PIWI-Interacting RNAs: Methods and Protocols

252 Pages·2014·3.596 MB·English

by  Hang Yin, Haifan Lin (auth.), Mikiko C. Siomi (eds.)

Methods in
Molecular Biology 1093
Mikiko C. Siomi Editor
PIWI-
Interacting
RNAs
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
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John M. Walker
School of Life Sciences
University of Hertfordshire

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PIWI-Interacting RNAs
Methods and Protocols
Edited by
Mikiko C. Siomi
Graduate School of Science, The University of Tokyo, Tokyo, Japan
Editor
Mikiko C. Siomi
Graduate School of Science
The University of Tokyo
Tokyo , Japan
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-693-1 ISBN 978-1-62703-694-8 (eBook)
DOI 10.1007/978-1-62703-694-8
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Pref ace
The study of small noncoding RNAs was accelerated by the discovery of RNA interference
(RNAi) in the nematode Caenorhabditis elegans in 1998, by two Nobel Prize laureates
Andrew Fire and Craig Mello. Researchers have subsequently focused on understanding
how short interfering RNAs (siRNAs) and microRNAs (miRNAs), which represent two
major RNAi-triggering small noncoding RNAs, actually induce gene silencing and what
mechanisms are responsible for producing them from double-stranded RNA precursors.
Studies revealed that RNAi is highly conserved in a wide variety of animals and plants, as
well as in unicellular organisms such as fi ssion yeast. They further demonstrated that in
addition to C. elegans , other model species such as Drosophila melanogaster , Arabidopsis
thaliana , and mice, along with cultured cell lines such as HeLa and D rosophila Schneider 2
(S2) could serve as useful tools for investigating the molecular nature of RNAi. Indeed,
these organisms have been used to address the fundamental questions of the biogenesis and
functions of RNAi-triggering small noncoding RNAs. It is clear that the ubiquity of RNAi
has a signifi cant impact on our understanding of the RNAi world.
PIWI-interacting RNAs (piRNAs) are the third and most-recently discovered group of
silencing-inducible small RNAs in animals. The delay in their discovery was mainly the
result of their expression predominantly in the gonads. Cultured HeLa and S2 cells, which
are otherwise excellent tools for studying ubiquitous RNAi, do not express piRNAs to a
detectable level. Although the limited expression of these piRNAs has meant they have
been less well studied than other small RNAs, it has conversely stimulated researchers’ curi-
osities. Comprehensive high-throughput sequencing analysis of piRNAs in embryos, testes,
and ovaries of D. melanogaster , as well as in mouse and rat testes, has raised the profi le of
piRNAs and thus further accelerated piRNA studies.
piRNAs associate with specifi c members of the Argonaute family of proteins to form
RNA-induced silencing complexes (RISCs), as with siRNAs and miRNAs, and piRNA-
containing RISCs (piRISCs) implement RNA silencing similarly to siRISCs and miRISCs,
though their mechanisms may vary. This indicates the equivalence of the various small
RNAs. However, piRNA precursors are thought to be single-stranded because piRNA pro-
cessing occurs independently of Dicer. piRNAs are several nucleotides longer than miRNAs
and siRNAs. Most piRNAs are complementary to transcripts of transposable elements and
represses the mobile genomic elements to maintain the integrity of the genome in the
germline. These piRNA-specifi c features further highlight the uniqueness of piRNAs,
prompting studies aimed at developing a comprehensive understanding of the small RNA
world. However, the process whereby piRNAs mature from their precursors remains
unknown. We still do not understand how piRNA precursors are selectively chosen or the
mechanisms responsible for silencing of target elements. Given these unanswered basic
questions, it is clear that the piRNA studies are still a long way from their fi nal goal. In
order to further progress towards this goal, this is an ideal time to gather and share our
v
vi Preface
expertise and knowledge on the experimental methods used to unveil the piRNA world.
This book “PIWI-interacting RNAs: Methods and Protocols” provides the most recent
methods and protocols for studying piRNAs in the gonads of a wide range of species, as
well as in any other organs where piRNAs may be detected. This book will help both estab-
lished researchers and newcomers to the fi eld to progress towards the ultimate goal of
understanding the mechanisms and actions of piRNAs.
Tokyo , Japan Mikiko C. Siomi
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Chromatin Immunoprecipitation Assay of Piwi in Drosophila. . . . . . . . . . . . . . 1
Hang Yin and Haifan Lin
2 Drosophila Germline Stem Cells for In Vitro Analyses
of PIWI-M ediated RNAi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Yuzo Niki, Takuya Sato, Takafumi Yamaguchi, Ayaka Saisho,
Hiroshi Uetake, and Hidenori Watanabe
3 RNAi and Overexpression of Genes in Ovarian Somatic Cells . . . . . . . . . . . . . 2 5
Kuniaki Saito
4 Making piRNAs In Vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Shinpei Kawaoka, Susumu Katsuma, and Yukihide Tomari
5 A Framework for piRNA Cluster Manipulation . . . . . . . . . . . . . . . . . . . . . . . . 47
Ivan Olovnikov, Adrien Le Thomas, and Alexei A. Aravin
6 Biochemical and Mass Spectrometric Analysis of 3′-End Methylation
of piRNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Takeo Suzuki, Kenjyo Miyauchi, Yuriko Sakaguchi, and Tsutomu Suzuki
7 HITS-CLIP (CLIP-Seq) for Mouse Piwi Proteins. . . . . . . . . . . . . . . . . . . . . . 7 3
Anastassios Vourekas and Zissimos Mourelatos
8 DNA Methylation in Mouse Testes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Satomi Kuramochi-Miyagawa, Kanako Kita-Kojima, Yusuke Shiromoto,
Daisuke Ito, Hirotaka Koshima, and Toru Nakano
9 Analysis of Small RNA-Guided Endonuclease Activity
in Endogenous Piwi Protein Complexes from Mouse Testes . . . . . . . . . . . . . . 111
Michael Reuter and Ramesh S. P illai
10 Small RNA Library Construction from Minute Biological Samples . . . . . . . . . 1 23
Jessica A. Matts, Yuliya Sytnikova, Gung-wei Chirn, Gabor L. Igloi,
and N elson C. Lau
11 Analysis of sDMA Modifications of PIWI Proteins. . . . . . . . . . . . . . . . . . . . . . 1 37
Shozo Honda, Yoriko Kirino, and Yohei Kirino
12 Analyses of piRNA-Mediated Transcriptional Transposon Silencing
in Drosophila: Nuclear Run-On Assay on Ovaries. . . . . . . . . . . . . . . . . . . . . . . 149
Sergey Shpiz and Alla Kalmykova
13 Combined RNA/DNA Fluorescence In Situ Hybridization
on Whole-Mount Drosophila Ovaries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Sergey Shpiz, Sergey Lavrov, and Alla Kalmykova
vii
viii Contents
14 Fast and Accurate Method to Purify Small Noncoding RNAs
from Drosophila Ovaries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Thomas Grentzinger and Séverine Chambeyron
15 Isolation of Zebrafish Gonads for RNA Isolation. . . . . . . . . . . . . . . . . . . . . . . 1 83
Hsin-Yi Huang and René F. Ketting
16 Small RNA Library Construction for High-Throughput Sequencing. . . . . . . . 1 95
Jon McGinn and Benjamin Czech
17 Analysis of Piwi-Loaded Small RNAs in Terahymena . . . . . . . . . . . . . . . . . . . . 2 09
Tomoko Noto, Henriette M. Kurth, and Kazufumi Mochizuki
18 Effective Gene Knockdown in the Drosophila Germline
by Artificial miRNA-Mimicking siRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 25
Hailong Wang, Haidong Huang, and Dahua Chen
19 Isolation and Bioinformatic Analyses of Small RNAs Interacting
with Germ Cell-Specific Argonaute in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
R eina Komiya and Ken-Ichi Nonomura
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Contributors
ALEXEI A. ARAVIN • Division of Biology, California Institute of Technology , Pasadena,
CA , USA
SÉVERINE CHAMBEYRON • Institut de Génétique Humaine, CNRS , Montpellier, Cedex, France
DAHUA CHEN • State Key Laboratory of Reproductive Biology, Institute of Zoology,
Chinese Academy of Sciences , Beijing, China
GUAN-WEI CHIRN • Department of Biology, Rosenstiel Basic Medical Science Research
Center , Brandeis University , Waltham , MA , USA
BENJAMIN CZECH • Cold Spring Harbor Laboratory , Cold Spring Harbor, NY , USA
THOMAS GRENTZINGER • Institut de Génétique Humaine, CNRS , Montpellier, France
SHOZO HONDA • Department of Biomedical Sciences, Cedars-Sinai Medical Center ,
Samuel Oschin Comprehensive Cancer Institute , Los Angeles, CA , USA
HAIDONG HUANG • State Key Laboratory of Reproductive Biology, Institute of Zoology,
Chinese Academy of Sciences , Beijing, China
HSIN-YI HUANG • Ontario Cancer Institute, Princess Margaret Hospital , Toronto ,
ON , Canada
GABOR L. IGLOI • Institute of Biology, University of Freiburg , Freiburg , Germany
DAISUKE ITO • Department of Pathology, Medical School, Osaka University , Osaka , Japan
ALLA KALMYKOVA • Institute of Molecular Genetics of Russian Academy of Sciences ,
Moscow , Russia
SUSUMU KATSUMA • Department of Agricultural and Environmental Biology, Graduate
School of Agricultural and Life Sciences, The University of Tokyo , Tokyo , Japan
SHINPEI KAWAOKA • Cold Spring Harbor Laboratory , Cold Spring Harbor, NY , USA
RENÉ F. KETTING • Institute of Molecular Biology (IMB), Faculty of Biology, University of
Mainz , Mainz , Germany
YOHEI KIRINO • Computational Medicine Center, Department of Biochemistry and
Molecular Biology, Thomas Jefferson University , Philadelphia, PA , USA
YORIKO KIRINO • Department of Biomedical Sciences, Cedars-Sinai Medical Center ,
Samuel Oschin Comprehensive Cancer Institute , Los Angeles, CA , USA
KANAKO KITA-KOJIMA • Department of Pathology, Medical School, Osaka University ,
Osaka , Japan
REINA KOMIYA • Experimental Farm, National Institute of Genetics (NIG) , Mishima,
Shizuoka, Japan
HIROTAKA KOSHIMA • Department of Pathology, Medical School, Osaka University ,
Osaka , Japan
SATOMI KURAMOCHI-MIYAGAWA • Department of Pathology, Medical School, Osaka University ,
Osaka, Japan
HENRIETTE M. KURTH • Viollier AG Spalenring , Basel , Switzerland
NELSON C. LAU • Department of Biology, Rosenstiel Basic Medical Science Research Center,
Brandeis University , Waltham , MA , USA
ix