Prevalent cytidylation and uridylation of precursor miRNAs in Arabidopsis

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Abstract

A key step in microRNA biogenesis is the processing of a primary precursor RNA by the microprocessor into a precursor miRNA (pre-miRNA) intermediate. In plants, little is known about the processes that act on pre-miRNAs to influence miRNA biogenesis. Here, we performed 3′ rapid amplification of complementary DNA ends sequencing to profile pre-miRNA 3′ ends in Arabidopsis. 3′ end heterogeneity was prevalent, and the three microprocessor components promoted 3′ end precision. Extensive cytidylation and uridylation of precise and imprecise pre-miRNA 3′ ends were uncovered. The nucleotidyl transferase HESO1 uridylated pre-miRNAs in vitro and was responsible for most pre-miRNA uridylation in vivo. HESO1, NTP6 and NTP7 contribute to pre-miRNA cytidylation. Tailing of pre-miRNAs tended to restore trimmed pre-miRNAs to their intact length to promote further processing. In addition, HESO1-mediated uridylation led to the degradation of certain imprecisely processed pre-miRNAs. Thus, we uncovered widespread cytidylation and uridylation of pre-miRNAs and demonstrated diverse functions of pre-miRNA tailing in plants.

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Fig. 1: Identification of pre-miRNA 3′ ends by 3′ RACE-seq.
Fig. 2: Uridylation and cytidylation of pre-miRNAs by HESO1.
Fig. 3: NTP6 and NTP7 cytidylate pre-miRNAs.
Fig. 4: Tailing repairs trimmed pre-miRNAs.
Fig. 5: Uridylation promotes the degradation of imprecisely processed pre-miRNAs.
Fig. 6: HESO1 exhibits nucleotidyl transferase activity on pre-miR166b in vitro.
Fig. 7: Proposed model for pre-miRNA tailing in Arabidopsis.

Data availability

The GEO accession number for the pre-miRNA 3' RACE-seq data is GSE134124. All other data are available in the main text or the Supplementary Information. The data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

We thank Y. Qi for the gift of soe1 and soe2 seeds, B. Zheng for sharing the vector of 35S:HYL1-YFP and Y. Dou for technical assistance. This work was funded by the Guangdong Innovation Team Project (grant no. 2014ZT05S078) and National Science Foundation of China (grant nos. 31571332 and 31560076).

Author information

J.S., X.W., B.M. and X.C. designed the study. J.S., X.W., X.M., L.Y., H.Y. and J.L. performed the experiments. J.S., X.W., B.S., L.G. and X.C. analysed the data. G.R. provided critical reagents and equipment for the study. J.S., X.W., B.M. and X.C. wrote the manuscript.

Correspondence to Beixin Mo or Xuemei Chen.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 DCL1, HYL1 and SE promote the precise generation of pre-miRNA 3’ ends.

a, Percentage of Class I pre-miRNAs with intact 3’ ends in different genetic backgrounds (replicate 2 and replicate 3). Each dot in the scatter plot represents one pre-miRNA. The reads whose genome-matched 3’ ends were consistent with the annotated ends with or without non-templated addition were defined as ‘intact’. The black and gray horizontal lines represent the median and the quartiles (first and third quartiles), respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Differences between WT (Col-0) and mutants were evaluated by Wilcox. test, two-sided, *P < 0.05; **P < 0.01. See Fig. 1 for biological replicate 1. b, The percentage of individual Class I pre-miRNAs with intact 3’ ends as determined by 3’ RACE-seq in different genetic backgrounds. The reads whose genome-matched 3’ ends were consistent with the annotated ends with or without non-templated addition were defined as ‘intact’. Error bars are s.e.m., n = 3. Differences between WT (Col-0) and mutants were evaluated by student t-tests (paired, two-sided). *P < 0.05; **P < 0.01. Source data

Extended Data Fig. 2 HESO1 confers uridylation and cytidylation of pre-miRNAs.

ac, HESO1 causes pre-miRNA uridylation and cytidylation. The pHESO1:HESO1 transgene complemented the reduction of pre-miRNA uridylation in heso1-1 (a). Levels of pre-miRNA uridylation (b) and cytidylation (c) in WT (Ler) and heso1-2. Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles, respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage of uridylation or cytidylation was calculated as [number of reads with U or C tails/number of total reads]x100. Differences between WT (Col-0) and heso1 were evaluated by Wilcox. test, two-sided, **P < 0.01. d, Site-specific uridylation analysis of Class I pre-miRNAs in WT (Col-0) and heso1-1. The diagram of positions is shown on the top. Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles, respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage of uridylation was calculated as [number of reads with U tails at a particular position/total number of reads ending at that position]x100. Differences between WT (Col-0) and heso1-1 were evaluated by Wilcox. test, two-sided, **P < 0.01. Source data

Extended Data Fig. 3 Uridylation and cytidylation of pre-miRNAs in Col-0 and heso1-1 (replicate 2 and replicate 3).

a, b, Pre-miRNA uridylation and cytidylation analysis. Levels of pre-miRNA uridylation (a) and cytidylation (b) in WT (Col-0) and heso1-1. c, Cytidylation analysis at 5 positions of Class I pre-miRNAs in WT (Col-0) and heso1-1. Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles (first and third quartiles), respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Differences between WT (Col-0) and heso1-1 were evaluated by Wilcox. test, two-sided, *P < 0.05; **P < 0.01. See Fig. 2 for biological replicate 1. Source data

Extended Data Fig. 4 HESO1 localizes in the nucleus.

Sub-cellular localization of HYL1-YFP and HESO1-mCherry in Nicotiana benthamiana epidermal cells. For each construct, 40-50 cells were observed and gave similar patterns of protein localization. YFP and mCherry fluorescence signals were observed by confocal microscopy 40-48 h after agrobacterial infiltration. Scale bar, 20 μm. Experiments were repeated three times and gave similar results. Source data

Extended Data Fig. 5 HESO1 confers site-specific cytidylation of pre-miRNAs.

Error bars are s.e.m., n = 3. Percentage cytidylation was calculated as [number of reads with C tails at a particular position/total number of reads ending at that position]x100. Differences between WT (Col-0) and heso1-1 were evaluated by student t-tests (paired, two-sided). *P < 0.05; **P < 0.01. N.D., not detected. Source data

Extended Data Fig. 6 NTP6 and NTP7 cytidylate pre-miRNAs (replicate 2).

a, Levels of Class I pre-miRNA cytidylation in WT (Col-0) and ten nucleotidyl transferase mutants (replicate 2). Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles (first and third), respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage cytidylation was calculated as [number of reads with C tailing/number of total reads from each pre-miRNA]x100. Differences between WT (Col-0) and mutant were evaluated by Wilcox. test, two-sided, **P < 0.01. b, Analysis of site-specific cytidylation of Class I pre-miRNAs in WT (Col-0), ntp6-1 and ntp7-1(replicate 2). Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles (first and third), respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage cytidylation was calculated as [number of reads with C tailing at a specific 3’ end position/total number of reads with the specific 3’ end position]x100. Differences between WT (Col-0) and mutants were evaluated by Wilcox. test, two-sided, **P < 0.01. c, Histograms showing the degree of cytidylation of individual Class I pre-miRNAs in WT (Col-0), ntp6-1 and ntp7-1. Percentage of cytidylation was calculated as [number of reads with C tails/number of total reads]x100. Error bars are s.e.m., n = 3. Differences between Col-0 and heso1-1 were evaluated by student t-tests (unpaired, two-sided). *P < 0.05. See Fig. 3 for biological replicate 1. Source data

Extended Data Fig. 7 Cytidylation and uridylation of Class I pre-miRNA in nucleotidyl transferase mutants.

a, Phylogenetic analysis of NTP proteins in Arabidopsis. A neighbor-joining (NJ) tree was constructed by MEGA, using the p-distance method with gaps treated by pairwise deletion and a 1,000 bootstrap replicate. b, c, Levels of Class I pre-miRNA cytidylation in WT (Col-0), ntp6 ntp7 and ntp2 ntp6 ntp7. Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles (first and third), respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage cytidylation was calculated as [number of reads with C tailing/number of total reads from each pre-miRNA]x100. Differences between WT (Col-0) and mutant were evaluated by Wilcox. test, two-sided, **P < 0.01. d, Levels of Class I pre-miRNA uridylation in WT (Col-0) and ten nucleotidyl transferase mutants. Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles, respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage of uridylation was calculated as [number of reads with U tailing/number of total reads from each pre-miRNA]x100. Differences between WT (Col-0) and mutant were evaluated by Wilcox. test, two-sided, **P < 0.01. Source data

Extended Data Fig. 8 Quantification of tailing at specific sites for all Class I pre-miRNAs in WT (Col-0) and heso1-1 (replicate 2 and replicate 3).

Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles (first and third), respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage of reads was calculated as [number of reads with specific end type/number of total reads from each pre-miRNA]x100. Differences between WT (Col-0) and heso1-1 were evaluated by Wilcox. test, two-sided, *P < 0.05; **P < 0.01. See Fig. 4 for biological replicate 1. Source data

Extended Data Fig. 9 Tailing status of pre-miRNAs and miRNAs in WT (Col-0) and hen1-8.

a, b, Analysis of uridylation (a) and cytidylation (b) of Class I pre-miRNAs and miRNAs in WT (Col-0) and hen1-8. Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles, respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage of uridylation or cytidylation was calculated to the total reads mapped to each pre-miRNA or miRNA. Mono: mono-uridylation or mono-cytidylation; All: total uridylation or cytidylation. c, d, Analysis of uridylation (c) and cytidylation (d) of individual Class I pre-miRNAs and miRNAs (miRNA locates in the 3 P strand) in WT (Col-0) and hen1-8. Error bars are s.e.m., n = 3. e, f, Percentage of 1-nt and 2-nt tails at position 1 (e) and 2 (f) of pre-miR158a and miR158a in WT (Col-0) and hen1–8. Percentage was calculated based on total tailed pre-miR158a and miR158a at position 1 (e) and 2 (f). The inset shows all cytidine-containing tails. Error bars are s.e.m., n = 3. Differences between WT (Col-0) and hen1–8 were evaluated by student t-tests (unpaired, two-sided). *P < 0.05. Source data

Extended Data Fig. 10 Tailing of pre-miRNAs and miRNAs in WT (Ler) and hen1-2.

a, b, Analysis of uridylation (a) and cytidylation (b) of Class I pre-miRNAs and miRNAs in WT (Ler) and hen1-2. Each dot represents a pre-miRNA. The black and gray lines represent the median and the quartiles, respectively. Values are Class I pre-miRNAs from one biological repeat (n = 24). Percentage of uridylation or cytidylation was calculated to the total reads mapped to each pre-miRNA or miRNA. Mono: mono-uridylation or mono-cytidylation; All: total uridylation or cytidylation. c, d, Analysis of uridylation (c) and cytidylation (d) of individual Class I pre-miRNAs and miRNAs (miRNA locates in the 3 P strand) in WT (Ler) and hen1-2. Error bars are s.e.m., n = 3. e, The uridylation/cytidylation ratio in the tails of pre-miRNAs and miRNAs in WT (Ler) and hen1-2. Tails composed of uridine or cytidine only were included in the quantification of uridylation or cytidylation. Percentage of uridylation or cytidylation was calculated to the total reads mapped to each pre-miRNA and miRNA. Error bars are SEM, n = 3. Differences between WT (Ler) and hen1-2 were evaluated by student t-tests (unpaired, two-sided). *P < 0.05; **P < 0.01. fh, Percentage of 1-nt and 2-nt tails at position 0 (f), 1 (g) and 2 (h) of pre-miR158a and miR158a in WT (Ler) and hen1-2. Percentage was calculated based on total tailed pre-miR158a and miR158a at position 0 (f), 1 (g) and 2 (h). The inset shows all cytidine-containing tails. Error bars are s.e.m., n = 3. Differences between WT (Ler) and hen1-2 were evaluated by student t-tests (unpaired, two-sided). *P < 0.05. Source data

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Song, J., Wang, X., Song, B. et al. Prevalent cytidylation and uridylation of precursor miRNAs in Arabidopsis. Nat. Plants 5, 1260–1272 (2019) doi:10.1038/s41477-019-0562-1

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