Microarray analysis shows that some microRNAs

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Microarray analysis shows that some microRNAs
downregulate large numbers of target mRNAs
LEE P. LIM, NELSON C. LAU,
PHILIP GARRETT-ENGELE ANDREW GRIMSON,
JANELL M. SCHELTER,
JOHN CASTLE, DAVID P. BARTEL,
PETER S. LINSLEY JASON M. JOHNSON

Presented by
Li-Chen Wu

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• What are MicroRNAs (miRNAs)?
• ? MicroRNAs are a growing family of small
  non-protein-coding regulatory
• genes ( 22 nucleotides) that regulate the
  expression of homologous
• target-gene transcripts.
• ? Bioinformatics studies predict that there are
  250 miRNAs encoded in
• the human genome. About a quarter of human
  miRNA genes are located
• in introns of pre-mRNAs the remaining
  miRNAs are clustered in the
• genome, predicting a multi-cistronic
  transcript.
• ? miRNAs are processed by the RNA-mediated
  interference machinery.
• ? They have been implicated in the control of
  cell death and proliferation
• in flies, haematopoietic lineage
  differentiation in mammals, neuronal
• patterning in nematodes, and leaf and
  flower development.

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Model of microRNA biogenesis
Step 1. Transcription of a miRNA gene produces a
primary nuclear transcript
(pri-miRNA). Step 2. The nuclear RNase III enzme
Drosha cleaves pri-miRNA to
generate a stem-loop pre-miRNA. (70
nucleotides pre-miRNA intermediate) Step 3. The
pre-miRNA directly associates with the exportin
Exp5 and Ran-GTP, and this complex
exits the nucleus. Hydrolysis of
Ran-GTP to Ran-GDP causes
dissociation of the export complex. Step 4. The
pre-miRNA associates with the cytoplasmic
RNase III enzyme Dicer, which cleaves it to
form a 22-nucleotide miRNA
intermediate. Step 5. The miRNA intermediate
rapidly unwinds as it assembles
into a miRNP complex, and one miRNA
strand is retained in the miRNP.
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Small RNAs act upstream of several effectors

• siRNA regulate histone methyltransferases
• (HMTs) in nuclear complexes to promote
• heterochromatin formation, also regulate
• the RISC (RNA-induced silencing complex)
• to degrade specific mRNA transcripts.
• miRNAs regulate miRNP complexes that
• degrade specific mRNAs or repress their
• translation.

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Direct Labeling and Hybridization of MicroRNA
Sample
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Quantitative northern blot analysis of
miR-1 and miR-124 expression
Supplementary Figure 2.
A. Northern blot of a human total RNA tissue
panel. B. Molecular abundance of upregulated
miRNAs and corresponding U6 snRNA in tissue
types, based on quantitation of signals from the
Northern blot in A).
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Figure 1. Tissue-specific gene expression
rankings for
downregulated genes
a. Microarray signature12h
after miR-124
transfection. (Significantly downregulated
probes are in green)
b.c. Cerebral cortex rankings for
LocusLink genes and the
set of genes downregulated
at both 12 and 24h following transfection
of miR124.
The 174 genes downregulated by miR-124
transfection are
significantly enriched for
genes that are expressed at lower levels in
cerebal cortex relative to
other tissues.
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Tissue-specific gene expression rankings for
downregulated genes
d. e. Log (base 10) P values are
plotted for each of the 46 tissues for
the miR124 (d) and miR-1 (e)
downregulated sets.
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Figure 2. Over-represented motifs in the
3' UTRs of downregulated genes.
a. b. miR-1 (a) and miR-124 (b) downregulated
genes. Motif nucleotides that were
complementary to the transfected miRNA are shown
in red base-pairing to the miRNA
sequence is shown in blue.
These results imply that knockdown of the
transcripts is caused mainly by direct binding
of the transfected miRNAs to the 3UTRs, with
binding at the 5 end of the miRNA being
particularly vital. The 5 region, and
particularly seed positions 2-8, is the most
conserved region of metazoan miRNAs and has
long been thought to play a key role in the
target recognition.
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Figure 3 Microarray analysis of the effects
of miRNA mutations.

• Sequences of 124mut9-10 duplex after
• transfected into HeLa cells, gave a
• downregulated signature 89 shared
• with that of the wild type, whereas for
• 124mut5-6 was almost completely distinct.

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Tissue analysis for mutant microRNAs
Supplementary Figure 2. As in Figure 1c and 1d,
log p-values are plotted for
each of the 46 tissues for the
downregulated sets from the
a. 124mut9-10, b. 124mut5-6, c.
chimiR-124/1, and
d. chimiR-1/124 transfections.
These results demonstrate that, for the mRNA
knockdown observed in this assay, positions 5 and
6 are much more crucial than positions 9 and 10.
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Figure 3 Microarray analysis of the effects
of miRNA mutations.

• Sequences of 124mut9-10 duplex after
• transfected into HeLa cells, gave a
• downregulated signature 89 shared
• with that of the wild type, whereas for
• 124mut5-6 was almost completely distinct.
• Sequences of wild type and chimaeric
• miRNA after transfected into HeLa cells,
• showed that the 5 ends of the miRNAs
• were sufficient for generating
  tissue-specific
• signatures.

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Figure 4. MicroRNA-directed repression of
renilla luciferase reporter
genes bearing 3' UTR segments
from predicted target genes.

• b. MiR-1(a) and miR-124(b) target genes.
• wild type (open bars), mutant
  plasmids (filled bars)

Results When cotransfected with the cognate
miRNA, six of the ten wild-type reports
exhibited significant repression
relative to the corresponding constructs with
mutant seed matches
cotransfection of the noncognate miRNA typically
had no effect.
Conclusion Pairing to the miRNA seed region
contributes directly to mRNA repression
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Expected and observed seed match counts in
different regions of miR-1 or miR-124
downregulated genes
Supplementary Figure 3.
Conclusion MiRNAs interact more frequently or
more effectively with 3
UTRs than with other regions of mRNAs.
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Conclusion

• Metazoan miRNAs are predominantly negative
  regulators of gene
• expression, they can reduce the levels of
  many of their target transcripts,
• not just the amount of protein deriving
  from these transcripts.
• MiR-1 and miR-124. and presumably other
  tissue-specific miRNAs, seem to
• downregulate a far greater number of
  targets than previously appreciated,
• thereby helping to define tissue-specific
  gene expression in humans.
• 3. Microarrays can be used to detect
  physiologically relevant miRNA.