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Regulation of gene
expression by small
RNAs
Garrett A. Soukup
Creighton University School of Medicine
Department of Biomedical Sciences
Objectives
• Appreciate that there are two related biochemical
pathways through which small RNAs can affect gene
expression
• Understand how each pathway through its small RNA
product (siRNA or miRNA) differently affects gene
expression
• Distinguish differences in biogenesis and action of
siRNAs and miRNAs
• Appreciate the biological roles and significance of
siRNAs and miRNAs
• Comprehend how small RNAs might be used as agents
for biotechnological or therapeutic manipulation of gene
expression
A tale of two pathways
• RNA interference (RNAi) pathway: produces small
interfering RNAs (siRNAs) that silence
complementary target genes
• MicroRNA pathway: produces microRNAs (miRNAs)
that silence complementary target genes
• Mechanisms involve transcriptional gene silencing
(TGS) and/or post-transcriptional gene silencing
(PTGS)
• Pathways are conserved among most all eukaryotic
organisms (fungi, protozoans, plants, nematodes,
invertebrates, mammals)
RNAi pathway
• Double-stranded RNA (dsRNA) is
processed by Dicer, an RNase III
family member, to produce 2123nt small interfering RNAs
(siRNAs)
• siRNAs are manipulated by a
multi-component nuclease called
the RNA-induced silencing
complex (RISC).
• RISC specifically cleaves mRNAs
that have perfect complementarity
to the siRNA strand
A brief history of RNAi
• RNAi was initially discovered and characterized in
the nematode worm, C. elegans
• It was observed that double-stranded RNA (dsRNA)
was 10-times more effectiv in silencing target gene
expression than antisense or sense RNA alone
• Genetic studies in C. elegans identified that the
effect requires two components: Dicer and
Argonaute
• Andrew Fire (Stanford) and Craig Mellow (U Mass)
were awarded the 2006 Nobel Prize in Medicine for
their discovery of RNAi
Core components of the RNAi pathway
• Dicer
Dicer family proteins contain an N-terminal helicase
domain, a C-terminal segment containing dual RNase
III domains, and one or more dsRNA-binding motifs.
Family members also contain a PAZ domain.
• Argonaute (RISC complex)
Argonaute family members are highly basic, ~100 kD
proteins that contain PAZ and PIWI domains.
Utility of RNAi for functional genomics
• siRNAs are powerful tools for manipulating gene
expression and determining gene function
Synthetic siRNAs
• Synthetic siRNAs that target any sequence can be
prepared by chemical synthesis
• In mammalian cells, siRNAs range in effectiveness at
knocking down target gene expression (50-95%)
• The effectiveness of an siRNA is dependent upon
target sequence
sense
5´-NNNNNNNNNNNNNNNNNNNUU-3´
|||||||||||||||||||
3´-UUNNNNNNNNNNNNNNNNNNN-5´
antisense
Example of siRNA knockdown
• siRNA targeting rev mRNA
sequence encoding revEGFP fusion protein
• Sense (S) or antisense (AS)
strand of siRNA alone does
not effect knockdown of revEGFP expression
• An irrelevant siRNA
sequence (IR) does not
effect knockdown of revEGFP expression
Nature did not exhaust billions of years of
evolution creating RNAi solely for the
benefit of modern day biologists!
Biological roles of RNAi
• Cellular immune response to viruses (some
organisms)
• Genetic stability
Immune response
• In certain organisms (especially plants), RNAi serves
as a first line of defense against viral infection, as
virus may contain or viral replication can produce
dsRNA
• To this point, a number of plant viruses encode
proteins that specifically bind and sequester siRNAs
as a means of countering the cellular immune
response of RNAi
Genetic stability
• RNAi represses transposable genetic elements in C.
elegans and S. pombe
• Disruption of Dicer or Argonaute increases the relative
abundance of transposon RNA and increases transposon
mobility
• RNAi is required to establish and maintain
heterochromatin formation and gene silencing at mating
type loci and centromeres in S. pombe
• Disruption of Dicer or Argonaute eliminates silencing,
decreases histone and DNA methylation, and causes
aberrant chromosome segregation
• Highly repetitive DNA is often associated with
heterochromatin which is transcriptionally silent
Mechanism effecting heterochromatin?
miRNA pathway
• miRNAs are the
products of
endogenous genes
• miRNAs function to
post-transcriptionally
repress target genes
by inhibiting
translation and/or
decreasing mRNA
half-life
• One miRNA may
effect many (e.g.
hundreds) of target
genes
A brief history of miRNAs
• C. elegans was discovered to possess small noncoding
RNAs (lin-4 and let-7) required to repress expression of
target genes (lin-28 and lin-41) that direct developmental
progress
• At that time, these 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 require Argonaute, and thus function through an
RNAi-related pathway
• With the subsequent discovery that there are many such
small RNAs throughout eukaryotic organisms, the entire
class was renamed microRNAs
Small but plenty
• To date, nearly 8600 miRNA genes have been identified among
73 eukaryotic organisms (plants and animals) and 15 viruses
• There are, for example 132 C. elegans, 78 Drosophila, 377
mouse, and 474 human miRNA genes
• Approximately one third of miRNA genes are intronic with
respect to protein coding genes
• Approximately two thirds of miRNA genes are intergenic
(independent genes)
• Many miRNA genes are conserved among species
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
miRNA is processed
by Dicer
miRNA gene transcription
• Most miRNA genes are transcribed by RNA Pol II
• miRNA genes can be arrayed and thus co-expressed
The machinery: PAZ domains bind
3´ends
The machinery: Dicer recognition and
cleavage of RNA
The machinery: Argonaute RNA binding
and function
The machinery: Accessory factors
Argonaute proteins
• Mammals possess 4 argonaute proteins (Ago1,
Ago2, Ago3, and Ago4)
• Only Ago2 has been demonstrated to possess RNA
cleavage or “Slicer” activity
• What, if any, are the distinctive roles of other Ago
proteins?
Potential
mechanisms
miRNA-target interaction
• miRNA binding sites reside within the 3´ UTRs of
target transcripts
• Seed-pairing hypothesis (animal miRNAs)
(miRNA nucleotides 2-7 and sometimes 8)
mRNA 5’
NNNNNNNA
|| |||
|||||||
miRNA 3´-NNNNNNNNNNNNNNNNNNNNN-5´
3´
seed
• An aside: plant miRNAs differ in that they are entirely
complementary to their target genes
Target gene identification
• 3´ UTRs are typically highly divergent (not conserved)
among otherwise highly conserved genes
• Rationale: If miRNAs are conserved among species, so
too should be their binding sites among conserved target
genes
• Based on the seed pairing hypothesis, bioinformatic
algorithms search for conserved miRNA binding sites
among conserved target genes
• Due to the minimal base-pairing requirement, predicted
target genes are numerous
• Therefore, elucidating miRNA functions based on
predicted target genes is difficult
miRNAs in development
• miRNAs play various roles in cell proliferation,
differentiation, fate determination, and differentiated
cell function
• miRNAs appear to contribute to transitions from
stem (precursor) cells to differentiated cell types by
refining/reinforcing desired gene expression profiles
• miRNAs appear to “sharpen” developmental
outcomes with regard to organogenesis,
morphogenesis, and histogenesis
Differential expression of miRNAs among
cell types: clues to function
• Different cells express different miRNAs (e.g. stem cells versus
differentiated cells)
• miRNA expression is typically examined by microarray analysis
or cloning and sequencing
• miRNA expression domains within an organism are revealed by
in situ hybridization (Locked Nucleic Acid probes)
miR-1
miR-100
miR-375
Dicer knockout organisms
• Knockout of Dicer disrupt RNAi and miRNA pathways
• Conditional knockout of Dicer enables analysis of RNAi effects
in specific tissues
• Dicer knockout is embryonic lethal in mice
Knockout embryos exhibit lack of stem cells, and cell proliferation is
decreased
• Conditional Dicer knockout mice display defects in
morphohistogenesis
Dicer knockout in certain tissues results in developmental delays,
cell death, and aberrant gene expression
miRNAs in disease
• Cancer cells exhibit distinct miRNA expression profiles
• Aberrant miRNA expression can contribute to
carcinogenesis
miRNAs as tumor suppressors and
oncogenes