Biology Reference
In-Depth Information
network. Importantly, since the same network is deployed
to achieve distinct cellular functions in a context-dependent
manner, it is essential to extend the systems biology
approaches to construct network models that incorporate
information regarding the dynamics of molecular interac-
tions within cells. Inferring the flow of information through
a network provides directionality to the edges between the
nodes of the network. This can be achieved by systemati-
cally perturbing nodes, either by gene knockouts or by
RNAi knockdown, followed by measurements of a wide
variety of quantitative phenotypes. The phenotypes
measured can include changes in gene expression, post-
translational modifications such as phosphorylation and
acetylation of key proteins, and/or in cellular morphology
or behavior. Quantitative phenotypic signatures provide
insight into the information processing function of
signaling networks, which is key to achieving a mecha-
nistic understanding of how various cellular processes are
regulated in time and space.
explosion of genome-wide cell-based RNAi data for
diverse biological processes, including signal transduction,
host
pathogen interactions and oncogenesis
[47]
.
When dsRNAs are introduced into cells, they are
recognized and degraded by the conserved RNAse III
family of nucleases known as Dicer
[52,56
e
60]
. Dicer
e
enzymes process the dsRNA into 21
23 nucleotide (nt)
short-interfering RNAs (siRNAs) that are incorporated into
a multi-protein RNA-induced silencing complex (RISC).
This complex directs the unwinding of the siRNAs con-
tained within RISC, and guides RISC to the corresponding
mRNA to eventually degrade the targeted transcript.
Different types of RNAi reagent have been developed to
knockdown target genes in different types of cells and
organisms. The four most commonly used RNAi reagents
include long dsRNAs (~500 nt), siRNAs (21
e
23 nt), short-
hairpin RNAs (shRNAs; 70 nt) that can be produced
exogenously or carried on an expression vector, and
endoribonuclease-prepared siRNA (esiRNAs)
e
63]
.
Typically, RNAi reagents are delivered into cells by virus-
mediated transduction for shRNAs, or by lipid-mediated
transfection or electroporation for shRNAs, siRNAs,
esiRNAs, and dsRNAs
[44,46,64,65]
. In the case of many
Drosophila tissue culture cells, dsRNAs are directly taken
up from the surrounding medium without the need for
transfection
[55,56]
.
The success of RNAi screening depends on the robust-
ness of the cell-based assay, especially its suitability to high-
throughput screening (HTS). Almost all HTS cell-based
assays provide a quantitative readout for the biological
process under study. Many assays use transcriptional
reporters where a well-characterized transcriptional regula-
tory element that is known to respond to the signaling
pathway under study is linked to a reporter such as lucif-
erase, green fluorescence protein (GFP) or the E. coli
b
-galactosidase (LacZ). The overall output of the reporter
can be rapidly measured using a standard plate reader
[66
[61
e
SYSTEMS APPROACHES TO IDENTIFY
THE 'PARTS' OF CELLULAR SIGNALING
NETWORKS
RNA Interference (RNAi)
In recent years, as full genome sequences have become
available for Drosophila
[36]
,human
[37,38]
and other
organisms, large-scale analyses of gene functions have given
rise to the field of 'functional genomics'. RNAi has emerged
as a unique and powerful functional genomics tool to effec-
tively suppress gene expression inmany animal systems
[39]
.
In contrast to other genomic-based approaches, RNAi
provides a direct link fromgene to function. The development
of genome-scale RNAi libraries that contain clones for most
genes in a genome in multiple organisms from Caeno-
rhabditis elegans, Drosophila, mouse and human cells, to the
flatworm Planaria
[40,41]
and Arabidopsis
[42]
, permits the
rapid identification of all genes involved in a particular
process
[43
70]
. Candidate RNAi hits are identified by their ability
to affect the basal or induced expression of the reporter
driven by the pathway responsive promoter. Transcriptional
reporter-based assays have been used to identify regulators
of individual transcription factors such as NF
k
B
[71]
,E2F
[72]
,andFoxO
[73]
. In addition, several transcriptional
reporter-based RNAi screens have been conducted to iden-
tify the regulatory network surrounding cellular signaling
pathways, including the Wnt pathway
[69,74
e
49]
. Because RNAi is applicable to high-
throughput genome-wide analyses it provides a tool to extract
functional information globally and comprehensively.
The phenomenon of RNAi was first identified in plants
and worms
[50]
.InC. elegans, the process of target gene
suppression by RNAi can be triggered by injecting long
dsRNAs (~500 nucleotides) into worms, by feeding them
bacteria that express the dsRNA or by simply soaking them
in solution containing the dsRNAs
[51
e
76]
,theHh
pathway
[7,70]
and the JAK-STAT pathway
[66,77]
.Further
screens based on Oct4 expression level or Oct4 driven GFP
expression level have been used to identify regulators of
stem cell identity
[78,79]
. Transcriptional reporter-based
assays have several advantages, including easy adaptability
to HTS, rapid and automated data collection, and the ability
to identify both positive and negative regulators of the
process under study. However, transcriptional reporter-based
e
53]
.InDrosophila,
dsRNAs can be delivered into embryos via injection or by
generating transgenic animals that carry RNAi hairpin
constructs for in vivo screens
[54]
. Importantly, the addition
of long dsRNAs to Drosophila tissue culture cells (dsRNA
bathing) can efficiently reduce the expression of target
genes
[55,56]
. Using RNAi in cell lines has led to an
e
