Pseudogenes: Functions and Protocols

308 Pages·2014·7.115 MB·English

by  Laura Poliseno (eds.)

Methods in
Molecular Biology 1167
Laura Poliseno Editor
Pseudogenes
Functions and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
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Pseudogenes
Functions and Protocols
Edited by
Laura Poliseno
Istituto Toscano Tumori, Pisa, Italy
Editor
Laura Poliseno
Istituto Toscano Tumori, Pisa, Italy
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-0834-9 ISBN 978-1-4939-0835-6 (eBook)
DOI 10.1007/978-1-4939-0835-6
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Prefa ce
Discovered a few decades ago, pseudogenes have been dismissed as “junk DNA” on the
basis of the wrong assumption that absence of full coding potential means complete lack of
functionality. However, in the past few years, pseudogenic DNA, RNA, and peptides/pro-
teins have been shown to be involved in complex regulatory circuits that can affect not only
their highly homologous parental genes but also unrelated genes. Therefore, it has become
evident that these previously neglected molecules can contribute to the understanding of
the physiological functions of the noncoding genome as well as to its alterations in disease,
and for these reasons they have risen to the status of a new class of regulatory long noncod-
ing RNAs.
The main purpose of this volume is to increase the awareness of molecular, cellular, and
computational biologists on the subject of pseudogenes, as well as to provide a list of meth-
ods that can be useful both to those who indeed want to study pseudogenes and to those
who actually want to avoid their inadvertent detection. The introductory chapters give an
overview of the functions that have been so far attributed to pseudogenes. After a section
collecting methods for pseudogene identifi cation, the volume describes methods for the
detection of pseudogene transcription and translation, both in the case that a specifi c pseu-
dogene is under study and in the case that a high-throughput approach is undertaken. Two
sections collecting methods to study the functions of pseudogenic RNA and proteins are
then presented. The last section is composed of methods to avoid pseudogene detection
when the targets of the study are their highly homologous parental counterparts.
It is our hope that this volume will contribute to maintaining the high interest of the scien-
tifi c community toward pseudogenes, while stimulating the conception of pseudogene-c entered
research projects and providing experimental protocols that can facilitate their execution.
Pisa, Italy Laura P oliseno
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I INTRODUCTORY CHAPTERS
1 Pseudogene Redux with New Biological Significance. . . . . . . . . . . . . . . . . . . . 3
Leonardo Salmena
2 C ontribution of Pseudogenes to Sequence Diversity . . . . . . . . . . . . . . . . . . . . 1 5
Mauno Vihinen
PART II METHODS TO IDENTIFY PSEUDOGENES
3 Computational Methods for Pseudogene Annotation Based
on Sequence Homology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 7
Paul M . Harrison
4 C omputational Methods of Identification of Pseudogenes Based
on Functionality: Entropy and GC Content . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Evgeniy S. Balakirev, V ladimir R . C hechetkin, Vasily V. Lobzin,
and Francisco J. A yala
5 M ethods of Identification of Pseudogenes
Based on Functionality: Hybridization of 18S rRNA
and mRNA During Translation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Chuanhua Xing
6 Whole-Genome Identification of Neutrally Evolving Pseudogenes Using
the Evolutionary Measure dN/dS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5
Lise O livia A ndrieux and D avid T orrents A renales
7 M ethods to Study the Occurrence and the Evolution
of Pseudogenes Through a Phylogenetic Approach. . . . . . . . . . . . . . . . . . . . . 87
Jacques Dainat and P ierre P ontarotti
PART III METHODS TO DETECT TRANSCRIBED PSEUDOGENES
OF A PARENTAL GENE OF INTEREST
8 Methods for Detecting Transcribed Pseudogenes: PCR on Regions
of High Sequence Similarity Followed by Cloning and Sequencing . . . . . . . . . 1 03
Wenyong D ing and Jianwu D ai
PART IV HIGH-THROUGHPUT METHODS TO DETECT
TRANSCRIBED PSEUDOGENES
9 RNA Amplification for Pseudogene Detection Using RNA-Seq. . . . . . . . . . . . 1 19
Stephen C. M. Tsoi and M ichael K . D yck
vii
viii Contents
10 GENCODE Pseudogenes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Adam Frankish and Jennifer H arrow
11 M ethods to Detect Transcribed Pseudogenes: RNA-Seq Discovery
Allows Learning Through Features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Camilo V aldes and Enrico C apobianco
PART V HIGH-THROUGHPUT METHODS TO DETECT TRANSLATED PSEUDOGENES
12 Proteomics Techniques for the Detection of Translated Pseudogenes. . . . . . . . 1 87
Nadia Ucciferri and Silvia R occhiccioli
PART VI METHODS TO STUDY TRANSCRIBED PSEUDOGENES
13 Pseudogenes as Competitive Endogenous RNAs:
Target Prediction and Validation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Florian A . Karreth, U go Ala, P aolo P rovero, and P ier Paolo P andolfi
14 P seudogenes: A Novel Source of trans-Acting Antisense RNAs. . . . . . . . . . . . 213
Per Johnsson, K evin V. Morris, and D an Grandér
15 Pseudogene-Derived Endogenous siRNAs and Their Function . . . . . . . . . . . . 2 27
Wen-Ling C han and Jan-Gowth C hang
PART VII METHODS TO STUDY TRANSLATED PSEUDOGENES
16 Methods to Study Translated Pseudogenes: In Vitro Translation,
Fusion with a Tag/Reporter Gene, and Complementation Assay. . . . . . . . . . . 243
Anne Parle-McDermott

PART VIII METHODS TO AVOID PSEUDOGENE DETECTION
17 Targeted and Robust Amplification of Mitochondrial DNA
in the Presence of Nuclear-Encoded Mitochondrial Pseudogenes
Using Φ29 DNA Polymerases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Jonci N . W olff
18 Discrimination of Pseudogene and Parental Gene DNA Methylation
Using Allelic Bisulfite Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Luke B . H esson and Robyn L . W ard
19 M utational Analysis of CYP21A2 Gene and CYP21A1P Pseudogene:
Long-range PCR on Genomic DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Hsien-Hsiung L ee
20 P MS2 Gene Mutational Analysis: Direct cDNA Sequencing
to Circumvent Pseudogene Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Katharina W immer and A nnekatrin W ernstedt
21 Dealing with Pseudogenes in Molecular Diagnostics
in the Next-Generation Sequencing Era . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 03
Kathleen B.M. Claes and Kim D e Leeneer
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 17
Contributors
UGO ALA • Molecular Biotechnology Center and Department of Biotechnology and Life
Sciences, U niversity of Turin , Turin, Italy
LISE OLIVIA ANDRIEUX • Joint IRB-BSC Program on Computational Biology,
Barcelona Supercomputing Center , B arcelona, S pain
DAVID TORRENTS A RENALES • Joint IRB-BSC Program on Computational Biology,
Barcelona Supercomputing Center , B arcelona, S pain ; I nstitució Catalana de Recerca i
Estudis Avançats (ICREA) , Barcelona, S pain
FRANCISCO J. A YALA • Department of Ecology and Evolutionary Biology, U niversity of
California , Irvine, CA , U SA
EVGENIY S. B ALAKIREV • Department of Ecology and Evolutionary Biology, U niversity of
California , I rvine, CA , USA ; A.V. Zhirmunsky Institute of Marine Biology, Far Eastern
Branch, Russian Academy of Science , Vladivostok, Russia
ENRICO CAPOBIANCO • Center for Computational Science, University of Miami , Miami, FL,
USA ; L aboratory of Integrative Systems Medicine (LISM), CNR, Institute of Clinical
Physiology , P isa, Italy
WEN-LING C HAN • Biomedical Informatics , Asia University , Taichung, Taiwan ; Epigenome
Research Center, China Medical University Hospital , T aichung, T aiwan ; S chool of
Medicine, China Medical University , Taichung, T aiwan
JAN-GOWTH CHANG • Epigenome Research Center, China Medical University Hospital ,
Taichung, T aiwan ; School of Medicine, China Medical University , T aichung, T aiwan ;
Department of Laboratory Medicine, China Medical University Hospital , Taichung,
Taiwan ; C ancer Center, Kaohsiung Medical University Hospital , K aohsiung, T aiwan
VLADIMIR R. CHECHETKIN • Theoretical Department of Division for Perspective
Investigations, T roitsk Institute of Innovation and Thermonuclear Investigations
(TRINITI), Troitsk , M oscow , R ussia ; Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences , M oscow , Russia
KATHLEEN B .M. CLAES • Center for Medical Genetics, Ghent University Hospital , G hent ,
Belgium
JIANWU D AI • State Key Laboratory of Molecular Developmental Biology,
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences ,
Beijing, C hina; Stem cell and Regenerative Medicine Laboratory, Institute of Genetics
and Developmental Biology, Chinese Academy of Sciences, Beijing, China
JACQUES DAINAT • Evolutionary Biology and Modeling Group , A ix-Marseille Université ,
Marseille, F rance
WENYONG DING • Department of Biochemistry , D alian Medical University , D alian, China
MICHAEL K. DYCK • Department of Agricultural, Food and Nutritional Science,
3-10C Agriculture/Forestry Centre, University of Alberta , Edmonton, A B, Canada
ADAM FRANKISH • Human and Vertebrate Analysis and Annotation Group , W ellcome Trust
Sanger Institute, Wellcome Trust Genome Campus , H inxton, U K
DAN G RANDÉR • Department of Oncology and Pathology , C ancer Center Karolinska R8:03,
Karolinska Institutet , S tockholm, Sweden
PAUL M . HARRISON • Department of Biology, McGill University , Montreal , Q C , C anada
ix