Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis

doi:10.1038/s42003-024-06142-4

. 2024 Apr 18;7(1):474.

doi: 10.1038/s42003-024-06142-4.

Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis

Li Feng^#¹, Wei Yan^#², Xianli Tang^#², Huihui Wu², Yajie Pan², Dongdong Lu¹, Qianyan Ling-Hu², Yuelin Liu², Yongqi Liu¹, Xiehai Song¹, Muhammad Ali¹, Liang Fang², Hongwei Guo³, Bosheng Li⁴

Affiliations

¹ Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agriculture Sciences in Weifang, Weifang, Shandong, 261325, China.
² Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China.
³ Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China. guohw@sustech.edu.cn.
⁴ Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agriculture Sciences in Weifang, Weifang, Shandong, 261325, China. bosheng.li@pku-iaas.edu.cn.

^# Contributed equally.

PMID: 38637717
PMCID: PMC11026412
DOI: 10.1038/s42003-024-06142-4

Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis

Li Feng et al. Commun Biol. 2024.

. 2024 Apr 18;7(1):474.

doi: 10.1038/s42003-024-06142-4.

Authors

Affiliations

¹ Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agriculture Sciences in Weifang, Weifang, Shandong, 261325, China.
² Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China.
³ Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China. guohw@sustech.edu.cn.
⁴ Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agriculture Sciences in Weifang, Weifang, Shandong, 261325, China. bosheng.li@pku-iaas.edu.cn.

^# Contributed equally.

PMID: 38637717
PMCID: PMC11026412
DOI: 10.1038/s42003-024-06142-4

Erratum in

Author Correction: Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis.
Feng L, Yan W, Tang X, Wu H, Pan Y, Lu D, Ling-Hu Q, Liu Y, Liu Y, Song X, Ali M, Fang L, Guo H, Li B. Feng L, et al. Commun Biol. 2024 May 6;7(1):534. doi: 10.1038/s42003-024-06241-2. Commun Biol. 2024. PMID: 38710925 Free PMC article. No abstract available.

Abstract

Coding transcript-derived siRNAs (ct-siRNAs) produced from specific endogenous loci can suppress the translation of their source genes to balance plant growth and stress response. In this study, we generated Arabidopsis mutants with deficiencies in RNA decay and/or post-transcriptional gene silencing (PTGS) pathways and performed comparative sRNA-seq analysis, revealing that multiple RNA decay and PTGS factors impede the ct-siRNA selective production. Genes that produce ct-siRNAs often show increased or unchanged expression and typically have higher GC content in sequence composition. The growth and development of plants can perturb the dynamic accumulation of ct-siRNAs from different gene loci. Two nitrate reductase genes, NIA1 and NIA2, produce massive amounts of 22-nt ct-siRNAs and are highly expressed in a subtype of mesophyll cells where DCL2 exhibits higher expression relative to DCL4, suggesting a potential role of cell-specific expression of ct-siRNAs. Overall, our findings unveil the multifaceted factors and features involved in the selective production and regulation of ct-siRNAs and enrich our understanding of gene silencing process in plants.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. RNA decay and PTGS factors regulate ct-siRNA production.**
a The percentage of siRNAs derived from various siRNA-generating loci, including protein-coding, structure RNA, non-coding RNA, pseudogenes & TE, and pri-miRNA. The mutant alleles used in this study are abbreviated as x2 (*xrn2-2*), x3 (*xrn3-3*), *x2×3*(*xrn2-2 xrn3-3*), s2 (*ski2-2*), h2 (*hen2-1*), e5 (*ein5-1*), *es3* (*ein5-1 ski2-3*), *dcp2* (*dcp2-1*), *dcl4* (*dcl4-2*), *x3d4* (*xrn3-3 dcl4-2*), *x2d4* (*xrn2-2 dcl4-2*), *fd4* (*fry1-6 dcl4-2*), *sd4* (*ski2-2 dcl4-2*), *hd4* (*hen2-1 dcl4-2*), *ed4* (*ein5-1 dcl4-2*), *dd4* (*dxo1-2 dcl4-2*), *ud4* (*urt1-1 dcl4-2*), *esa1-27* (*ein5-1 ski2-3 ago1-27*), *esa1-45* (*ein5-1 ski2-3 ago1-45*), *sda1-27* (*ski2-2 dcl4-2 ago1-27*), *sda-45* (*ski2-2 dcl4-2 ago1-45*), *hda1-27* (*hen2-1 dcl4-2 ago1-27*), *hda1-45* (*hen2-1 dcl4-2 ago1-45*), *eda1-27* (*ein5-1 dcl4-2 ago1-27*), *eda-45* (*ein5-1 dcl4-2 ago1-45*), *rdr6* (*rdr6-11*), *res3* (*ein5-1 ski2-3 rdr6-11*), *rres3* (*ein5-1 ski2-3 rdr1-1 rdr6-11*), *hr6* (*hen2-1 rdr6-11*), *hdr6* (*hen2-1 dcl4-2 rdr6-11*), *dcl2* (*dcl2-1*), *hd2* (*hen2-1 dcl2-1*), *sd2* (*ski2-2 dcl2-1*), *ed2* (*ein5-1 dcl2-1*), *esd2* (*ein5-1 ski2-3 dcl2-1*), *d4d2* (*dcl4-2 dcl2-1*), *hdd* (*hen2-1 dcl4-2 dcl2-1*), *edd* (*ein5-1 dcl4-2 dcl2-1*), *sdd* (*ski2-2 dcl4-2 dcl2-1*), *es3dd* (*ein5-1 ski2-3 dcl4-2 dcl2-1*), and *fdd* (*fry1-6 dcl2-1 dcl4-2*). b The percentage of ct-siRNAs with lengths ranging from 20-nt to 24-nt. Only reads produced from the antisense strand of protein-coding genes, representing ct-siRNAs, were calculated.

**Fig. 2. Accumulation and distribution of ct-siRNAs in mutants deficient in RNA decay and PTGS factors.**
a Pie charts ranking the top10-scoring 22-nt ct-siRNA-producing loci by accumulated ct-siRNA abundance. b Heatmap depicting the expression of ct-siRNAs produced by 52 hotspot genes. c Dot plot of the relative expression levels (log2) of hotspot genes in *ed4* (*ein5 dcl4-2*) or *sd4* (*ski2-2 dcl4-2*) mutant versus Col-0 plants. d An Integrated Genome View (IGV) illustrating the distribution of 21-nt and 22-nt ct-siRNAs accumulated at specific genes. e Clustering of samples based on the relative expression of ct-siRNAs in mutants versus Col-0. f A Gene Ontology (GO) annotation network illustrating the function categories of ct-siRNA source genes influenced by different groups of RNA decay factors.

**Fig. 3. Impact of source gene characteristics on ct-siRNA production.**
a The sequence length distribution of source genes. b The 5’ UTR length distribution of source genes. c The abundance of ct-siRNAs derived from genes with low, medium, and high GC content, as well as their 1Kb upstream and downstream regions. A total of 3838 genes exhibited differential accumulation of 22-nt ct-siRNAs in the eight selected mutants when compared to Col-0, with a *padj* < 0.05 and log2FC (Fold Change) > 1. The curves figures in the left and right represent 21-nt and 22-nt ct-siRNAs, respectively.

**Fig. 4. Transgenes of truncated *NIA1* and *NIA2* fragments effectively induce both gene silencing.**
a Schematic illustration of the consecutive truncated 600-nt *NIA1* and *NIA2* fragments. b GFP fluorescence in transgenic plants expressing truncated *NIA1* and *NIA2* fragments. Scale bar = 100 μm. c Fluorescence intensity was detected in transgenic plants. d Abundance of 21-nt and 22-nt ct-siRNAs (TPM, tags per million) accumulated at *NIA1* and *NIA2* in transgenic plants. n = 2 biologically independent samples. FL, full-length. e Northern blotting of ct-siRNAs produced from GFP, *NIA1*, and *NIA2* in transgenic plants. f Distribution of 21-nt and 22-nt ct-siRNAs generated from *NIA1* and *NIA2* in transgenic plants. g Sequence similarity between truncated *NIA1* and *NIA2* fragments. h GC contents of full-length and truncated *NIA1* and *NIA2* fragments.

**Fig. 5. Dynamic accumulation of ct-siRNAs in *ein5 dcl4* and *ski2 dcl4* plants across different stages of growth and development.**
**a, b** The accumulation and percentage of 20-nt to 24-nt ct-siRNAs in *ed4* (*ein5-1 dcl4-2*) and *sd4* (*ski2-2 dcl4-2*) plants across different days. The 7th and 10th days mark the earliest time points at which homozygous mutants of *sd4* and *ed4* can be distinguished from heterozygous mutants. Both mutants die on the 21st day. c The percentage of top-scoring 22-nt ct-siRNA source genes ranked by accumulated 22-nt ct-siRNA abundance in each mutant. d Clustering analysis of genes with differentially accumulated 22-nt ct-siRNAs by their abundance in *ed4* and *sd4* plants. 583 and 423 genes with 22-nt ct-siRNA abundance TPM > 10 in at least two stages and an absolute log2FC > 1 when comparing any two stages were used.

**Fig. 6. Cell-specific expression of ct-siRNA source genes.**
a UMAP visualization of seedling cell types. Each dot represents an individual cell, with color represents the respective cell type. Corresponding seedling clusters are indicated on the right. “n” indicates cell numbers. b UMAP visualization of Col-0, *ed4* (*ein5-1 dcl4-2*), *sd4* (*ski2-2 dcl4-2*), *and hd4* (*hen2-1 dcl4-2*) samples as shown in a. c Expression of *NIA1* and *NIA2* in each sample at the single-cell level visualized by UMAP. d Average expression of *NIA1* and *NIA2* and relative expression of *DCL2* versus *DCL4* in eight subtypes of mesophyll cells.

**Fig. 7. Proposed model for the selective generation of ct-siRNAs.**
RNA decay, PTGS, and associated factors synergistically influence ct-siRNA production, exerting either inhibitory or promotional effects. The accumulation of ct-siRNAs correlates with sequence length, GC content, and 5’ UTR length of their source genes. Concerning the expression of these genes, fluctuations induced by plant growth and development, combined with cell-level specificity, dictate ct-siRNA selective production. We hypothesize that the impact of each factor is proportional to the strength exhibited when considered individually.

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References

1. Elkind Y, et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. Proc. Natl. Acad. Sci. USA. 1990;87:9057–9061. doi: 10.1073/pnas.87.22.9057. - DOI - PMC - PubMed
1. Borges F, Martienssen RA. The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 2015;16:727–741. doi: 10.1038/nrm4085. - DOI - PMC - PubMed
1. Ferrer-Orta C, Agudo R, Domingo E, Verdaguer N. Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase. Curr. Opin. Struct. Biol. 2009;19:752–758. doi: 10.1016/j.sbi.2009.10.016. - DOI - PubMed
1. Bologna NG, Voinnet O. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev. Plant Biol. 2014;65:473–503. doi: 10.1146/annurev-arplant-050213-035728. - DOI - PubMed
1. Liu L, Chen X. RNA quality control as a key to suppressing RNA silencing of endogenous genes in plants. Mol. Plant. 2016;9:826–836. doi: 10.1016/j.molp.2016.03.011. - DOI - PMC - PubMed

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[1] Elkind Y, et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. Proc. Natl. Acad. Sci. USA. 1990;87:9057–9061. doi: 10.1073/pnas.87.22.9057. - DOI - PMC - PubMed

[2] Elkind Y, et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. Proc. Natl. Acad. Sci. USA. 1990;87:9057–9061. doi: 10.1073/pnas.87.22.9057. - DOI - PMC - PubMed

[3] Borges F, Martienssen RA. The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 2015;16:727–741. doi: 10.1038/nrm4085. - DOI - PMC - PubMed

[4] Borges F, Martienssen RA. The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 2015;16:727–741. doi: 10.1038/nrm4085. - DOI - PMC - PubMed

[5] Ferrer-Orta C, Agudo R, Domingo E, Verdaguer N. Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase. Curr. Opin. Struct. Biol. 2009;19:752–758. doi: 10.1016/j.sbi.2009.10.016. - DOI - PubMed

[6] Ferrer-Orta C, Agudo R, Domingo E, Verdaguer N. Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase. Curr. Opin. Struct. Biol. 2009;19:752–758. doi: 10.1016/j.sbi.2009.10.016. - DOI - PubMed

[7] Bologna NG, Voinnet O. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev. Plant Biol. 2014;65:473–503. doi: 10.1146/annurev-arplant-050213-035728. - DOI - PubMed

[8] Bologna NG, Voinnet O. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev. Plant Biol. 2014;65:473–503. doi: 10.1146/annurev-arplant-050213-035728. - DOI - PubMed

[9] Liu L, Chen X. RNA quality control as a key to suppressing RNA silencing of endogenous genes in plants. Mol. Plant. 2016;9:826–836. doi: 10.1016/j.molp.2016.03.011. - DOI - PMC - PubMed

[10] Liu L, Chen X. RNA quality control as a key to suppressing RNA silencing of endogenous genes in plants. Mol. Plant. 2016;9:826–836. doi: 10.1016/j.molp.2016.03.011. - DOI - PMC - PubMed

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Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis

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Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis

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