Dissecting arabidopsis thaliana dicer function in small rna processing, gene silencing and dna methylation patterning

ABSTRACT Small RNAs have several important biological functions1. MicroRNAs (miRNAs) and _trans_-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs

cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation2. Here, we study the

role of the four _Arabidopsis thaliana DICER-LIKE_ genes (_DCL1_–_DCL4_) in these processes. Sequencing of small RNAs from a _dcl2 dcl3 dcl4_ triple mutant showed markedly reduced tasiRNA

and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous

inverted repeats. _DCL2_, _DCL3_ and _DCL4_ showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also

suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs. Access through your institution Buy or subscribe This is a preview of subscription content,

access via your institution ACCESS OPTIONS Access through your institution Subscribe to this journal Receive 12 print issues and online access $209.00 per year only $17.42 per issue Learn

more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS

OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS AN SIRNA-GUIDED ARGONAUTE PROTEIN DIRECTS RNA

POLYMERASE V TO INITIATE DNA METHYLATION Article Open access 08 November 2021 ANALYSIS OF MRNA-DERIVED SIRNAS IN MUTANTS OF MRNA MATURATION AND SURVEILLANCE PATHWAYS IN _ARABIDOPSIS

THALIANA_ Article Open access 27 January 2022 SUMO PROTEASE FUG1, HISTONE READER AL3 AND CHROMODOMAIN PROTEIN LHP1 ARE INTEGRAL TO REPEAT EXPANSION-INDUCED GENE SILENCING IN _ARABIDOPSIS

THALIANA_ Article 19 April 2024 ACCESSION CODES ACCESSIONS GENBANK/EMBL/DDBJ * NP_171612 * NP_189978 * NP_197532 * NP_498761 * NP_566199 * NP_683750 * NP_803187 * NP_912466 * NP_922059 *

XP_463595 * XP_961898 * XP_963538 REFERENCES * Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. _Nature_ 431, 343–349 (2004). Article  CAS  Google Scholar  *

Chan, S.W., Henderson, I.R. & Jacobsen, S.E. Gardening the genome: DNA methylation in _Arabidopsis thaliana_. _Nat. Rev. Genet._ 6, 351–360 (2005). Article  CAS  Google Scholar  * Xie,

Z. et al. Genetic and functional diversification of small RNA pathways in plants. _PLoS Biol._ 2, E104 (2004). Article  Google Scholar  * Xie, Z., Allen, E., Wilken, A. & Carrington,

J.C. DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in _Arabidopsis thaliana_. _Proc. Natl. Acad. Sci. USA_ 102, 12984–12989 (2005).

Article  CAS  Google Scholar  * Yoshikawa, M., Peragine, A., Park, M.Y. & Poethig, R.S. A pathway for the biogenesis of trans-acting siRNAs in _Arabidopsis_. _Genes Dev._ 19, 2164–2175

(2005). Article  CAS  Google Scholar  * Gasciolli, V., Mallory, A.C., Bartel, D.P. & Vaucheret, H. Partially redundant functions of _Arabidopsis_ DICER-like enzymes and a role for DCL4

in producing trans-acting siRNAs. _Curr. Biol._ 15, 1494–1500 (2005). Article  CAS  Google Scholar  * Dunoyer, P., Himber, C. & Voinnet, O. DICER-LIKE 4 is required for RNA interference

and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. _Nat. Genet._ 37, 1356–1360 (2005). Article  CAS  Google Scholar  * Schauer, S.E.,

Jacobsen, S.E., Meinke, D.W. & Ray, A. DICER-LIKE1: blind men and elephants in _Arabidopsis_ development. _Trends Plant Sci._ 7, 487–491 (2002). Article  CAS  Google Scholar  * Hamilton,

A., Voinnet, O., Chappell, L. & Baulcombe, D. Two classes of short interfering RNA in RNA silencing. _EMBO J._ 17, 4671–4679 (2002). Article  Google Scholar  * Qi, Y., Denli, A.M. &

Hannon, G.J. Biochemical specialization within _Arabidopsis_ RNA silencing pathways. _Mol. Cell_ 19, 421–428 (2005). Article  CAS  Google Scholar  * Liu, B. et al. Loss of function of

OsDCL1 affects microRNA accumulation and causes developmental defects in rice. _Plant Physiol._ 139, 296–305 (2005). Article  CAS  Google Scholar  * Margulies, M. et al. Genome sequencing in

microfabricated high-density picolitre reactors. _Nature_ 437, 376–380 (2005). Article  CAS  Google Scholar  * Allen, E., Xie, Z., Gustafson, A.M. & Carrington, J.C. microRNA-directed

phasing during trans-acting siRNA biogenesis in plants. _Cell_ 121, 207–221 (2005). Article  CAS  Google Scholar  * Allen, E. et al. Evolution of microRNA genes by inverted duplication of

target gene sequences in _Arabidopsis thaliana_. _Nat. Genet._ 36, 1282–1290 (2004). Article  CAS  Google Scholar  * Cam, H.P. et al. Comprehensive analysis of heterochromatin- and

RNAi-mediated epigenetic control of the fission yeast genome. _Nat. Genet._ 37, 809–819 (2005). Article  CAS  Google Scholar  * Li, J., Yang, Z., Yu, B., Liu, J. & Chen, X. Methylation

protects miRNAs and siRNAs from a 3′-end uridylation activity in _Arabidopsis_. _Curr. Biol._ 15, 1501–1507 (2005). Article  CAS  Google Scholar  * Yu, B. et al. Methylation as a crucial

step in plant microRNA biogenesis. _Science_ 307, 932–935 (2005). Article  CAS  Google Scholar  * Shen, B. & Goodman, H.M. Uridine addition after microRNA-directed cleavage. _Science_

306, 997 (2004). Article  CAS  Google Scholar  * Cao, X. & Jacobsen, S.E. Role of the _Arabidopsis_ DRM methyltransferases in _de novo_ DNA methylation and gene silencing. _Curr. Biol._

12, 1138–1144 (2002). Article  CAS  Google Scholar  * Chan, S.W. et al. RNA silencing genes control _de novo_ DNA methylation. _Science_ 303, 1336 (2004). Article  CAS  Google Scholar  *

Soppe, W.J. et al. The late flowering phenotype of _fwa_ mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene. _Mol. Cell_ 6, 791–802 (2000). Article  CAS  Google

Scholar  * Jacobsen, S.E., Sakai, H., Finnegan, E.J., Cao, X. & Meyerowitz, E.M. Ectopic hypermethylation of flower-specific genes in _Arabidopsis_. _Curr. Biol._ 10, 179–186 (2000).

Article  CAS  Google Scholar  * Jackson, J.P., Lindroth, A.M., Cao, X. & Jacobsen, S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. _Nature_ 416,

556–560 (2002). Article  CAS  Google Scholar  * Malagnac, F., Bartee, L. & Bender, J. An _Arabidopsis_ SET domain protein required for maintenance but not establishment of DNA

methylation. _EMBO J._ 21, 6842–6852 (2002). Article  CAS  Google Scholar  * Lu, C. et al. Elucidation of the small RNA component of the transcriptome. _Science_ 309, 1567–1569 (2005).

Article  CAS  Google Scholar  * Yoder, J.A., Soman, N.S., Verdine, G.L. & Bestor, T.H. DNA (cytosine-5)-methyltransferases in mouse cells and tissues. Studies with a mechanism-based

probe. _J. Mol. Biol._ 270, 385–395 (1997). Article  CAS  Google Scholar  * Pradhan, S., Bacolla, A., Wells, R.D. & Roberts, R.J. Recombinant human DNA (cytosine-5) methyltransferase. I.

Expression, purification, and comparison of _de novo_ and maintenance methylation. _J. Biol. Chem._ 274, 33002–33010 (1999). Article  CAS  Google Scholar  * Zilberman, D. et al. Role of

_Arabidopsis_ ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. _Curr. Biol._ 14, 1214–1220 (2004). Article  CAS  Google Scholar  * Valoczi, A. et al. Sensitive and

specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. _Nucleic Acids Res._ 32, e175 (2004). Article  Google Scholar  Download references

ACKNOWLEDGEMENTS We thank S. Chan for the _dcl2 dcl3_ double mutant, J. Carrington for drawing our attention to the _IR-71_ locus and members of the Jacobsen laboratory for critical reading

of the manuscript. I.R.H. was supported by a European Molecular Biology Organization (EMBO) Long Term Postdoctoral Fellowship. S.E.J. is an investigator of the Howard Hughes Medical

Institute. This work was supported by US National Science Foundation grants 0439186 and 0548569 to P.J.G. and B.C.M., and NIH grant GM60398 and funds from the Howard Hughes Medical Institute

to S.E.J. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, 90095, California, USA Ian R

Henderson, Xiaoyu Zhang, Lianna Johnson & Steven E Jacobsen * Delaware Biotechnology Institute and Department of Plant and Soil Sciences, University of Delaware, Newark, 19714, Delaware,

USA Cheng Lu, Blake C Meyers & Pamela J Green * Howard Hughes Medical Institute, University of California, Los Angeles, 90095, California, USA Steven E Jacobsen Authors * Ian R

Henderson View author publications You can also search for this author inPubMed Google Scholar * Xiaoyu Zhang View author publications You can also search for this author inPubMed Google

Scholar * Cheng Lu View author publications You can also search for this author inPubMed Google Scholar * Lianna Johnson View author publications You can also search for this author inPubMed

 Google Scholar * Blake C Meyers View author publications You can also search for this author inPubMed Google Scholar * Pamela J Green View author publications You can also search for this

author inPubMed Google Scholar * Steven E Jacobsen View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Steven E

Jacobsen. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIG. 1 Repeat structure and siRNA accumulation

at the large inverted repeat _IR-71_. (PDF 272 kb) SUPPLEMENTARY FIG. 2 Distribution of methylation at repeat loci. (PDF 200 kb) SUPPLEMENTARY TABLE 1 Composition of small RNAs sequenced

from wild-type Columbia (Col) and the _dcl2-1 dcl3-1 dcl4-2_ triple mutant. (PDF 55 kb) SUPPLEMENTARY TABLE 2 Sodium bisulfite sequencing analysis of repeat loci. (PDF 44 kb) SUPPLEMENTARY

TABLE 3 Adaptor, primer and probe sequences. (PDF 43 kb) SUPPLEMENTARY TABLE 4 Small RNA sequences cloned from Columbia that show a perfect match to the _Arabidopsis_ genome. (PDF 417 kb)

SUPPLEMENTARY TABLE 5 Small RNA sequences cloned from _dcl2 dcl3 dcl4_ that show a perfect match to the _Arabidopsis_ genome. (PDF 285 kb) RIGHTS AND PERMISSIONS Reprints and permissions

ABOUT THIS ARTICLE CITE THIS ARTICLE Henderson, I., Zhang, X., Lu, C. _et al._ Dissecting _Arabidopsis thaliana_ DICER function in small RNA processing, gene silencing and DNA methylation

patterning. _Nat Genet_ 38, 721–725 (2006). https://doi.org/10.1038/ng1804 Download citation * Received: 09 March 2006 * Accepted: 19 April 2006 * Published: 14 May 2006 * Issue Date: 01

June 2006 * DOI: https://doi.org/10.1038/ng1804 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is

not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative