microRNA miRNA

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microRNA (miRNA)
Small non-coding RNA with Big Impact in Biology

• Hua-Chien Chen Ph.D

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Contents in this lecture

• Operational definition of microRNA (miRNA)
• Discovery history
• miRNA biogenesis
• miRNA and target genes interaction
• Pathophysiological functions of miRNA

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Central Dogma of Molecular Biology
Genome

• RNA merely plays an accessory role
• Complexity is defined by proteins encoded in the
  genome

mRNA
tRNA
rRNA
Protein
Cellular responses
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Central Dogma of Molecular Biology
Genome

• Small non-coding RNAs play an important role in
  gene regulation
• Human genes
• protein coding genes
  non-coding regulatory RNAs

miRNA siRNA
rRNA tRNA
mRNA
Protein
Cellular responses
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Basic units of DNA and RNA molecules
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Types of RNA molecules
RNA
mRNA Protein-coding RNA
ncRNA Non-coding RNA. Transcribed RNA with a
structural, functional or catalytic role
tRNA Transfer RNA Interface between mRNA amino
acids
snRNA Small nuclear RNA Incl. RNA that form
part of the spliceosome
snoRNA Small nucleolar RNA Found in nucleolus,
involved in modification of rRNA
rRNA Ribosomal RNA Participate in protein
synthesis
RNAi RNA interference Small non-coding RNA
involved in regulation of expression
Other Including large RNA with roles in
chromotin structure and imprinting
siRNA Small interfering RNA Active molecules in
RNA interference
miRNA MicroRNA Small RNA involved in regulation
of expression
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Operative definition of microRNA

• short (20-25nt) RNA molecules
• Encoded in genome in most eukaryotic organisms
• transcripted as a precursor RNA molecule by RNA
  polymerase II
• Mature miRNAs are process from precursors that
  contain hairpin structure
• Regulates gene expression at post-transcriptional
  level
• target mRNA for cleavage or translational
  repression

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Discover history of miRNA
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The discovery of RNA interference

• An Unexpected Result
• petunias surprisingly developed areas of
  hypopigmentation when transduced with the gene
  encoding an enzyme required for pigment
  synthesis.
• The phenomena was called Co-suppression
• Similar effects seen in fungi. called Quelling

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C. elegans lin-4 first identified microRNA
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MicroRNAs the story so far.

• Second microRNA identified at year 2000
• let-7
• 21 nt
• 3 UTR of lin-41 and hbl-1
• Larval stage 4 to adult
• Orthologues detected in throughout metazoans
  (2000)

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More than 3,000 miRNAs in public databases

• Homo sapiens (462)
• Mus musculus (358)
• Rattus norvegicus (234)
• Drosophila melanogaster (78)
• Caenorhabditis elegans (114)
• Arabidopsis thaliana (118)
• Epstein Barr virus (23)
• Human cytomegalovirus (11)

From miBase Release 8.2 (Jun 2006)
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Biogenesis of miRNA
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Genomic Organization of miRNAs
(Intronic miRNAs)
(34.7)
(Intergenic miRNAs)
(61.8)
(Exonic miRNAs)
(3.5)
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Processing of intronic miRNA
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Chromosome organization of human miR-17-92
clusters
Total 53 miRNA clusters have been identified
within human geneme
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Biogenesis of miRNA
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miRNA biogenesis player Drosha
Pro-rich
RS-rich
RIIIDa
RIIIDb
dsRBD
1,374 aa

• Processes pri-miRNA into pre-miRNA
• Leaves 3 overhangs on pre-miRNA
• Nuclear RNAse-III enzyme Lee at al., 2003
• Tandem RNAse-III domains
• How does it identify pri-miRNA?
• Hairpin terminal loop size
• Stem structure
• Hairpin flanking sequences
• Not yet found in plants
• Maybe Dicer does its job?

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miRNA biogenesis player Dicer
1,922 aa

• Cleaves dsRNA or pre-miRNA
• Leaves 3 overhangs and 5 phosphate groups
• Cytoplasmic RNAse-III enzyme
• Functional domains in Dicer
• Putative helicase
• PAZ domain
• Tandem RNAse-III domains
• dsRNA binding domain
• Multiple Dicer genes in Drosophila and plants
• Functional specificity?

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Working hypothesis of Dicer
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RNA Induced Silencing Complex (RISC)

• RNAi effector complex
• Critical for target mRNA degredation or
  tranlslation inhibition
• Not well characterized 4 subunits? More?
• Activities associated with RISC
• Helicase
• Endonuclease and exonuclease Slicer (or is it
  Dicer?)
• homology seeking/RNA binding
• Preferentially incorporates one strand of unwound
  RNA
• Antisense
• How does it know which is which?

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RISC Preference for Antisense RNA

• Helps ensure specificity for target
• 5 stability of siRNA and miRNA duplex strands
  often different
• The strand with less 5 stability usually
  incorporated into RISC
• Due to easier unwinding from one end?
• If strand stability is similar (rare), strands
  incorporated at similar frequency

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miRNA Target Gene Interaction
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From base pairing to gene silencing
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Seed sequence hypothesis
The 5 region, and particularly seed positions
2-8, is the most conserved region of miRNAs and
has been shown to play a key role in the target
recognition
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Two classes of miRNA binding sites in animal
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mRNA Degradation

• Perfect complementarity between miRNA in RISC and
  the target mRNA
• Cleavage by RISC Slicer activity
• Could be Dicer?
• Other endo/exonucleases?
• Recruitment of other components?

Novina and Sharp, 2004c
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Translational Inhibition

• Imperfect match between miRNA in RISC and target
  mRNAs
• RISC usually binds 3 UTR
• Mechanism of inhibition... ????

He and Hannon, 2004
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miRNA target prediction programs
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Take home messages

• miRNAs are negative regulators for protein coding
  genes
• miRNAs regulate expression of target genes
  through base-pairing interaction
• Each miRNA may regulate tens to hundreds of
  target genes
• Each protein coding gene may be regulated by one
  or multiple miRNAs
• It is estimated that more than 30 of protein
  coding genes are regulated by miRNAs

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Pathophysiological functions of miRNA
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Physiological Roles of miRNAs

• Organ (or tissues) development
• Stem cell differentiation and maturation
• Cell growth and survival
• Metabolic homeostasis
• Oncogenic malignancies and tumor formation
• Viral infection
• Epigenetic modification

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microRNAs and hematopoesis
Genome Biol. 20056(8)R71. Epub 2005 Aug 1
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microRNAs and stem cells

• Drosophila germline stem cells
• dcr-1 mutant has fewer clonal cysts than wt.
• Effect via Dap.

Hatfield et. al., Nature 2005
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microRNAs and cardiogenesis

• Excess miR-1-1 ? decreased cardiomyocytes
• Effect is via miR-1-1 targeting of Hand2
  transcription factor

Zhao et al. Nature 2005
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Tissue-specific miRNA expression
Brain and spine code
Muscle
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microRNA and Cancer
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miRNA frequently located at chromosome fragile
sites
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Examples of miRNAs located in chromosome fragile
sites
D deleted region A amplified region
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miR-17-92 cluster is overexpressed in human lung
cancer cells

• miR-17-92 cluster (containing miR-19a and miR-20)
  is markedly overexpressed in lung cancer cell
  lines

Cancer Research (2005) 65 9628
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A microRNA polycistron as a potential human
oncogene

• Overexpression of the mir-17-19b cluster
  accelerates c-myc-induced lymphomagenesis in mice
• Em-myc/mir-17-19b tumors show a more disseminated
  phenotype compared with control tumor

Nature (2005) 435 828
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miRNAs associated with human cancers
TS tumor suppressor OG oncogene
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miRNAs function as oncogenes or tumor suppressor
genes
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Take home messages

• There are gt500 miRNAs in the human genome
• miRNAs are regulators just as important as
  transcription factors
• Many miRNAs play central roles in normal tissue
  development as well as in disease pathogenesis

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Viral encoded miRNAs
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EBV virus-encoded miRNAs