regulation of gene expression by small rnas garrett a. soukup
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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 - PowerPoint PPT PresentationTRANSCRIPT
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Regulation of gene expression by small
RNAs
Garrett A. Soukup
Creighton University School of Medicine
Department of Biomedical Sciences
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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
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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)
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RNAi pathway• 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 multi-component nuclease called the RNA-induced silencing complex (RISC).
• RISC specifically cleaves mRNAs that have perfect complementarity to the siRNA strand
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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
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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.
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Utility of RNAi for functional genomics• siRNAs are powerful tools for manipulating gene
expression and determining gene function
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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
5´-NNNNNNNNNNNNNNNNNNNUU-3´ |||||||||||||||||||3´-UUNNNNNNNNNNNNNNNNNNN-5´
sense
antisense
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Example of siRNA knockdown• siRNA targeting rev mRNA
sequence encoding rev-EGFP fusion protein
• Sense (S) or antisense (AS) strand of siRNA alone does not effect knockdown of rev-EGFP expression
• An irrelevant siRNA sequence (IR) does not effect knockdown of rev-EGFP expression
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Nature did not exhaust billions of years of evolution creating RNAi solely for the
benefit of modern day biologists!
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Biological roles of RNAi• Cellular immune response to viruses (some
organisms)
• Genetic stability
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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
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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
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Mechanism effecting heterochromatin?
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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
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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
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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
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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
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miRNA gene transcription• Most miRNA genes are transcribed by RNA Pol II• miRNA genes can be arrayed and thus co-expressed
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The machinery: PAZ domains bind 3´ends
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The machinery: Dicer recognition and cleavage of RNA
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The machinery: Argonaute RNA binding and function
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The machinery: Accessory factors
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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?
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Potential mechanisms
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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)
• An aside: plant miRNAs differ in that they are entirely complementary to their target genes
5’ NNNNNNNA 3´ || ||| ||||||| 3´-NNNNNNNNNNNNNNNNNNNNN-5´
mRNA
miRNAseed
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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
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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
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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
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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
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miRNAs in disease• Cancer cells exhibit distinct miRNA expression profiles
• Aberrant miRNA expression can contribute to carcinogenesis
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miRNAs as tumor suppressors and oncogenes