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Nat Plants. 2018 Mar;4(3):181-188. doi: 10.1038/s41477-017-0100-y. Epub 2018 Jan 29.

RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.

Liu W1,2, Duttke SH3,4,5,6, Hetzel J3,4,5, Groth M2, Feng S2,7, Gallego-Bartolome J2, Zhong Z2,8, Kuo HY2, Wang Z8, Zhai J2,9, Chory J3,4,5, Jacobsen SE10,11,12,13.

Author information

1
Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA, USA.
2
Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA.
3
Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA.
4
Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA.
5
Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, CA, USA.
6
Department of Cellular & Molecular Medicine, School of Medicine, University of California at San Diego, La Jolla, CA, USA.
7
Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California at Los Angeles, Los Angeles, CA, USA.
8
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, China.
9
Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, China.
10
Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA, USA. jacobsen@ucla.edu.
11
Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA. jacobsen@ucla.edu.
12
Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California at Los Angeles, Los Angeles, CA, USA. jacobsen@ucla.edu.
13
Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, CA, USA. jacobsen@ucla.edu.

Abstract

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4-siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5' adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

PMID:
29379150
PMCID:
PMC5832601
DOI:
10.1038/s41477-017-0100-y
[Indexed for MEDLINE]
Free PMC Article

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