CHAPTER 16
SMALL RNA DISCOVERY AND
CHARACTERISATION IN EUKARYOTES USING
HIGH-THROUGHPUT APPROACHES
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School of Computing Sciences, University of East Anglia, Norwich, UK
*Corresponding Author: Vincent Moulton——Email: [email protected]
Abstract:
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noncoding RNAs, or small RNAs (sRNAs). Regulatory interactions are established
based on nucleotide sequence complementarity between the sRNAs and their
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sRNAs can produce new sRNAs or the protein encoded by the target mRNA can
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and their targets.
INTRODUCTION
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systems eukaryotic cells have in place to accomplish this task is RNA silencing, a process
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RNA Infrastructure and Networks, edited by Lesley J. Collins .
©2011 Landes Bioscience and Springer Science+Business Media.
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RNA INFRASTRUCTURE AND NETWORKS
or transcription. The RNA molecules present usually contain less than 30 nucleotides
and are commonly called small RNAs (sRNAs). In this chapter we will adopt this
convention, although we note that there are other noncoding small RNAs that are not
involved in RNA silencing (e.g., tRNAs, snoRNAs) but they will not be described here.
In prokaryotic organisms there are also noncoding RNAs that regulate gene expression,
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comparison to conventional Sanger sequencing, these new technologies are characterised
by producing shorter reads with very high-throughput. Researchers in the RNA silencing
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sequencing and other high-throughput methods, such as microarrays, have also been
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describe the small RNAs that are involved in this process and how high-throughput
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THE MECHANISMS OF RNA SILENCING
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RNAaseIII-type enzymes called Dicers are able to recognise these precursors and process
them to produce double stranded sRNAs. These are then incorporated into an Argonaute
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mRNA destabilisation without cleavage, inhibit protein translation or cause DNA and
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determines the class a sRNA belongs to (Fig. 1).
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Figure 1. 6PDOO 51$ SURGXFWLRQ SDWKZD\V 5HSULQWHG ZLWK SHUPLVVLRQ IURP 3KLOOLSV -5 'DOPD\ 7 %DUWHOV ' FEBS Letters 2007; 581(19):3592-3597.
SMALL RNA DISCOVERY AND CHARACTERISATION IN EUKARYOTES
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RNA INFRASTRUCTURE AND NETWORKS
individual small RNA conserved between animals and plants has been discovered.14,15
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diversity in sRNA producing mechanisms than in animals.7
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extensively describe miRNA biogenesis in animals,18-21 plants17,22 and both.15,23
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Dicer enzyme.10,14,24,25VL51$VFDQWDUJHWERWKWKHORFLZKHUHWKH\DUHSURGXFHGIURPDQG
other loci with high sequence homology. siRNAs can act both by DNA transcriptional
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to be involved in transposon silencing.26
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complementarity between the sRNA and its target. The rules governing the interaction
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SMALL RNA DISCOVERY AND CHARACTERISATION IN EUKARYOTES
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with protein coding genes, control can be exerted at many levels and with distinct
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promoter region.39,QSDUWLFXODUWKHSURPRWHUUHJLRQVRIPL51$VKDYHEHHQH[WHQVLYHO\
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USING MICROARRAYS AND DNA SEQUENCING TO MEASURE RNA
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other means.42,43+RZHYHUWKHVHPHWKRGVDUHYHU\WLPHFRQVXPLQJDQGWKHUHIRUHFDQ
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approaches that can be used to measure RNA, have been developed: microarrays and
RNA sequencing (RNAseq).
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SUREHVDWWDFKHGWRDVROLGVXUIDFHDQGWKHVDPSOHRILQWHUHVW44 The probes are designed
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244
RNA INFRASTRUCTURE AND NETWORKS
probe target. This approach has two limitations: it is possible to measure only molecules
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case hybridization to the probe might still occur.
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to be developed is called massively parallel signature sequencing (MPSS)45 but is now
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)/;V\VWHP46 Illumina’’s Genome Analyzer47IRUPHUO\NQRZQDV6ROH[DVHTXHQFLQJ
and succeeded by Illumina’’s more recent model the HiSeq 2000) and ABI’’s SOLiD.48
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sequences. Using the Genome Analyzer technology each nucleotide is sequenced
individually, eliminating the problem that 454 technology has with homopolymeric
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the 3vHQGRIORQJHUVHTXHQFHVPHDQLQJWKDWVHTXHQFHOHQJWKLVDOLPLWLQJIDFWRURIWKLV
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with sequencing it is possible to measure both known and unknown products and in
principle the measurements are independent. Furthermore, in high-throughput sequencing
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highlighted by two recent studies that indicate that there might be biases on the number
RIWLPHVDVHTXHQFHLVUHDG49,50 That is, the expression levels, as measured by the count
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sRNAs is limited to ligation compatible sRNAs, in general with a 5v mono-phosphate
group and a 3v hydroxyl group. This means that there are probably unknown sRNAs that
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HIGH-THROUGHPUT APPROACHES FOR THE DISCOVERY
AND FUNCTIONAL CLASSIFICATION OF sRNAs
sRNA Discovery
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computationally predict miRNA genes.30,51-550RVWRIWKHVHPHWKRGVWU\WR¿QGJHQRPLF
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SMALL RNA DISCOVERY AND CHARACTERISATION IN EUKARYOTES
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under a certain model. miRNA candidates display predicted secondary structures with
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predictions, many algorithms30,53,54,58 employ a conservation rule, i.e., a candidate miRNA
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This process was adopted in ArabidopsisULFHDQGSRSODUDQGFRPSDULVRQRIPL51$
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least not widely conserved in related organisms. For example, this has been used to
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novel miRNAs in Arabidopsis.71 Subsequently, 454 pyrosequencing,59,61,63,72,73, Illumina’’s
Genome Analyzer74-76 and ABI’’s SOLiD77,78SODWIRUPKDYHEHHQXVHGWRGLVFRYHUV51$V
Although high-throughput techniques have revolutionised sRNA sequencing they
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PLOOLRQVRIUHDGVWRDHXNDU\RWLFJHQRPHLVDFRPSXWDWLRQDOO\LQWHQVLYHWDVNDQGDQXPEHU
RIVRIWZDUHSDFNDJHVKDYHEHHQGHYHORSHGWRSHUIRUPWKLVWDVNRSWLPDOO\79 These new
methods run in less time using less memory than other alignment tools, such as BLAST
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Bowtie and BWA80,81LQGH[WKHUHIHUHQFHJHQRPHXVLQJD%XUURZV:KHHOHU7UDQVIRUP
7KH3DW0D1SURJUDPLQGH[HVDOOUHDGVLQWRDVXI¿[WUHHDQGXVHVDPRGL¿HGYHUVLRQRIWKH
$KR&RUDVLFNDOJRULWKPWRDOLJQWKHVXI¿[WUHHWRWKHJHQRPH82 Additionally, in contrast
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246
RNA INFRASTRUCTURE AND NETWORKS
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RIWKHVDPHVL]HDVDW\SLFDOPL51$SUHFXUVRUDQGFRPSDWLEOHZLWKWKHSURGXFWLRQRID
mature miRNA in the same position where the read was mapped to. These regions are
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package, miRanalyser, was released.857KLVWRROSURGXFHVH[SUHVVLRQSUR¿OHVRINQRZQ
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high-throughput sequencing data.766HT%XVWHUDUHFHQWO\SXEOLVKHGVRIWZDUHSDFNDJH87
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sRNA sequencing to 14,197 in the latest release (15.0). With next generation sequencing
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PRUH PL51$V ZLOO EH FKDUDFWHULVHG RYHU WKH FRPLQJ \HDUV 7KH UHDO FKDOOHQJH IRU
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will be covered later in this chapter.
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a single-stranded RNA transcript which is targeted in two positions by a miRNA which
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ta-siRNAs.91 This imprecise Dicer processing can give rise to highly complex sRNA
ORFLZKHUHODUJHQXPEHUVRIV51$VHTXHQFHVDUHUHSUHVHQWHGE\ORZDEXQGDQFHUHDGV
even in high-throughput data sets. In order to try to overcome problems when comparing
SMALL RNA DISCOVERY AND CHARACTERISATION IN EUKARYOTES
247
Table 1.3HUIRUPDQFHRIVHTXHQFLQJWHFKQRORJLHV1XPEHUVREWDLQHGIURPPDQXIDFWXUHU¶V
ZHEVLWHVRQ0D\7KHQXPEHUVIRUWKHKLJKWKURXJKSXWSODWIRUPVZLOOFKDQJHDV
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Technology
Read Length
Nucleotides
Read Per Day
Sanger sequencing
(ABI3730)
Up to 900nt
8SWRA
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com
)/;7LWDQLXP
series)
Up to 500nt
8SWRA
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Genome Analyzer (IIe)
Up to 100nt
8SWRA
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SOLiD (version 4)
Up to 50nt
8SWRA
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com
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Table 2.%LRLQIRUPDWLFVRIWZDUHUHVRXUFHVWRDQDO\VHKLJKWKURXJKSXWVHTXHQFLQJGDWD
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Name
Organism
Functionalities
5HIHUHQFH
miRDeep
Animals
Find miRNAs
83
miRCat
Plants and animals
Find miRNAs
84
miRanalyzer
Animals
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85
miRExpress
Animals and plants
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Animals and plants
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121
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tool
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84
SeqBuster
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87
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NiBLS
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loci
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sRNAs into transcriptional units or loci using either genome or transcript annotations.
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sRNAs and such that the gap between two consecutive sRNAs is below a maximum gap.84
More recently the NiBLS algorithm, based on graph properties, has been developed.92
This method builds a graph where its nodes are individual sRNAs and edges are created
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coordinates among the sequences in each subset.
248
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a very time-consuming and expensive process. In plants, sRNA mediated target site
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to distinguish between cleavage products and other mRNA degradation products.
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cleavage products. Additionally, the 3v cleavage product contains a 5v mono-phosphate
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cleavage position.93 Traditionally this has been done using a process called 5v Rapid
$PSOL¿FDWLRQRIF'1$(QGVv RACE). In essence this process allows the sequencing
RIFOHDYDJHSURGXFWVIURPWKHWUDQVFULSWSUHGLFWHGWREHWDUJHWHGE\DJLYHQV51$7KH
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was predicted to occur.
Recently a new high-throughput approach has been described which allows researchers
to carry out a high-throughput target validation analysis.94,95 This degradome sequencing
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validation.977KH¿UVWRQHUHTXLUHVWKHDELOLW\WRPDQLSXODWHWKHFRQFHQWUDWLRQRUOHYHORI
DFWLYLW\RIWKHV51$)RUUHDOWDUJHWVWKHUHVSHFWLYHSURWHLQFRQFHQWUDWLRQOHYHOVVKRXOG
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The target site should contain not only the region complementary to the sRNA but also
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is taken into account: sequence complementarity between miRNAs and target sites,
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PRUHW\SLFDORISODQWVDQGWDUJHWVLWHVZLWKORZGHJUHHRIFRPSOHPHQWDULW\PRUHXVXDO
LQDQLPDOV)RUWKHIRUPHUWKHPRVWXVHGPHWKRGLVDUXOHEDVHGDOJRULWKPSUHVHQWHGLQ
a paper by Schwab and colleagues.29 For the latter TargetScan98 and PicTar99 have been
XVHGWRSURGXFHSUHGLFWLRQVRIFRQVHUYHGPL51$WDUJHWV7ZRRWKHUPHWKRGV51$
and Pita are widely used to predict nonconserved targets.100,101 The predictions generated
E\WKHVHPHWKRGVLQFOXGHPDQ\IDOVHSRVLWLYHVDQGIDOVHQHJDWLYHVZLWKWKHVHUDWHVPXFK
higher in the low complementarity predictions.
Alternative hybrid target prediction methods using microarrays or high-throughput
VHTXHQFLQJKDYHDOVREHHQGHYHORSHG2QHVHWRIDSSURDFKHVUHOLHVRQPHDVXULQJWKH
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SMALL RNA DISCOVERY AND CHARACTERISATION IN EUKARYOTES
249
contain potential target sites. mRNA concentrations can be estimated using microarrays
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FRQFHQWUDWLRQVLVDOVRSRVVLEOHDOWKRXJKZLWKWKHFXUUHQWWHFKQRORJ\RQO\DIUDFWLRQRI
proteins can be measured.102,103
,QWKH¿UVWH[SHULPHQWWKDWXVHGP51$PHDVXUHPHQWVREWDLQHGDIWHUPL51$DFWLYLW\
PDQLSXODWLRQWKHDEXQGDQFHVRIPL5DQGPL5ZHUHLQFUHDVHGE\WUDQVIHFWLQJFHOOV
ZLWKVL51$VZLWKLGHQWLFDOVHTXHQFHVRIWKRVHWZRPL51$V104(YLGHQFHIRUZLGHVSUHDG
WDUJHWLQJDWWKHP51$OHYHOZDVIRXQGE\DQDO\VLQJWKHVHTXHQFHVRIGRZQUHJXODWHG
JHQHV ,Q SDUWLFXODU WKH VHTXHQFH FRPSOHPHQWDU\ WR WKH ¿UVW HLJKW QXFOHRWLGHV RI WKH
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UHSUHVHQWHGDPRQJWKHVHJHQHV,QRWKHUH[SHULPHQWVWKHDFWLYLW\RILQGLYLGXDOPL51$V
was decreased, either by suppressing activity or deleting the miRNA gene.105-108 In both
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RIWKHWDUJHWHGP51$VDQGDFRQVHTXHQWLQFUHDVHLQP51$FRQFHQWUDWLRQV)LQDOO\LQD
VXEVHTXHQWH[SHULPHQWWKHHIIHFWVRIRYHUH[SUHVVLQJDQGVXSSUHVVLQJWKHVDPHPL51$
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computational methods.
$QRWKHUDSSURDFKXVLQJKLJKWKURXJKSXWPHDVXUHPHQWRIP51$VKDVDOVREHHQ
GHYHORSHGLQUHFHQW\HDUV7KLVDSSURDFKUHOLHVRQWKHLPPXQRSUHFLSLWDWLRQRI5,6&V
ERXQGWRWDUJHWHGP51$VDQGVXEVHTXHQWSUR¿OLQJRIWKHVHP51$VXVLQJPLFURDUUD\V110-113
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P51$VWRLGHQWLI\WKHSXWDWLYHWDUJHWVLWHVDQGV51$VWKDWWDUJHWHDFKP51$0RUH
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complexes were immuno-precipitated in the same sample and both mRNAs and sRNAs
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DFFXUDWHGHWHUPLQDWLRQRIV51$P51$UHJXODWRU\LQWHUDFWLRQV114
Further sRNA Characterisation
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the targeting pathways in which each sRNAs is involved.
High-throughput technologies have also been used to study the transcriptional
UHJXODWLRQRIV51$V7KLVFDQEHGRQHE\LPPXQRSUHFLSLWDWLQJWUDQVFULSWLRQIDFWRUVDQG
WKH'1$WKH\DUHERXQGWRIROORZHGE\'1$SUR¿OLQJUHVRUWLQJHLWKHUWRPLFURDUUD\V
or high-throughput sequencing. This method has been used to determine transcription
start sites and promoter regions116 and to establish transcriptional regulatory interactions
EHWZHHQ WUDQVFULSWLRQ IDFWRUV DQG V51$V39 7KHVH W\SHV RI VWXGLHV KDYH DOORZHG WKH
FRQVWUXFWLRQRIUHJXODWRU\QHWZRUNVLQYROYLQJERWKV51$VDQGUHJXODWRU\SURWHLQV117,118
250
RNA INFRASTRUCTURE AND NETWORKS
CONCLUSION
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V51$VQDPHO\IRUWKHGLVFRYHU\DQGFODVVL¿FDWLRQRIWKHJHQHVWKDWHQFRGHV51$VWKH
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