Volume 30, Issue 2, Pages 166-176 (July 2014) Long Noncoding RNA Modulates Alternative Splicing Regulators in Arabidopsis  Florian Bardou, Federico Ariel, Craig G. Simpson, Natali Romero-Barrios, Philippe Laporte, Sandrine Balzergue, John W.S. Brown, Martin Crespi  Developmental Cell  Volume 30, Issue 2, Pages 166-176 (July 2014) DOI: 10.1016/j.devcel.2014.06.017 Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 AtNSRs Localize in Nuclear Speckles and Relocalize into Cytoplasmic Bodies When Coexpressed with the lncRNA ENOD40 (A) The NSRs from Medicago truncatula (MtRBP1/MtNSR1: Medtr6g034835.1) and Arabidopsis thaliana (AtNSRa and AtNSRb: At1g76940 and At1g21320, respectively) have a conserved gene structure (exons 1 to 5), an RNA recognition motif in the C-terminal part of the proteins (RRM), and a nuclear localization signal (NLS, represented by a red arrow in exon 1). The AtNSRb gene is expressed from two different transcription start sites, and the NSRb.2 mRNA isoform contains exon 1 but not exon 1′. The two transcripts have different translational start codons (indicated by blue arrows). (B and C) 35S-AtNSRa-GFP (B) and 35S-AtNSRb-GFP (C) colocalize with splicing-related SR protein AtSR34 (35S-SR34-RFP; AT1G02840) in nuclear “speckles” (colocalizations are in yellow). (D) 35S-AtNSRb-GFP fusion proteins localize in nuclear speckles (left panel), and coexpression of MtENOD40 RNA (from a 35S-ENOD40 construct) results in relocalization from the nucleus to punctuate cytoplasmic regions (fluorescent dots) after transient expression in tobacco leaves (right panel). n, nucleus (circled by a red line); c, cytoplasmic dots. Scale bars, 8 μm. See also Figure S1. Developmental Cell 2014 30, 166-176DOI: (10.1016/j.devcel.2014.06.017) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 AtNSRs Are Expressed during LR Formation (A and B) AtNSRa (pAtNSRs-GUS) (A) and the two AtNSRb promoters [pAtNSRb1-GUS and pAtNSRb2-GUS, schematized in (B)] fused to GUS are active in vascular tissues and during LR formation in dividing primordia until lateral root emergence. Scale bars, 100 μm. (C) Expression of the translational fusion pNSRa-NSRa-GFP labels nuclear particles (higher magnification images of single nuclei are provided) during all steps of LR formation (I and II, IV, VII, and VIII according to Malamy and Benfey, 1997). Arrows indicate nuclei; yellow arrows show nuclei after migration during early steps of LR formation. See also Figure S2. Developmental Cell 2014 30, 166-176DOI: (10.1016/j.devcel.2014.06.017) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 The nsra/nsrb DM Shows a Reduced Number of Auxin-Induced LRs (A) A kinetics of AtNSRb induction by auxin treatment (1 μM NAA) is shown (white bars), whereas AtNSRa is constitutively expressed (black bars). (B–D) The nsra/nsrb DMs show a reduced number (C) and length (D) of auxin-induced LRs (NAA, 100 nM). These phenotypes are more accentuated in response to auxin where LRs are formed all along the primary root, as shown in (B) corresponding to a WT and DM plant 7 days after germination in auxin-containing medium. (E) In contrast, no variation in primary root length was observed at this time point. n > 35; Error bars indicate confidence interval 5%; stars indicates Student’s t test α = 0.05. (F) Transcriptome analysis of the Atnsra/nsrb DM showed 535 genes differentially regulated compared to WT (−NAA), whereas an auxin treatment revealed 2,214 genes with expression changes under the same criteria when compared to WT (+NAA; CATMA arrays V6). (G) The lncRNA lnc34 is strongly induced by auxin, an accumulation significantly lost in the DM. (H) The ASCO-RNA is upregulated in the nsra/nsrb DM in both control and auxin conditions. Error bars indicate SD. See also Figures S3 and S4. Developmental Cell 2014 30, 166-176DOI: (10.1016/j.devcel.2014.06.017) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Modulation of AS in the nsra/nsrb Mutant during Auxin Response (A) Relative proportion and number of genes showing a significant change in AS from the analysis using the high-resolution AS real-time PCR panel of the nsra/nsrb DM with and without 1 μM of auxin in comparison to WT (based on Table S1). An important class (43 genes) corresponds to auxin-dependent AS changes requiring AtNSRs (light gray). Genes with auxin-specific (green) and NSR-specific (blue) effects on AS are represented. The threshold used for the analysis was >3% change with a p value of < 0.05; detailed data and analysis are provided in Table S1. (B) The ATPase1 (At1G27770) is mainly transcribed as a single mRNA in control conditions or during auxin treatment (1 μM) whereas, in the Atnsra/nsrb mutant treated with auxin, a second isoform accumulates (an IR event, black bars; see Figure S5E). Arrows indicate the fully spliced (white) and AS (black) isoforms in PAGE gels, and their shading correspond to its quantification (relative to the amount of the same isoform in Col-0 during control conditions) in the accompanying bar graph panels. See also Figures S5 and S6 and Table S1. Developmental Cell 2014 30, 166-176DOI: (10.1016/j.devcel.2014.06.017) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 An NSR Complex Binds to Alternatively Spliced Targets and to ASCO-RNA In Vivo and In Vitro (A and B) RIP assays using HA-tagged NSRa or NSRb on total cell lysates (Total RNA) or nuclear extracts (Nuclear RNA) of 10-day-old seedlings treated with 10 μM NAA for 24 hr. Results of quantitative real-time PCR are expressed as the percentage of the respective input signal (INPUT: total signal before RIP). Genes analyzed in (A), with accession numbers, are as follows: housekeeping (HKeeping): At1G13320 (Czechowski et al., 2005); Fbox: At4G27050; PIWI factor: At2G29210; and auxin-regulated gene: At2G33830. In (B), the following lncRNAs were tested: lnc34, 43, 72, 78, 351 (ASCO-RNA), 375, and 536 (Ben Amor et al., 2009). The only significant enrichment was detected for ASCO-RNA (lnc351, At1G67105); the other RNAs gave values similar to those of housekeeping genes. (C) Quantitative real-time PCR showing the relative expression of U6 (a known nuclear RNA) and ASCO-RNA in total or nuclear RNA. Enrichment in the nuclear RNA fraction at higher values than U6 was observed for the ASCO-RNA. Errors bars indicate SD. (D) In vitro competition assay; the graph represents the average quantification of dot blots in three biological replicates (relative to the amount of the same dilution without competitor RNA [w/o]). These results indicate that the ASCO-RNA has the capacity to compete the binding of NSR to an AS target. The lncRNA72 (a lncRNA that does not bind to NSRs) was not able to compete NSR-AS target binding. Error bars indicate SD. See also Figure S7. Developmental Cell 2014 30, 166-176DOI: (10.1016/j.devcel.2014.06.017) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 The ASCO-RNA Modulates AS of NSR-Dependent AS Targets In Vivo, and Its Overexpression Reduced the Number of Auxin-Induced LRs (A) Plants overexpressing the ASCO-RNA showed changes in isoform distribution of the auxin-related protein (At2G33830) (an IR event; black bars). (B) The ATPase1 (At1G27770) is transcribed as a single mRNA in control conditions, whereas a second isoform (an IR event; black bars) is detectable in ASCO-RNA overexpressing plants, a pattern similar to the one observed in the nsra/nsrb DM treated with auxin (Figure 4B). Arrows indicate each AS isoform in PAGE gels, and their shading corresponds to its quantification (relative to the amount of the same isoform in Col-0 during control conditions) in the accompanying bar graph panels. (C and D) Two ASCO-RNA overexpressing lines (35S-ASCO.1 and 35S-ASCO.2) do not present any major variation in primary root length (C) but present a reduced LR density after and auxin treatment (100 nM NAA) 7 days after germination (D). For comparison, the nsra/nsrb phenotype is shown. n > 20; error bars indicate SD; asterisks indicate Kruskal-Wallis test α = 0.05. See also Figure S5. Developmental Cell 2014 30, 166-176DOI: (10.1016/j.devcel.2014.06.017) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 ASCO-RNA Interacts with the NSRs to Modulate AS Model for the action of NSRs. NSRs are splicing regulators that bind to pre-mRNA targets and modulate the ratio between different isoforms. In addition, NSRs can bind lncRNAs such as the endogenous ASCO-RNA in the nucleus to alter AS. Our results support the hypothesis that lncRNAs can hijack NSRs to affect their binding to AS targets and modulate AS regulatory activity of the NSRs. These results indicate a link between alternative splicing regulation and the action of lncRNA. Developmental Cell 2014 30, 166-176DOI: (10.1016/j.devcel.2014.06.017) Copyright © 2014 Elsevier Inc. Terms and Conditions