodon-stem loop of tRNA, Ψ39 is involved in the stabilization of the A31-Ψ39 base pair at the interface between the anticodon loop and stem region16. The consecutive Ψ38 and Ψ39 residues in tRNAHis provide stacking stabilization for bases on the 3′ side of the tRNA anticodon loop17. Also, the conserved Ψ modifications in helix 69 (H69) from large subunit ribosomal RNA have been shown to modulate the structure and conformational behavior of the stem-loop region by stabilizing the loop-closing base pair and promoting base stacking in the 3′ half of the loop19 and an additional A-Ψ base pair within the loop20. The UV melting data revealed that the Ψ residues in the stem region provided thermodynamic stabilization to the RNA, whereas individual Ψs located in the loop region contributed to a slight destabilization22. Moreover, structural studies of the P6.1 hairpin derived from the human telomerase RNA activation domain revealed that two Ψ modifications located in the loop increased both base-stacking and hydrogen-bonding interactions within the loop compared with the unmodified construct, resulting in higher thermodynamic stability. Here, the insertion of two Ψs in the loop increased the RNA thermodynamic stability more than the insertion of two Ψs in the stem23. The presence of Ψ modification within the branch-site recognition region of splicesomal U2 snRNA induces a bulged conformation of the branch-point adenosine25. Replacing uridines with Ψ in RNAs containing toxic CUG or CCUG repeats that form helical conformations induced structural stabilization26. Furthermore, Ψ is known to modulate codon-anticodon interactions between mRNA and tRNA17 allowing purine-purine base pairing28. The effect of Ψ modification on structure and thermodynamic stability is commonly attributed to the stabilization of the C3′-endo conformation, increased base stacking properties12, and the presence of an additional N1 H-bond donor that coordinates structural water molecules30, and all these components are considered to be interdependent17.Pseudouridine is an isomer of the nucleoside uridine in which the uracil is attached to the sugar via the C5-C1′ instead of the N1-C1′ glycosidic bond  than those in duplexes with other motifs . Moreover, in the duplex with the 5′-AΨU/3′-UAA motif, the replacement of U with Ψ stabilized the duplex 3 kcal/mol less than what was predicted using the available nearest-neighbor parameters32.We previously reported the influence of pseudouridylation on the thermodynamic stability of RNA duplexes with Ψ modifications where the Ψ was located at various positions and formed base pairs with A, G, U and C31, namely, duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. To understand how the replacement of U with Ψ changes the stacking interactions, we applied high-level ab initio methods and performed calculations for eight unique base pair steps for the RNA structures derived from MD simulations.In this work, we determined the NMR structures of two 9-mer RNA duplexes with 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs, to examine whether structural factors could be responsible for the differences observed in the increment of thermodynamic stabilities. To further elucidate how the replacement of the middle U-A base pair with Ψ-A in different sequence contexts influences the structure, dynamics and hydration of RNA duplexes, we performed a systematic study using molecular dynamics (MD) simulations. We carried out MD simulations in explicit solvent for all duplexes with internal Ψ-A base pairs studied in previous workThe RNA duplexes considered in this study are listed in Table 2O) used for nonexchangeable proton assignment revealed NOE connectivity typical of A-form geometry. Expanded contour plots of the NOESY spectra corresponding to the interactions between the base H6/H8 and H1′/H5 protons with sequential pathways traced for both duplexes are shown in Fig. 33. Similar to 1H, the 13C chemical shifts of C1′-Ψ5 differ significantly from the ranges observed for the other anomeric carbon resonances  that fall within the distributions of canonical A-RNA duplexes35. This result implied the anti-orientation of all the nucleobases and C3′-endo sugar ring conformation with calculated average pseudorotation angles and puckering amplitudes of approximately 11° and 42°, respectively. The calculated C1′-C1′ distances averaged over all the base pairs in all the models were 10.75 ± 0.05 Å for both duplexes. A detailed comparison of the two duplexes with the use of rigid body parameters that describe the geometry of the base pairs and sequential base pair steps  for sugar C1′-H1′ vectors. We found that in the duplexes with Ψ modification, the backbone flexibility was only marginally lower than that in the corresponding reference duplexes. For the duplexes with the central RΨY motif (R-purine and Y-pyrimidine), the decrease in RMSF was mainly localized in residues 5 and 6, whereas for the YΨR motif, this effect propagated along the entire strand  per residue for the backbone atoms  and the order parameters  for suga34  compared with that of U5 (~205°) in all the duplexes studied  around the selected atoms in the modification region. The RDF plot of the water oxygen atoms with respect to the HN1-Ψ5 atom  method to study stacking interaction energies for eight base pair steps containing the Ψ-A and the reference U-A base pairs. The calculations were performed using the geometries of the stacked base pairs derived from explicit solvent MD simulations of the respective RNA duplexes . Our MM-based calculations were performed over the trajectories, thus assessing the dynamic nature of the systems. On the average, the variability of the stacking energy for the base pair steps along the RNA trajectory was approximately 2.2 kcal/mol. For four base pair steps  where the calculations at the QM level (ΔEQM) showed stabilization in the stacking interaction energies from the Ψ modification, the MM-based calculations (ΔEMM) displayed the same stabilization order  compared with unmodified U and thus could influence the base stacking at base pair steps in the duplex31 showing a more favorable thermodynamic effect for Ψ modification of duplex-CΨG than that of duplex-GΨC , rRNA and in U2 snRNA-branch point duplexes . Different scenarios of interactions mediated by the pseudouridine-bound water molecule were observed: a single water molecule bridging the HN1, OP2 atoms of the Ψ and OP2 of the preceding residue29 , an intra-residue bridge only (Ψ2621 in PDB ID: 1S72) or two water molecules between the HN1 and OP2 atoms of the Ψ and the OP2 of the preceding residue (PDB ID: 3CGP). Ψ intra-residue water bridge was present in all of the above structures whereas inter-residue water bridge to the OP2 atom of the 5′ residue was formed at the loop or stem/loop interface structural context. In our duplexes, both from the NMR structures and from the MD trajectories, the distance between the HN1 atom of the Ψ and the OP2 atom of the 5′ residue was too large to make a single water bridge  the transition of single strands from an unstacked to a stacked, ordered state and (ii) the association of two preorganized single-stranded helices to form a duplex46. The preference for the C3′-endo sugar pucker conformation of Ψ appears to play a more important role in the process (i). The stabilization of the C3′-endo sugar pucker conformation in single stranded RNAs has been demonstrated experimentally12. In addition, stacking interactions and structural water likely contribute to the enhanced preorganization of single stranded RNA with pseudouridine. The contribution of Ψ to single strand preorganization has recently been supported by experimental data and MD studies47. In effect, Ψ modification resulted in preorganization of single strands with nucleotides in the C3′-endo, anti and axial conformations, thereby decreasing the entropic cost of duplex formation and increasing the thermodynamic stability of the duplex. The modifications that operate by the mechanism of reducing unfavorable entropy when going from a single-stranded to a double-stranded molecule include 2′-O-methyl-modified residues, LNA nucleosides48 and 2-thiouridine46. In process (ii), i.e., helix association, Ψ does not play significant role because it does not change the hydrogen bonding, and the structural water molecule is not at the binding interface of the two strands. To test if the differences in the thermodynamics of the duplex formation of the Ψ-modified and unmodified duplexes can be obtained by computational predictions, we employed the MM-PBSA method. The relative binding free energies calculated for the Ψ-modified and unmodified duplexes were between 0.61 to −1.47 kcal/mol and did not follow the experimental trend  were synthesized on an Applied Biosystems DNA/RNA synthesizer, deprotected and purified according to previously published procedures1H resonances were recorded at 25 °C. To facilitate peak assignments and obtain additional structural information 2D NOESY spectra at 30 and 35 °C were also acquired for duplex-GΨC and -CΨG, respectively, with the broad spectral width of ~7002 Hz. High resolution DQF-COSY spectra were acquired with narrow spectral width of 2520 Hz. Natural abundance 1H-13C HSQC experiments (25 °C) were performed within the spectral width of 7002 Hz in the 1H dimension and 28248 Hz in the 13C dimension. To increase the resolution 1H-13C HSQC spectra were acquired with narrowed spectral range of 4901 Hz in 1H dimension and 10570 Hz in 13C dimension and 64 scans per t1 increment. Proton detected 1H-31P COSY spectra were recorded at 25 °C within 2102 Hz spectral range in the 1H dimension and 1417 Hz in the 31P dimension. To aid 1H resonance assignment a two-dimensional 1H-31P hetero TOCSY-NOESY experiments were recorded at 25 °C with a DIPSI-2 sequence55 using 20 ms spin-lock period and 500 ms NOESY mixing time. The spectra were acquired with a spectral width of 10504 Hz in 1H and 1701 Hz in the 31P dimension. Exchangeable 1H resonances were analyzed using the 2D NOESY spectra obtained at 15 °C with a 100 ms mixing time. The residual water peak for homonuclear experiments in D2O was suppressed using low-power presaturation whereas solvent suppression for samples in 90% H2O/10% D2O was achieved by applying a pulse sequence using excitation sculpting with gradients56. A sodium 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) was used as an internal chemical shift reference. Collected spectra were processed with TopSpin  and analyzed with Felix  software.The NMR spectra were acquired on a Bruker Avance III 700 MHz spectrometer, equipped with a QCI CryoProbe. The two-dimensional NOESY experiments involving non-exchangeable 2O) with mixing times of 100 and 150 ms recorded at 25 °C using the Isolated Spin Pair Approximation (ISPA) approach. An average pyridine H5-H6 peak volumes were used as a reference with a distance of 2.45 Å. Error bounds for the restraints were set to −15% (lower) and +30% (upper) of the calculated interproton distance. The details for the identification of Watson-Crick base pairs and hydrogen bonding pattern between Ψ and A bases, constraints for endocyclic and backbone torsion angles α, β, γ, ε and ζ, sugar pucker conformations and orientations of the nucleobases with respect to the sugar rings are summarized in Supplementary Methods, Section NOE restraints for non-exchangeable protons were obtained from analysis of 2D NOESY spectra . The Ψ modification was incorporated at the 5th position of the initial structures by rearranging the atom positions of the uridine residue.All molecular mechanics (MM) calculations and molecular dynamic (MD) simulations were performed with AMBER 14 simulation package and AmberTools1562. During the simulated annealing calculations, restrained MD simulations were performed using NOE structural restraints collected from the analysis of NMR specta  were carried out with the starting coordinates in the A-RNA conformation by the simulated annealing methodet al.63  for distance restraints. Among these sorted structures, 20 structures, without any restraint violations higher than 0.1 Å and with minimum number of restraint violations less than 0.1 Å, were selected for the next cycle of simulated annealing run. In the second cycle, these 20 structures were further refined to extract an ensemble of 10 final structures applying the similar violation criteria  chosen in the first cycle.The energy minimized structure of each RNA duplex was subjected to two cycles of a simulated annealing procedure following Chen 64 water molecules in truncated octahedral boxes with minimal distance of 10 Å from the solute border and periodic boundary conditions were applied. The systems were neutralized with Na+ ions and KCl excess salt was added to obtain the concentration of 1.0 M by using Joung and Cheatham ion parameters65 to resemble conditions of UV melting experiments. The solvated systems were minimized and equilibrated. For each system, during the production run constant pressure Langevin dynamics was performed at 300 K for a total 500 ns with a 2 fs time step and trajectory files were written at each 10 ps. The pressure was regulated using Berendsen barostat66 at reference pressure of 1 atm. Electrostatics was handled with PME67 method with a direct space cutoff of 10.0 Å  the donor-acceptor distance was ≤3.0 Å, and (ii) the donor-hydrogen-acceptor angle was ≥135.0°. The lie utility in the cpptraj program was used for the calculation of the molecular mechanics (MM) force field-based base stacking energies as a summation of pairwise electrostatic and van der Waals interaction energies between the base atoms only. Cluster analysis was performed with cpptraj using the average-linkage clustering algorithm  were calculated for C1′-H1′ vector using the ired utility in the cpptraj program  approach at the B97D/Def2TZVPP level of theory using the GAUSSIAN09 software suite. The intra- and interstrand base stacking energies were calculated following Svozil et al.70 , where [E]ADBC represents the interaction energy of the complete four-base stacked system, and [E]AD and [E]BC represent hydrogen bonding interaction energies in the Watson-Crick base pairs. The interaction energies were calculated by subtracting the monomer total energies from the corresponding dimer/tetramer energies corrected for basis set superposition error (BSSE) via the counterpoise method implemented in the GAUSSIAN09 software suite. The intrastrand ([E]AB and [E]CD) and interstrand ([E]AC and [E]BD) interaction energies were also reported in Supplementary Table The Supplementary information


The perception of choice in becoming a caregiver may impact on caregiver psychological and physical health. We determined the proportion of spousal dementia caregivers who felt they had a choice, and examined whether lack of choice in taking up the caregiving role and the perceived degree of choice in caregiving predicted caregiver health and wellbeing and care-recipient placement in long-term care at 1-year follow-up.We performed secondary analyses of data from DeStress, a longitudinal study of 251 spousal dementia caregivers in Ireland. We used multivariate logistic and linear regression analyses to examine whether lack of choice (a dichotomous item) and/or the perceived degree of choice  at baseline predicted caregiver health  and wellbeing  and care status (continued care at home or placement in long-term care) at follow-up.SD = 3.22; Median = 9; Mode = 9), indicating they provided care voluntarily. Caregivers who reported a greater degree of choice were more likely to still be providing care at home at follow-up and to identify benefits from providing care. Neither choice nor degree of choice predicted any other caregiver outcomes.The vast majority of caregivers (82%) reported that they had no choice in taking up the caregiving role. Nevertheless, nearly three-quarters (74%) responded above the midpoint on the rating scale (Mean = 6.82, For the vast majority of spousal dementia caregivers, taking up the caregiving role is not perceived as a choice; yet, most report performing this role voluntarily. Thus, facilitating greater choice may not necessarily diminish the key contribution family caregivers make to the care system. Although we found no evidence that caregiver choice predicted more positive caregiver health and wellbeing, the perception of choice is important in and of itself, and may benefit caregivers by facilitating the identification of positive aspects of care and be a factor in delaying care-recipient placement in long-term care. Future research should be especially mindful of how caregiver choice is assessed and how this may affect the resulting prevalence of choice. Unpaid family members provide the vast majority of care for the increasing numbers of people living with dementia worldwide . In IrelThe Irish National Dementia Strategy acknowledges the excessive demands placed on family dementia caregivers and recognizes the need to safeguard their own health, psychological wellbeing, and social life . Indeed,We define caregiver choice as the extent to which an individual believes they had the freedom to choose to take up the responsibility of care . While iA large United States study on a mixed sample of informal caregivers found that over half of caregivers (54%) reported they had a choice in taking up the caregiver role . A similPrevious studies on broader caregiver samples suggest that the absence of caregiver choice is a significant predictor of poorer caregiver outcomes on measures including life-satisfaction, happiness, quality of life, and capability ; emotionTo our knowledge, only one previous study has explicitly examined the role of choice among spousal dementia caregivers specifically; In addition to impacting on caregiver health and wellbeing – or perhaps through these factors -, perceptions of caregiver choice may also play a role in the premature cessation of care in the community. Indeed, previous research has indicated that caregiver “role captivity” – fulfilling a role because of an obligation rather than by choice – predicts nursing home placement among persons with Alzheimer’s disease . HoweverGiven that the prevalence of caregiver choice among dementia caregivers is not known, our aim was to determine the proportion of caregivers who felt they had a choice in taking up the responsibility of care for their spouse with dementia. Furthermore, building on previous cross-sectional research, we sought to examine whether the perception of choice in taking up the caregiving role and the degree of choice predicted a variety of caregiver health and wellbeing outcomes, as well as care-recipient placement in long-term care, at 1-year follow-up. We hypothesized that lack of choice and a lower degree of choice would predict poorer caregiver outcomes.Participants were recruited for a longitudinal study (the DeStress study) on the relationship between caregiver stress and cognitive functioning . CaregivParticipants were recruited from all over Ireland through a broad range of channels, including media advertisements, as well as community gatekeepers and organizations for caregivers, dementia and/or older people. Of the 370 persons identified who were eligible, 252 (68%) participated. All data, unless otherwise specified, were collected at two study time-points (baseline and 1-year follow-up) using a combination of a telephone health survey, a postal questionnaire, and a face-to-face assessment.The study protocol was approved by the Trinity College Dublin School of Psychology Ethics Committee. Caregivers’ participation in the study was completely voluntary and participants were free to withdraw from the study at any time. Participants were also free to decline to provide some or all information requested as part of the study. All participants gave written informed consent.RUD4  were recorded at baseline. Items from the RUD4  were useneuropsychiatric inventory questionnaire (NPI-Q)  was assessed using the  (NPI-Q) . The NPI (NPI-Q) .Bristol Activities of Daily Living Scale (BADLS) (The  (BADLS)  was used (BADLS) , as well (BADLS) .Perceived choice in caregiving was assessed with a single yes/no item (“Do you feel you had a choice in taking on the responsibility of caring for your spouse”?), which has been used in previous research . An addiChristensen health screening questionnaire  ( (PSS-4) , a widel (PSS-4) .Anxiety was assessed using the 7 anxiety items on the Hospital Anxiety and Depression Scale (HADS-A) ((HADS-A) . The HAD(HADS-A) .Depression was assessed using the Centre for Epidemiological Studies Depression (CES-D) scale (D) scale . The CESD) scale .Self-efficacy for symptom management was assessed using the Fortinsky dementia-specific caregiver self-efficacy scale . The relationships between caregiver and care-recipient characteristics and the caregiver choice measures were examined using 2 likelihood ratio statistics. Caregiver age, sex and education; the duration of caregiving; and care-recipient level of functional impairment and BPSD severity were included as covariates.Multivariate logistic regression analysis was carried out to examine whether caregiver choice and/or the perceived degree of choice at baseline predicted placement in long-term care at follow-up amongst those who were not bereaved in the intervening period, and whose care status was known. Model fit was evaluated with the Hosmer-Lemeshow test and compared with standard χRelationships between the hypothesized predictors (caregiver choice and degree of choice) and caregiver health and well-being variables  were examined at baseline and at follow-up using Pearson correlations amongst those still providing care at home. In order to minimize potential confounds, only those still providing care at follow-up were included in this analysis; however, results were equivalent when those who were bereaved and those whose spouse was in long-term care at follow-up were included. Multivariate linear regression analysis was conducted to test whether baseline lack of choice and/or perceived degree of choice predicted caregiver health and wellbeing outcomes at follow-up. In addition to the covariates included in the logistic regression models, baseline measures of the relevant health, and well-being outcome was controlled in each model.The participant socio-demographic characteristics and the care-recipient characteristics are presented in SD = 3.22; Median = 9; Mode = 9; see p > 0.05 (results not presented).Less than a fifth of caregivers (18%) reported that they had a choice in taking up the caregiving role see . Neverthn = 121) gave the same response to this item at baseline and follow-up, 7.4% (n = 11) of caregivers reported at follow-up that they had no choice when they had previously reported choice and 11.4% (n = 17) reported choice at follow-up when they had previously reported no choice. The perceived degree of caregiver choice did not change significantly from baseline to follow-up , t(145) = 0.81, p < 0.05.Among those who were still providing care at home 1 year later, 23.3% agreed at follow-up that they had a choice in taking up the caregiving role. While 81.2% .At follow-up 177 participants were still providing care for their spouse at home and 41 had moved their spouse into LTC, giving a combined sample of 218 participants. Bivariate correlations indicated that neither caregiver choice nor perceived degree of choice at baseline were significantly associated with placement in LTC at follow-up , the model explained little additional variance and the percentage of cases correctly classified did not increase substantially over a model that included only the demographic covariates.Caregiver choice at baseline was not a significant predictor of placement in LTC in a logistic regression model that adjusted for covariates (results not presented); however, the perceived degree of choice was see . SpecifiOf the 177 caregivers who were still providing care at home at follow-up, 151 (85%) participated in the follow-up assessment. Bivariate correlations between baseline caregiver choice and degree of choice and follow-up caregiver health and wellbeing are presented in F = 4.34, p < 0.001. The addition of baseline positive aspects of caregiving in step 2 explained a further 34% of the variance, F = 19.71, p < 0.001. Finally, the addition of degree of choice in step 3 increased the explained variance by 2%, F = 18.45, p < 0.001.In multivariate models, caregiver choice did not significantly predict any caregiver health and well-being outcomes (results not presented); while the perceived degree of choice only significantly predicted the perception of positive aspects of care. Specifically, caregivers who reported greater voluntariness in taking up the caregiving role were more likely to identify benefits as a result of providing care at follow-up see . BaselinOur findings indicate that the vast majority of caregivers – less than a fifth – felt that they had a choice in taking up the responsibility of care for their spouse with dementia. This was consistent at follow-up among those still providing care at home. The high prevalence of perceived lack of choice is striking, yet perhaps not totally unsurprising given the heavy reliance on family caregivers as the main providers of dementia care in the social care system in Ireland, and the limited availability of dementia services and supports that are a prerequisite for choice.The substantially lower prevalence of caregiver choice we observed compared to previous studies  is likelPrevious studies also included non-relative caregivers, while our sample consisted exclusively of spousal caregivers whose perceived freedom to choose is likely to be more constrained by a sense of reciprocity and responsibility than other types of caregivers. This is supported by previous findings that spousal and parental relationships with a care-recipient were significantly associated with a lack of choice . FurtherCaregiver choice is important; yet, enabling unpaid caregivers to exercise free choice with regards to caregiving presents a significant problem if it leads to decreases in unpaid caregiving activities . The proAlthough the lack of choice in taking up the caregiving role was not associated with placement in long-term care at follow-up in our sample, caregivers who perceived a lower degree of choice in respect to their caregiving at baseline were less likely to still be caring at home 1 year later. While this may seem like a contradiction, that caregivers cease a role they feel they have no choice in carrying out, it is clear that caregiving typically does not end when the care-recipient moves into long-term care, and the decision to institutionalize is often made at crisis points, such as during hospitalization, and is contingent on many extraneous factors including the availability of long-term beds and the financial means to avail of acceptable alternative care options . Our finNevertheless, while the degree of choice was a significant predictor, it explained little of the variance in the model. This may be in part because less than 20% of the sample had moved into long-term care at follow-up; further research with a larger sample and a longer follow-up time is needed to examine whether caregiver choice decreases the likelihood of institutionalization and the mechanisms through which this may take place. For example, lack of choice may impact negatively on the quality of care for the care-recipient as caregiver burden and distress is associated with worsening BPSDs and a greater likelihood of mistreatment and abusive behaviors toward the care-recipient . Alternaperceiving caregiving to be their choice may benefit caregivers by functioning as a coping strategy that gives them a greater sense of control and enables them to view caregiving as in line with their personal values rather than as a duty they are obliged to carry out (The finding that lack of choice, both in terms of taking up the caregiving role and the perceived degree of choice in caregiving, was not associated with poorer caregiver health, and well-being outcomes was unexpected. It is, however, in line with one study which found that willingness to care did not significantly predict quality of life or strain . It is parry out . This maarry out , more auFuture research should be especially mindful of the way in which choice is assessed; our findings highlight that a dichotomous item is likely to give a different impression of the prevalence of choice and how choice relates to caregiver outcomes from a multiple option degree-of-choice measure. Since few people are likely to have an entirely unconstrained choice regarding whether or not to provide care that can be captured using a yes/no item, a more nuanced exploration of motivations to care, and the organizational and contextual factors that constrain choice would be of greater value.Although our findings do not support the idea that limiting caregiver choice is related to poorer outcomes, this does not take away from the importance of choice in and of itself. It is a sad reflection of the social care system that the vast majority of caregivers did not feel they had a choice in taking up the responsibility of care and this points to the need for health and social care providers to be more mindful of caregivers’ perceptions of choice and how they might help to facilitate greater autonomy. For example, the availability and accessibility of current and accurate information on available service options is a prerequisite to exercising choice in relation to caregiving . NeverthWhile a stated objective of the Irish National Dementia Strategy  is to suEven when alternative options for care are available, caregivers may constrain their own choices because of feelings of obligation or duty to the care-recipient or by imposing limits on what they consider appropriate or necessary to get help with. For example, some caregivers may not consider it acceptable for someone else to carry out personal care tasks, while others may not be comfortable getting support with more general household chores . FurtherOur data are based on a relatively large sample of dementia caregivers and present the first prevalence data regarding spousal dementia caregiver choice. Furthermore, the inclusion of longitudinal data, and the examination of relationships between caregiver choice and caregiver outcomes over time is another strength of this study. Nevertheless, our findings are limited by the lack of data on the context in which caregivers took on their caregiving role or the actual alternative options that were available to them. We measured choice in an oversimplified manner using only a dichotomous item and a Likert scale. Therefore we have no information on participants’ positive motivations for caregiving or the reasons for their perceived lack of choice. Our findings are also limited by the homogenous nature of our sample, which included only spousal caregivers. The experiences of adult children or other relatives or friends caring for someone with dementia are likely to be very different, particularly if they perceive a lack of choice in adopting a caregiving role. In addition, while placement in long-term care and the identification of positive aspects of care at follow-up were predicted by degree of choice at baseline in our data, these findings should be interpreted with caution given the number of outcomes measures we examined and, therefore, the greater possibility that these findings emerged based on chance alone.For the vast majority of spousal dementia caregivers in Ireland, taking up the caregiving role is not perceived as a choice; yet, most caregivers report performing this role voluntarily, suggesting that facilitating greater choice would not necessarily diminish the key contribution family caregivers currently make to the dementia care system. Although, we found no evidence that a greater degree of choice predicted more positive health and wellbeing outcomes for caregivers over time, the perception of choice for caregivers is important in and of itself, and this should be reflected in dementia care policy. Health and social care professionals should give due consideration to caregivers’ perception of choice and facilitate caregiver preferences wherever possible by carefully outlining and exploring the care options available and by working with caregivers to increase their confidence in providing care. Providing caregivers with greater choice with regards to care may benefit caregivers by facilitating the identification of positive aspects of care and could potentially delay care-recipient placement in long-term care.The datasets for this study will not be made publicly available because the participants did not give consent for their data to be shared outside of the research team.This study was carried out in accordance with the recommendations of the Trinity College Dublin School of Psychology Ethics Committee with written informed consent from all participants. All participants gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Trinity College Dublin School of Psychology Ethics Committee.MP, SB, IR, and BAL conceived and designed the study. MP performed the analysis. MP and AS-R wrote the first draft of the manuscript. All authors revised the manuscript and approved it for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.