1. 5-Methylcytosine RNA Methylation in Arabidopsis Thaliana
Xuean Cui, Zhe Liang, Lisha Shen, Qian Zhang, Shengjie Bao, Yuke Geng, Bin Zhang, Vonny Leo, Leah A. Vardy, Tiegang Lu, Xiaofeng Gu, Hao Yu 
Molecular Plant 
Volume 10, Issue 11, Pages (November 2017)
DOI: /j.molp
Copyright © 2017 The Author Terms and Conditions

2. Figure 1 Detection of m5C in Arabidopsis and Other Plants.
(A) Chemical structure of m5C RNA modification with standard numbering of cytosines.
(B) Dot blot analysis shows that the anti-m5C antibody specifically recognizes an m5C-modified RNA oligo. The upper panel shows quantification of the dot blot results of three biological replicates by ImageJ, while the lower panel shows representative blots.
(C–E) Dot blot (left panels) and LC–MS/MS (right panels) assays show detection and quantification of m5C levels in Arabidopsis in different RNA species of 9-day-old wild-type whole seedlings (C), in mRNA from various tissues of wild-type plants, including rosette leaves (RL), cauline leaves (CL), stems (St), flower buds (FB), 9-day-old seedlings (D9), open flowers (OF), roots (Rt), and siliques (Sil) (D), and in mRNA from developing wild-type seedlings (3- to 15-day-old; D3 to D15) (E). Error bars, mean ± SD; n = 3.
(F and G) LC–MS/MS assays show detection (F) and quantification (G) of m5C levels in different plant species. Error bars, mean ± SD; n = 3.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

3. Figure 2 Transcriptome-Wide Mapping of m5C in Arabidopsis.
(A) Schematic diagram of the m5C-RIP-seq procedure.
(B) An example of m5C enrichment at At5g Blue or red color represents input or IP reads, respectively. The transcript structure is shown beneath, with thick and thin boxes representing exons and introns, respectively.
(C) Distribution of m5C-modified or non-modified mRNAs relative to mRNA abundance.
(D) Verification of m5C-RIP-seq results. m5C-RIP-qPCR was performed on 9-day-old wild-type seedlings harvested independently of the m5C-RIP-seq assays. Error bars, mean ± SD; n = 3. Asterisk indicates a significant difference in enrichment fold of a selected transcript compared with that of TUB2 indicated by a red dashed line (two-tailed paired Student's t-test, P < 0.05).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

4. Figure 3 m5C Methylome in Arabidopsis.
(A) List of the numbers of m5C-modified genes in different gene categories. snoRNA, small nucleolar RNA; snRNA, small nuclear RNA.
(B) List of the numbers of genes bearing 1, 2, 3, or >3 m5C peaks.
(C) Distribution of m5C peaks within protein-coding gene bodies divided into 5′ UTRs, CDSs, 3′ UTRs, and intronic and intergenic regions.
(D) Comparison of the numbers of observed m5C peaks versus expected peaks in transcript segments divided into 5′ UTRs, CDSs, and 3′ UTRs. Asterisk indicates a significant difference in the numbers of observed m5C peaks versus expected peaks (binomial test, P < 10−5).
(E) Distribution of m5C peaks in transcript segments divided into 5′ UTRs, CDSs, and 3′ UTRs.
(F) Sequence logo representations of the consensus motifs identified by MEME-ChIP. The number of occurrences of each motif relative to the total number of m5C peak summits and the corresponding P value generated by MEME-ChIP are shown under the logo.
(G) Gene ontology (GO) biological processes enrichment analysis of genes bearing m5C peaks. Statistical analysis was performed using the DAVID tool. P < 0.05.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

5. Figure 4
Monosomes with Low Translation Activity Are Associated with High m5C Levels.
(A) Polysome profile of 9-day-old seedlings of Arabidopsis. Twelve fractions were collected.
(B) Dot blot assay shows that the fractions containing mRNA associated with ribosomal subunits (40S and 60S; faction 3) and monosomes (80S; fraction 4) have the highest m5C levels. The number on the top of each dot indicates its fraction number shown in (A). The blot shown is a representative result of three independent replicates.
(C) Quantification of dot blot results of three biological replicates as shown in (B) by ImageJ. The highest level is set as 100%. Error bars, mean ± SD; n = 3.
(D) Quantification of the m5C percentage relative to cytosine (m5C/C ratio) determined by LC–MS/MS assays of mRNA associated with various collected fractions. Error bars, mean ± SD; n = 3.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

6. Figure 5 m5C Levels Decrease in trm4b Mutants.
(A) Ion chromatograms of m5C levels in m5C methylation assays. RNA containing the m5C motifs (RNA) or without cytosines (RNA w/o C) served as the input and was incubated with GST or GST-TRM4B. m5C levels of RNA input and RNA purified from incubation with GST protein or GST-TRM4B were measured by LC–MS/MS assays.
(B) Quantification of the m5C percentage relative to cytosine (m5C/C ratio) in m5C methylation assays shown in (A). Error bars, mean ± SD; n = 3.
(C) m5C/C ratios determined by LC–MS/MS assays of mRNA purified from root tissues (upper panel) and aerial tissues (lower panel) of 9-day-old wild-type, trm4b, and trm4b pTRM4B:TRM4B-3HA plants. Error bars, mean ± SD; n = 3. Asterisks indicate significant differences between wild-type and trm4b plants (two-tailed paired Student's t-test, P < 0.05).
(D) List of the numbers of m5C-modified genes in different gene categories in trm4b-4 seedlings.
(E) Cumulative distribution function of the log2 peak intensity of m5C peaks in trm4b-4 and wild-type seedlings.
(F) List of the numbers of genes bearing 1, 2, 3, or >3 m5C peaks in trm4b-4 seedlings.
(G) Distribution of m5C peaks within protein-coding gene bodies divided into 5′ UTRs, CDSs, 3′ UTRs, and intronic and intergenic regions in trm4b-4 seedlings.
(H) Comparison of the numbers of observed m5C peaks versus expected peaks in transcript segments divided into 5′ UTRs, CDSs, and 3′ UTRs in trm4b-4 seedlings. Asterisk indicates a significant difference in the numbers of observed m5C peaks versus expected peaks (binomial test, P < 10−5).
(I) Distribution of m5C peaks in transcript segments divided into 5′ UTRs, CDSs, and 3′ UTRs in trm4b-4 seedlings.
(J) Sequence logo representations of the consensus motifs identified by MEME-ChIP in trm4b-4 seedlings. The number of occurrences of each motif relative to the total number of m5C peak summits and the corresponding P value generated by MEME-ChIP are shown under the logo.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

7. Figure 6
TRM4B Simultaneously Affects m5C and Transcript Levels of Key Genes Involved in Root Development.
(A) Heatmap of differentially expressed genes in wild-type and trm4b-4 seedlings revealed by RNA-seq.
(B) Number of up- or downregulated genes in trm4b-4 revealed by RNA-seq.
(C) Overlap between m5C-RIP-seq and RNA-seq datasets. P value of the overlap between the two datasets was calculated using the hypergeometric distribution with the total number of Arabidopsis genes as the reference.
(D) Number of up- or downregulated genes in trm4b-4 that contain m5C modifications.
(E) Quantitative real-time PCR analyses of SHY2 and IAA16 expression in wild-type, trm4b, and trm4b pTRM4B:TRM4B-3HA seedlings. Gene expression levels in wild-type seedlings are set as 1. Error bars, mean ± SD, n = 3. *P < 0.05, two-tailed paired Student's t-test.
(F) m5C-IP-qPCR showing that m5C modification of SHY2 and IAA16 mRNA occurs in wild-type plants but is compromised in trm4b-4. Error bars, mean ± SD, n = 3. *P < 0.05, two-tailed paired Student's t-test.
(G) Quantitative real-time PCR analyses of transcripts levels of SHY2, IAA16, and TUB2 in roots of 9-day-old wild-type, trm4b, and trm4b pTRM4B:TRM4B-3HA plants treated with actinomycin D versus mock treated for 0, 8, and 24 hr. The relative expression of each gene at different time points was calculated by normalizing the gene expression in actinomycin D-treated samples against that in mock-treated samples. Error bars, mean ± SD, n = 3. *P < 0.05, two-tailed paired Student's t-test.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions