Characterization of 3′ extensions. (A) Northern blot of mRNAs and 3′ extensions of At1g16410 and At3g45160. Positions of probes used are shown in the illustration of the mRNA and the 3′ extension. Samples from two biological replicates are loaded on each blot. TUB8 mRNA and ribosomal RNA were used as internal controls. (B) Self-ligation–mediated RT-PCR analysis. The experimental procedure is shown in Figure S1C. ACT2 mRNA was used as an internal control. CIP, calf intestinal phosphatase; TAP, tobacco acid pyrophosphatase.

The major activity of XRN3 is to eliminate 3′ extensions. (A) Comparison of accumulation profiles in wild-type and each mutant in the range from the 3′ end of genes to 500 nt downstream. RPKM values of all expressed transcripts in wild-type or each mutant were classified into four categories. Total numbers are 18,431, 17,247, 18,480, and 15,323 in wild-type or xrn3-3, xrn2xrn3, xrn3xrn4, and xrn2xrn4, respectively. Averages RPKM values of two RNA-Seq replicates were used. Tissues use for wild-type (WT), xrn3-3, xrn3xrn4, and xrn2xrn4 were 2-week-old plants grown on nutrient medium, whereas xrn2xrn3 and the control wild-type (WT3w) were 3-week-old plants grown in soil. (B) Venn diagrams of the overlap between 3′ extensions identified in fry1-6 and those identified in xrn3-3, xrn2xrn3, and xrn3xrn4, and the overlap between in xrn3-3 and in xrn3 double mutants. (C) Quantitative RT-PCR analysis of four 3′ extensions and the corresponding 5′ adjacent mRNAs. Positions of primers used are shown in the illustration of the mRNA and the 3′ extension. Vertical axes show relative accumulation normalized against ACT2 expression. x2x3 = xrn2xrn3; x2x4 = xrn2xrn4; x3x4 = xrn3xrn4. Additional qRT-PCR data are presented in Figure S3C.

FRY1 and XRN3 repress 3′ remnants of miRNA precursors. (A) Examples of pri-miRNAs (pri-miR156c and pri-miR172a) showing increased accumulation. (B) Comparison of accumulation profiles of 151 expressed pri-miRNAs. Averages of RPKM values of three replicates of RNA-Seq in wild-type and fry1-6 and two replicates of RNA-Seq in xrn3-3, xrn3xrn4, 3-week-old wild-type, and xrn2xrn3 were used. (C) Quantitative RT-PCR analysis of six pri-miRNA_3′s and the pri-miRNA_5′s. Positions of primers used are shown in the illustration of pri-miRNA. Vertical axes show relative accumulation normalized against ACT2 expression. x2x3 = xrn2xrn3; x2x4 = xrn2xrn4; x3x4 = xrn3xrn4.

Identification of novel transcripts extending the 3′ ends of mRNAs. (A) Example (At1g16410 locus) of novel 3′ extended signals (3′ extensions) identified by the ARTADE program (Toyoda and Shinozaki 2005) in tiling array data from fry1-6. Vertical red bars indicate signal intensities from each probe. Horizontal blue and light blue regions are exons and introns, respectively, in the gene model of At1g16410. The horizontal orange region indicated by the arrow is a predicted 3′ extension. The length of black scale bar is 500 nt. (B) Distribution of estimated lengths of 171 3′ extensions identified in the tiling array analysis of fry1-6. (C) A schematic of the 500 nt downstream region used for calculation of RPKM values. (D) Comparison of accumulation profiles in the range from the 3′ ends of genes to 500 nt downstream between wild-type and fry1-6 (RNA-Seq). RPKM values of all expressed transcripts (total 19,104) in wild-type or fry1-6 were classified into four categories. (E) A scatter plot of average RPKM values between wild-type and fry1-6. The 3′ regions with RPKM values less than 500 were plotted. (F) Examples of 3′ extensions identified in fry1-6 by filtering through four parameters [Fold (fry1-6/WT) > 5, RPKM average (fry1) > 1, P-value < 0.1, non-pri-miRNA]. Pink regions are 3′ extensions. The length of the black scale bar is 500 nt.

Many actively transcribed genes possess 3′ RNA extensions. Comparison of accumulation profiles of 2230 3′ extensions and the 5′ mRNAs between wild-type and fry1-6. RPKM values of 3′ extensions and the mRNAs were classified into four and six categories, respectively. (B) Distribution of RPKM values of mRNAs tailed with 2230 3′ extensions. The population “all_gene” comprising mRNAs with RPKM values in fry1-6 was used for control of comparison. ext, extension.

With few exceptions, 3′ extensions do not lead to de novo DNA methylation of the 3′ extension originating loci. (A) Examples of RNA-Seq and MethylC-Seq data from loci that exhibit overlapping transcripts. Pink regions are 3′ extensions. (B) At1TE93275 locus. The length of black scale bar is 500 nt. (C) Quantitative RT-PCR analysis of the antisense transcript of At1TE93275. Vertical axes show relative accumulation normalized against ACT2 expression. x2x3 = xrn2xrn3; x2x4 = xrn2xrn4; x3x4 = xrn3xrn4.

Lithium induces accumulation of 3′ extension transcripts. (A) Model for inhibition of FIERY1 activity by lithium. (B) Quantitative RT-PCR analysis of 3′ extensions and pri-miRNA_3′s under some stressful conditions. Wild-type seedlings were soaked in water, 200 mM LiCl, 200 mM NaCl, 100 μM abscisic acid (ABA), or 300 mM mannitol solutions, and incubated for 5 hr.