Innate, translation‐dependent silencing of an invasive transposon in Arabidopsis
2021; Springer Nature; Volume: 23; Issue: 3 Linguagem: Inglês
10.15252/embr.202153400
ISSN1469-3178
AutoresStefan Oberlin, Rajendran Rajeswaran, Marieke Trasser, Verónica Barragán‐Borrero, Michael A. Schon, Alexandra Plotnikova, Lukas Loncsek, Michael D. Nodine, Arturo Marí‐Ordóñez, Olivier Voinnet,
Tópico(s)Plant Molecular Biology Research
ResumoArticle21 December 2021Open Access Source DataTransparent process Innate, translation-dependent silencing of an invasive transposon in Arabidopsis Stefan Oberlin Stefan Oberlin orcid.org/0000-0003-4577-9946 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Search for more papers by this author Rajendran Rajeswaran Rajendran Rajeswaran orcid.org/0000-0002-4842-1256 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Search for more papers by this author Marieke Trasser Marieke Trasser orcid.org/0000-0001-8507-5770 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria Search for more papers by this author Verónica Barragán-Borrero Verónica Barragán-Borrero orcid.org/0000-0002-0718-1858 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Michael A Schon Michael A Schon orcid.org/0000-0002-4756-3906 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Alexandra Plotnikova Alexandra Plotnikova orcid.org/0000-0002-7370-2041 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Lukas Loncsek Lukas Loncsek orcid.org/0000-0003-0772-6587 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Michael D Nodine Michael D Nodine orcid.org/0000-0002-6204-8857 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands Search for more papers by this author Arturo Marí-Ordóñez Corresponding Author Arturo Marí-Ordóñez [email protected] orcid.org/0000-0002-4416-6382 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Olivier Voinnet Corresponding Author Olivier Voinnet [email protected] orcid.org/0000-0001-6982-9544 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Search for more papers by this author Stefan Oberlin Stefan Oberlin orcid.org/0000-0003-4577-9946 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Search for more papers by this author Rajendran Rajeswaran Rajendran Rajeswaran orcid.org/0000-0002-4842-1256 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Search for more papers by this author Marieke Trasser Marieke Trasser orcid.org/0000-0001-8507-5770 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria Search for more papers by this author Verónica Barragán-Borrero Verónica Barragán-Borrero orcid.org/0000-0002-0718-1858 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Michael A Schon Michael A Schon orcid.org/0000-0002-4756-3906 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Alexandra Plotnikova Alexandra Plotnikova orcid.org/0000-0002-7370-2041 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Lukas Loncsek Lukas Loncsek orcid.org/0000-0003-0772-6587 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Michael D Nodine Michael D Nodine orcid.org/0000-0002-6204-8857 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands Search for more papers by this author Arturo Marí-Ordóñez Corresponding Author Arturo Marí-Ordóñez [email protected] orcid.org/0000-0002-4416-6382 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Olivier Voinnet Corresponding Author Olivier Voinnet [email protected] orcid.org/0000-0001-6982-9544 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Search for more papers by this author Author Information Stefan Oberlin1,5, Rajendran Rajeswaran1, Marieke Trasser2,3, Verónica Barragán-Borrero1,2, Michael A Schon2, Alexandra Plotnikova2, Lukas Loncsek2, Michael D Nodine2,4, Arturo Marí-Ordóñez *,1,2 and Olivier Voinnet *,1 1Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland 2Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Vienna, Austria 3Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria 4Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands 5Present address: Department of Microbiology and Immunology, UCSF Diabetes Center, University of California, San Francisco, CA, USA *Corresponding author. Tel: +43 1 79044 9901; E-mail: [email protected] *Corresponding author. Tel: +41 0 44 633 93 60; E-mail: [email protected] EMBO Reports (2022)23:e53400https://doi.org/10.15252/embr.202153400 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Co-evolution between hosts' and parasites' genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral-like silencing response. To investigate this response's activation, we studied the single-copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full-length genomic flGAG-POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG-POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5'OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto-unrecognized "translation-dependent silencing" (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern. SYNOPSIS Analyzing the initiation of RNA silencing of the Arabidopsis transposon EVADE (EVD) reveals a ribosome stalling event during EVD translation that correlates with the production of small RNAs involving RNA-dependent RNA polymerase 6 (RDR6). Only a cytoplasmic and translated mRNA isoform of EVD triggers RDR6-dependent siRNA production. An intense and discrete ribosome stalling event coincides with the onset of EVD siRNA production from this isoform. Atypical 5'-OH RNA cleavage fragments overlap with the ribosome stalling site and possibly serve as RDR6 substrates. Introduction Transposable elements (TEs) colonize and threaten the integrity of virtually all genomes (Huang et al, 2012). Chromosomal rearrangements caused by their highly repetitive nature (Fedoroff, 2012) are usually circumvented by cytosine methylation and/or histone-tail modifications at their loci of origin. The ensuing heterochromatic DNA is not conducive to transcription by RNA Pol II, bringing TEs into an epigenetically silent transcriptional state (Allshire & Madhani, 2018). This "transcriptional gene silencing" (TGS) is observed at the majority of TE loci in plants, including the model species Arabidopsis thaliana, and causes, over evolutionary times, accumulating mutations resulting in mostly degenerated, non-autonomous entities (Vitte & Bennetzen, 2006; Civáň et al, 2011). Nonetheless, the genome invasiveness of these remnants remains evident by their methyl cytosine-marked DNA, which is perpetuated over generations by METHYL-TRANSFERASE 1 (MET1), among other factors. MET1 reproduces symmetrical methylation sites from mother to daughter strands during DNA replication (Kankel et al, 2003) aided by the (hetero)chromatin remodeler DEFICIENT IN DNA METHYLATION 1 (DDM1) (Saze et al, 2003; Zemach et al, 2013). Loss of MET1 or DDM1 functions in Arabidopsis leads to genome-wide demethylation, transcriptional reactivation of many TE remnants, and mobilization of a small portion of intact, autonomous TEs (Mirouze et al, 2009; Tsukahara et al, 2010). Their proliferation together with genome-wide deposition of aberrant epigenetic marks likely explains why met1 and ddm1 mutants accumulate increasingly severe genetic and phenotypic burdens over inbred generations (Vongs et al, 1993). However, such secondary events can be avoided by backcrossing the first homozygous generation of ddm1- or met1-derived mutants with wild-type plants, upon which continuous selfing of F2 plants creates "epigenetic recombinant inbred lines" (epiRILs). These harbor only mosaics of de-methylated DNA while maintaining wild-type (WT) MET1 and DDM1 functions (Reinders et al, 2009; Teixeira et al, 2009). One such met1 epiRIL, epi15, endows epigenetic reactivation of the autonomous, long terminal repeat (LTR) retroelement EVADÉ (EVD) in theTy1/Copia family, which is one of the most proliferative families in plants (Vitte & Panaud, 2005). Of the two EVD copies in the Arabidopsis Col-0 genome, only one is reactivated in epi15 (Marí-Ordóñez et al, 2013). By providing a proxy for a de novo genomic invasion, this reactivation granted a unique opportunity to grasp how, over multiple inbred generations, newly invasive TEs might be detected and eventually epigenetically silenced (Marí-Ordóñez et al, 2013). We found that EVD is initially confronted to post-transcriptional gene silencing (PTGS) akin to that mounted against plant viruses (Voinnet, 2005; Marí-Ordóñez et al, 2013). Antiviral RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) produces cytosolic, long double-stranded (ds)RNAs from EVD-derived transcripts, which are then processed by DCL4 or DCL2, two of the four Arabidopsis Dicer-like RNase-III enzymes, into populations of respectively 21- and 22-nt small interfering (si)RNAs. However, despite their loading into the antiviral PTGS effectors ARGONAUTE1 and ARGONAUTE2 (AGO1/2), they do not suppress expression of EVDs increasingly more abundant genomic copies. This ultimately gives way to DCL3, instead of DCL4/2, to process the RDR6-made long dsRNAs into 24-nt siRNAs. In association with AGO4-clade AGOs, these species guide RNA-directed DNA methylation (RdDM) of EVD copies. Initially localized within the EVD gene body, it later spreads into the LTRs to eventually shut down the expression of EVD genome-wide via TGS (Marí-Ordóñez et al, 2013). A key, unsolved question prompted by this proposed suite of events pertains to the mechanisms whereby RDR6 is initially recruited onto EVD, and more generally on newly invasive TEs, during the primary antiviral-like silencing phase. "Homology-" or "identity"-based silencing entails sequence complementarity between TE transcripts and host-derived small RNAs. Loaded into AGOs, they likely attract RDR6 concomitantly to silencing execution. One such type of PTGS occurs with TEs reactivated in ddm1/met1 mutants, which, by displaying complementarity mostly to host-encoded microRNAs, spawn "epigenetically activated siRNAs" (easiRNAs) in an AGO1-dependent manner (Creasey et al, 2014). easiRNA production likely entails substantial co-evolution between host and TE genomes (Sarazin & Voinnet, 2014) because miRNAs usually target short and highly conserved TE regions, including the primer-binding sites required for retroelements' reverse transcription (RT; Šurbanovski et al, 2016; Borges et al, 2018). Another form of acquired immunity underlying identity-based silencing is conferred by siRNAs derived from relics of previous genome invasions by the same or sequence-related TE(s) (Fultz & Slotkin, 2017). New intruder TEs are unlikely to engage either form of identity-based silencing, as indeed noted for EVD (Creasey et al, 2014). Thus, RDR6-dependent PTGS initiation should involve intrinsic features of the TEs themselves (Sarazin & Voinnet, 2014). In the yeast Cryptococcus neoformans, stalled spliceosomes on suboptimal TE introns provide an opportunity for an RDR-containing complex to co-transcriptionally initiate such innate PTGS (Dumesic et al, 2013). Studies of transgene silencing in plants (Luo & Chen, 2007; Thran et al, 2012) have advocated other possible mechanisms, though none has yet been linked to epigenetically reactivated TEs. These studies describe how uncapped, prematurely terminated or non-polyadenylated transcripts might stimulate RDR activities when they evade or overwhelm RNA quality control (RQC) pathways that normally degrade these "aberrant" RNAs (Herr et al, 2006; Gy et al, 2007; Parent et al, 2015). A recent model also contends that widespread translation-coupled RNA degradation as a consequence of suboptimal codon usage and low GC content might trigger RDR-dependent silencing in plants (Kim et al, 2021). Initiation of innate PTGS in the context of EVD likely ties in with an unusual process of splicing-coupled premature cleavage and polyadenylation (PCPA) shared by Ty1/Copia retroelements to optimize protein expression from their compact genomes (Oberlin et al, 2017). On the one hand, an unspliced and full-length (fl) GAG-POL isoform codes for a polyprotein processed into protease, integrase/reverse-transcriptase RNase, and GAG nucleocapsid components. On the other hand, a spliced and prematurely terminated short (sh) GAG subgenomic isoform is solely dedicated to GAG production. Though less abundant than the flGAG-POL mRNA, shGAG is substantially more translated (Oberlin et al, 2017). This presumably results in a molar excess of structural GAG for viral-like particle (VLP) formation compared to Pr-IN-RT-RNase required for reverse transcription (RT) and, ultimately, mobilization (Oberlin et al, 2017; Lee et al, 2020). Supporting the notion that genome expression of Ty1/Copia elements influences PTGS initiation, EVD-derived RDR6-dependent siRNAs do not map onto the unspliced flGAG-POL mRNA, but instead specifically onto the spliced shGAG transcript of which, intriguingly, they only cover approximately the 3' half (Oberlin et al, 2017). Here, we show that differential subcellular distribution of the two mRNA isoforms due to splicing-coupled PCPA accounts for the peculiar EVD siRNA distribution and activity patterns. While the flGAG-POL isoform remains largely nuclear, the shGAG mRNA is enriched in the cytosol and endows vastly disproportionate translation over flGAG-POL. However, a previously uncharacterized innate PTGS process accompanies active shGAG translation, manifested as a discrete and unusually intense ribosome stalling event independent of codon usage or GC content, among other tested parameters. Ribosome stalling coincides precisely with the starting point of shGAG siRNA production and maps to the 5' ends of discrete, shGAG-derived RNA breakage fragments. These harbor unconventional 5'OH termini that prevent their RQC-based degradation via 5'P-dependent XRN4 action (Stevens, 2001; Peach et al, 2015). Based on the well-documented substrate competition between XRN4 and RDR6 (Gazzani, 2004; Gy et al, 2007; Gregory et al, 2008; Moreno et al, 2013; Martínez-de-Alba et al, 2015), we suggest that the 5'OH status of breakage fragments contributes to their conversion into dsRNA by RDR6, thereby initiating PTGS of EVD. We further show that splicing-coupled PCPA suffices to recapitulate this "translation-dependent silencing" (TdS) in reporter-gene settings. Given that Ty1/Copia retroelements share a PCPA-based genome expression strategy (Oberlin et al, 2017), the phenomenon discovered here with EVD might constitute a more generic primal defense that shapes the siRNA patterns initially associated with Ty1/Copia TEs. Results shGAG is the main source and target of EVD-derived siRNAs Arabidopsis lines constitutively overexpressing an LTR-deficient but otherwise intact form of EVD driven by the 35S promoter (35S:EVDwt) recapitulate the restriction of EVD siRNA to the 3' part of the shGAG sequence (Marí-Ordóñez et al, 2013; Oberlin et al, 2017; Fig 1A and B). We explored EVD transcripts levels in 35S:EVDwt in WT (siRNA-proficient) as opposed to rdr6 (siRNA-deficient) background (Fig 1B, Appendix Fig S1A). Both in RNA blot and qRT-PCR analyses, the spliced shGAG mRNA levels were increased in rdr6 compared to WT, whereas those of unspliced flGAG-POL were globally unchanged (Fig 1C and D). Accordingly, accumulation of the GAG protein—mainly produced via shGAG translation (Oberlin et al, 2017)—was higher in rdr6 compared to WT background (Fig 1E). Essentially identical results were obtained upon epigenetic reactivation of endogenous EVD in non-transgenic Arabidopsis with the ddm1 single- versus ddm1 rdr6 double-mutant background (Appendix Fig S1B–E). Following EVD mobilization from an early (F8) to a more advanced (F11) epi15 inbred generation (Marí-Ordóñez et al, 2013) revealed that its progressively increased copy number correlates with progressively higher steady-state levels of EVD-derived transcripts and EVD-derived siRNAs (Appendix Fig S1F and G). Again, these siRNAs disproportionately target the shGAG relative to flGAG-POL mRNA from F8 to F11 (Appendix Fig S1H). Collectively, these results indicate that PTGS activated de novo by EVD is both triggered by, and targeted against, the spliced shGAG mRNA. Therefore, features associated with shGAG, but not flGAG-POL, likely stimulate RDR6 recruitment, which we explored by testing current models for PTGS initiation from TEs and transgenes. Figure 1. EVD shGAG is both a trigger and a target of RDR6-dependent but miRNA-independent siRNAs A. EVD flGAG-POL and spliced shGAG mRNAs are distinguishable using specific PCR primer sets (arrows) for quantification and northern analysis. (35S) Cauliflower Mosaic Virus 35S promoter, (Pr) protease, (IN) integrase, (RT-RNase) reverse-transcriptase RNase; red squares: stop codons. B. sRNA-seq reads profile of EVD expressed from 35S:EVDWT in WT (black) or rdr6 (red). (RPM) Reads per million. Positions are indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGAG and GAG-POL 3' ends. C. Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 (ACT2) as a loading control. D. qPCR quantification of shGAG and flGAG-POL normalized to ACT2 and to GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT (GAPC) levels. qPCR was performed on n = 3 biological replicates; bars: standard error. **P < 0.01 (two-sided t-test between indicated values). E. Western analysis of GAG and RDR6 with Coomassie (coom.) staining as a loading control. Arrow indicates cognate RDR6 protein band. F, G. sRNA-seq profiles from EVD de-repressed in the ddm1 (F) or ddm1 dcl1 (G) backgrounds. Different siRNA size categories are stacked. Nomenclature as in (B). Source data are available online for this figure. Source Data for Figure 1 [embr202153400-sup-0002-SDataFig1.zip] Download figure Download PowerPoint shGAG siRNA production is miRNA-independent Though unlikely (Creasey et al, 2014; Sarazin & Voinnet, 2014), we first considered that production of RDR6-dependent siRNAs from shGAG might require its cleavage by miRNAs via the easiRNA pathway (Creasey et al, 2014). Arabidopsis miRNA biogenesis depends on DCL1 and the dsRNA-binding protein HYL1, among other factors (Brodersen & Voinnet, 2006). Analyses of publicly available sRNA-seq data (Creasey et al, 2014) showed, however, that epigenetically reactivated EVD spawns qualitatively and quantitatively identical shGAG-only siRNAs in both ddm1 single and ddm1 dcl1 double mutants (Fig 1F and G). Moreover, levels of shGAG siRNA, shGAG mRNA, and GAG protein remained unchanged in 35S:EVDwt plants with either the WT, hypomorphic dcl1-11, or loss-of-function hyl1-2 background (Appendix Fig S2A–D). By contrast and as expected, production of trans-acting (ta)siRNAs, which is both miRNA and RDR6 dependent, was dramatically reduced and the levels of tasiRNA precursors and target transcripts enhanced in both mutant backgrounds (Appendix Fig S2A–I). Therefore, RDR6 recruitment to the spliced shGAG mRNA is unlikely to involve endogenous miRNAs via an identity-based mechanism. We then explored known innate processes of PTGS initiation instead. Splicing-coupled premature cleavage and polyadenylation suffices to generate EVD-like siRNA accumulation and activity patterns Some cases of transgene-induced PTGS correlate with a lack of polyadenylation due to aberrant RNA transcription (Luo & Chen, 2007). We ruled out that this feature underlies EVD-derived siRNA production because shGAG displays no overt polyadenylation defects regardless of the onset of PTGS (Appendix Fig S3A–E). Next, we considered splicing defects, such as inaccurate splicing or spliceosome stalling, and premature transcriptional termination as possible PTGS triggers, two processes previously independently linked to innate, RDR-dependent siRNA production in plants and fungi (Dumesic et al, 2013; Dalakouras et al, 2019). Ty1/Copia elements have introns that are significantly longer than those of Arabidopsis genes. Moreover, shGAG undergoes atypical splicing-coupled PCPA (Oberlin et al, 2017). When engineered between the GFP and GUS sequences of a translational fusion, the shGAG intron and proximal PCPA signal spawn unspliced flGFP-GUS and spliced GFP-only (shGFP) mRNAs in the Arabidopsis line 35S:GFP-EVDint/ter-GUS (Oberlin et al, 2017) (Fig 2A and B; Appendix Fig S4A). Since this artificial system recapitulates the production of respectively flGAG-POL and shGAG, we asked whether an EVD-like siRNA pattern was likewise reproduced. Figure 2. The EVD intron and terminator suffice to initiate PTGS The 35S:GFP-EVDint/ter-GUS fusion was made by introducing the EVD intron and proximal shGAG terminator (including the premature cleavage and polyadenylation site; PCAP) between the GFP and GUS coding sequence. Like EVD, it spawns full-length unspliced and short-spliced mRNAs. Red squares: stop codons. Expression levels of shGFP (spliced) and GFP-EVDint/ter-GUS (unspliced) transcripts, relative to ACT2 and AT4G26410 (RHIP1), in the WT or rdr6 background. qPCR was performed on three biological replicates and error bars represent the standard error on. *P < 0.05 (two-sided t-test against corresponding controls). sRNA-seq profile mapped on the genomic 35S:GFP-EVDint/ter-GUS locus. (RPM) Reads per million. Positions indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGFP and GFP-GUS 3' ends. Low-molecular-weight RNA analysis of the GFP- and GUS-spanning regions. tasiRNA255 is a control for the rdr6 mutation and miR159 provides a loading control. Source data are available online for this figure. Source Data for Figure 2 [embr202153400-sup-0003-SDataFig2.zip] Download figure Download PowerPoint The majority of RDR6-dependent 21-nt siRNAs mapped to the GFP, but not the GUS region downstream of the PCPA signal (Fig 2C and D) suggesting that, just like shGAG in EVD, the spliced shGFP mRNA is the main source of siRNAs in GFP-EVDint/ter-GUS. Accordingly, and similar to EVD (Oberlin et al, 2017), many siRNAs spanned the exon–exon junction of GFP-EVDint/ter-GUS (Appendix Fig S4B). Moreover, shGFP, unlike flGFP-GUS, over-accumulated in GFP-EVDint/ter-GUS plants with the rdr6 background (Fig 2B, Appendix Fig S4A), indicating that only shGFP is efficiently targeted by PTGS (Fig 2B). Therefore, in the reconstituted setting, the intron and PCPA signal found in shGAG suffice to spawn RDR6-dependent siRNAs displaying accumulation and activity patterns resembling those generated in the authentic EVD context (Fig 1A–D). Neither splicing nor intron-retention per se initiate RDR6 recruitment The above result prompted us to investigate a potential facilitating role for splicing in shGAG siRNA biogenesis or, conversely, a role for intron retention in inhibiting RDR6 recruitment to flGAG-POL. We used previously engineered Arabidopsis EVD-overexpression lines with a point-mutated U1 snRNP-binding site (35S:EVDmU1) or a fully deleted intron (35S:EVDΔi) (Oberlin et al, 2017; Fig 3A–C). 35S:EVDΔi spawns fully matured shGAG transcripts that do not associate with the spliceosome, leading exclusively to prematurely terminated and polyadenylated mRNA species with a stop codon (Oberlin et al, 2017; Fig 3B,D,E, Appendix Fig S5A). However, lack of the intron, and hence splicing, did not prevent RDR6-dependent siRNA production from 35S:EVDΔi, which was comparable to that of 35S:EVDwt (Fig 3E). Moreover, the shGAG mRNA and GAG protein levels from 35S:EVDΔi were higher in an rdr6 compared to WT background (Fig 3D–F), indicating that EVD's unconventional splicing is unlikely to underpin shGAG siRNA production. Figure 3. Impact of splicing and premature termination on EVD silencing A–C. Constructs and isoforms transcribed from 35S:EVDwt (A), 35S:EVDΔintron (B) and 35S:EVDmU1 (C). Probes for northern analysis of GAG exon 1 (GAG-ex1), intron (GAG-in), exon2 (GAG-ex2), and the POL region are depicted with black lines. Red squares: stop codons. D. Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2. qPCR was performed on three biological replicates and error bars represent the standard error. (ns.) = non-significant, *P < 0.05, **P < 0.01, (two-sided t-test between indicated samples/targets). E. High- and low-molecular-weight RNA analysis of EVD GAG (GAG-ex1) and EVD intron (GAG-in) in two independent T1 bulks from each indicated line. The filled arrows on the right-hand side or with an asterisk on the blots correspond to the transcripts depicted in (A-C). ACT2: loading control for mRNAs; tasiR255, miR173, and U6: loading controls for sRNAs. Hybridizations for GAG-ex2 and POL probes are found in Appendix Fig S5A. F. Western blot analysis of the GAG protein with Coomassie (coom.) staining as a loading control. Source data are available online for this figure. Source Data for Figure 3 [embr202153400-sup-0004-SDataFig3.zip] Download figure Download PowerPoint To test the alternative possibility that intron-retention or specific sequences within the EVD intron prevent siRNA biogenesis from flGAG-POL, we analyzed the siRNAs from 35S:EVDmU1. Impeding U1 binding and its inhibitory action on PCPA causes a complete lack of splicing in EVDmU1 (Fig 3C and D). This generates short unspliced transcripts, alternatively terminated at the cognate shGAG terminator or at an intronic cryptic site previously mapped by 3' RACE (Oberlin et al, 2017), both detected here by northern analysis (Fig 3C–E, Appendix Fig S5A). Both alternatively terminated transcripts likely undergo translation, albeit largely unproductively (Fig 3F), because low levels of cryptic GAG translation products were detectable in rdr6 compared to WT (Appendix Fig S5B). EVDmU1 bestowed RDR6-dependent siRNA production expanding—as expected from its non-spliceable nature—into the retained intron sequence (Fig 3E). The near-complete lack of siRNAs downstream of the intron (Appendix Fig S5A), by contrast, suggested that both cryptically terminated shGAG transcripts are mainly involved in recruiting RDR6. Therefore, even though the shGAG intron and PCPA signal suffice to trigger PTGS from EVD and GFP-EVDint/ter-GUS (Figs 1 and 2), neither splicing nor intron-retention per se seem to initiate PTGS. This suggests that splicing-coupled PCPA does not co-transcriptionally condition the sensitivity of shGAG to RDR6 but, rather, downstream in the gene expression pathway. RDR6 recruitment onto shGAG likely requires translation Splicing-coupled PCPA, conserved among Arabidopsis Ty1/Copia
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