RNA: methods and protocols - a new series.
ABSTRACT This month, Silence launches a new series on methods and protocols to study silencing pathways and analyze nucleic acids and proteins.
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ABSTRACT: BACKGROUND: Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis. RESULTS: We introduced a dual 35S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nt siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression. CONCLUSION: We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation.Silence. 06/2012; 3(1):6.
EDITORIAL Open Access
RNA: methods and protocols — a new series
Phillip D. Zamore*
This month, Silence launches a new series on methods and protocols to study silencing pathways and analyze
nucleic acids and proteins.
This month, Silence launches a new series on methods
and protocols to study silencing pathways and analyze
nucleic acids and proteins. The first article in this series,
from Xuemei Chen and coworkers , reports the devel-
opment of a novel reporter system to enable forward
genetic screens for transcriptional silencing in plants.
This article exemplifies the type of methods we plan to
highlight: robust, quantitative tools and protocols that
make it easier to study silencing phenomena in particu-
lar, but also the regulation of gene expression more
broadly. We welcome not only articles describing novel
techniques and tools, but
improvements that advance existing methods beyond
their current use.
As Silence provides open access to all its articles, the
methods and protocols published in the journal are freely
available to all. New methods, for example, to detect RNA
molecules — in eukaryotes or in prokaryotes, whether
short or long, individually or genome-wide — published in
this series will therefore reach a wide audience and enable
new and better research. It is particularly gratifying to in-
augurate the series with a report detailing the construc-
tion, validation, and use of a new tool for genetic
screening, since the known silencing phenomena in bac-
teria, plants, fungi, and animals were all discovered by
classical forward genetics. We look forward to publishing
other genetic tools and screening methods, as well as tech-
niques for mapping novel alleles. The study of gene regula-
tion desperately needs new protocols for measuring and
sequencing RNA that decrease data bias, require fewer
cells for reliable detection, accelerate the time invested in
sample preparation, and reduce assay cost. Genome
sequencing and viral discovery are increasingly afford-
able, but improvements in assays and analysis are clearly
needed. High throughput methods currently enable the
detection of nearly all the RNA or DNA sequences
bound to a specific protein, but the techniques and ana-
lytical tools clearly can stand improvement. Silence
looks forward to reporting advances in such established
methods alongside descriptions of entirely novel tools
To submit your manuscript, please use our online
submission system and indicate in your cover letter that
you would like it to be considered for the series; alterna-
tively, send a pre-submission enquiry to editorial@silence-
Received: 2 May 2012 Accepted: 7 June 2012
Published: 7 June 2012
1.Won SY, Li S, Zheng B, Zhao Y, Li D, Zhao X, Yi H, Gao L, Dinh TT, Chen X:
Development of a luciferase-based reporter of transcriptional gene
silencing that enables bidirectional mutant screening in Arabidopsis
thaliana. Silence 2012, in press.
Cite this article as: Zamore: RNA: methods and protocols — a new
series. Silence 2012 3:7.
Howard Hughes Medical Institute and Department of Biochemistry and
Molecular Pharmacology, University of Massachusetts Medical School,
364 Plantation Street, Worcester, MA 01605, USA
© 2012 Zamore; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Zamore Silence 2012, 3:7