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observed with siRNAs. The other PPD family members are specifi-
cally expressed in germline and stem cells, 11,12 which prompted
researchers to investigate whether these proteins too are associated
with small regulatory RNAs. Cloning and sequencing of libraries
constructed from immunoprecipitates of PIWI proteins indeed led to
the discovery of a second class of small regulatory RNAs, the Piwi-
interacting RNAs (piRNAs), 13-15 which so far have been implicated in
transposon silencing. 16-18
In the following, we give an overview of the biogenesis and function
of small RNAs, and the computational tools that have been developed to
support the research on these topics. The focus will be mostly on
miRNAs, which up to this point have been better characterized than
other small regulatory RNAs.
2. Identification of Small Regulatory RNAs
The first miRNA was discovered using the so-called “forward genetics”
approach, which aims to identify mutations that produce specific pheno-
types. Frequently, mutagens are used to generate a panel of mutants that
are screened for the phenotype of interest; the genes responsible for the
phenotype are then identified with well-established molecular biology
techniques. An immediate advantage of this approach is that once the
gene is identified, information about its function is already available in
the form of the phenotype of the mutant. On the other hand, only genes
responsible for dramatic, easily observable phenotypes can be identified
this way.
The vast majority of miRNAs known today are studied using “reverse
genetics” approaches, in which one starts from the gene sequence rather
than from the phenotype in order to characterize gene function. The
classical approach to miRNA (and generally to regulatory RNA) identifi-
cation has been cloning and sequencing. The RNA population within a
selected size range (obtained by fractionation) or binding a specific pro-
tein (obtained from immunoprecipitates of that protein) is ligated to
short RNA adapters, amplified by reverse transcription-polymerase chain
reaction (RT-PCR), concatamerized, and sequenced. Such an approach
was used in the first studies aiming to identify fly, worm, and human
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