Download RNA interference - Creighton University

siRNA / microRNA
epigenetics
stem cells
RNA interference (RNAi)
• RNAi is a cellular process whereby double-stranded RNA
(dsRNA) induces silencing of complementary target genes
• RNAi is central to mechanisms of post-transcriptional gene
silencing (PTGS), but can also effect transcriptional gene
silencing (TGS)
• RNAi is highly conserved among all eukaryotic organisms
(fungi, protozoans, plants, nematodes, invertebrates, mammals)
Overview of RNAi
• Double-stranded RNA (dsRNA) is
processed by Dicer, an RNase III family
member, to produce 21-23nt small
interfering RNAs (siRNAs)
• siRNAs are manipulated by a multicomponent nuclease called the RNAinduced silencing complex (RISC).
• RISC specifically cleaves mRNAs that
have perfect complementarity to an
siRNA strand
A brief history of RNAi
• RNAi was initially discovered and characterized in the C.
elegans
• It was observed that antisense or sense RNA could equally
effect silencing (knock-down) of target gene expression
• A later study demonstrated that antisense and sense RNA
combined (dsRNA) is 10-times more effective in silencing target
gene expression
• Genetic studies in C. elegans have helped to identify genes
encoding RNAi machinery
Biological roles of small RNAs
• Cellular immune response to viruses (siRNAs)
• Genetic stability and heterochromatin maintenance (siRNAs)
• Development and cell differentiation (microRNAs)
Genetic stability
• siRNAs repress the mobility of transposable genetic elements
in C. elegans and S. pombe
• Disruption of Dicer, argonaute, or RdRP increases the relative
abundance of transposon RNA and increases transposon
mobility
• Transposable elements comprise a substantial fraction of the
highly repetitive DNA in eukaryotic genomes (another being
centromeres)
• Highly repetitive DNA is often associated with heterochromatin
which is transcriptionally silent
Genetic stability
• siRNAs are required to establish and maintain heterochromatin formation and gene
silencing at mating type loci and centromeres in yeast (S. pombe)
• Disruption of
Dicer, argonaute,
and RdRP
eliminate
silencing,
decrease
histone and DNA
methylation, and
cause aberrant
chromosome
segregation
• To this point,
siRNAs isolated
from eukaryotic
organisms
correspond to
centromeric
repeats and
transposable
elements
siRNAs and transcriptional gene
silencing
• The role of siRNAs in transcriptional gene silencing appears to
be important among all eukaryotic organisms
• However, the precise mechanism(s) of siRNA-mediated
transcriptional gene silencing are poorly understood
Small RNAs in development
• Small RNAs play a critical role in development through the
action of a specific class of small dsRNAs termed microRNAs
(miRNAs)
• miRNAs are the products of endogenous genes! They are
processed in part by Dicer but function to effect posttranscriptional gene silencing in a manner distinct from that of
siRNAs
• The importance of miRNAs in regulating cellular processes
through effects on target gene expression is reflected in the
fact that many miRNA genes are highly conserved among
eukaryotic organisms
A brief history of miRNAs
• C. elegans was discovered to possess small noncoding RNAs
(let-7 and lin-4) that negatively regulate expression of target
genes (lin-41 and lin-28) which direct developmental progress
• At that time, the so-called small temporal RNAs (stRNAs) were
found to repress translational of the target mRNAs by
interacting with complementary sites in their 3’ untranslated
regions (UTRs)
• It was later appreciated that the stRNAs are processed by Dicer
and thus function through related pathway
• Disruption of the stRNAs, Dicer, or argonaute genes result in
similar developmental abnormalities
• With the subsequent discovery that there are many such small
RNAs that function through the RNAi pathway, the entire class
was renamed microRNAs
Small but plenty
• To date, over 10,000 miRNA genes have been identified among
over 80 eukaryotic organisms (plants and animals)
• There are, for example 174 C. elegans, 157 Drosophila, 579
mouse, and 721 human miRNA genes
• About 10 viruses are known to express their own miRNAs
• Approximately one third of miRNA genes are intronic with
respect to protein coding genes
• Approximately two thirds of miRNA genes are intergenic
• miRNA genes by their earliest definition should be conserved
among species, but there are a growing number of species
specific miRNAs
Conservation of miRNA sequence and
structure
• Certain miRNAs are
highly conserved and
thus evolutionarily
ancient (e.g. let-7)
• Sequence conservation
must fulfill the require to
form a dsRNA hairpin
from which the identicle
miRNA is processed
miRNA gene transcription
• miRNA genes are typically transcribed by RNA pol II
• miRNA genes can be arrayed (i.e. clustered and coexpressed)
miRNA biogenesis
Quantitative real time
RT-PCR
Real time quantitative RT-PCR
From Wikipedia:
“amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification
(as absolute number of copies or relative amount when normalized to DNA input or additional normalizing
genes) of a specific sequence in a DNA sample.”
“amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Two
common methods of quantification are the use of fluorescent dyes that intercalate with double-stranded
DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary
DNA.”
“Frequently, real-time PCR is combined with reverse transcription polymerase chain reaction to quantify low
abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a
particular time, or in a particular cell or tissue type.”
Real time quantitative RT-PCR