1. microRNAs Small Non-coding RNAs with Big Impact in Biology
Hua-Chien Chen Ph.D

2. Lecture Content Non-coding RNAs and discovery of microRNAs
microRNA biogenesis
Tanscription of miRNA primary transcripts
microRNA processing and maturation
microRNA action
microRNA and target interaction
microRNA function
Normal physiological function
miRNA and human diseases

3. The genetic basis of developmental complexity
Yeast
Warm
Human
Genome: 15Mb
Genes: 6,000
Cell: 1
Genome: 100Mb
Genes: 18,500
Cell: 1,100
Genome: 3,200Mb
Genes: 25,000
Cell: 1X1012
Humans (and other vertebrates) have approximately the same number of protein-coding genes (~20,000) as C. elegans, and less than those of plants (Arabidopsis ~28,000, rice ~40,000) and protozoa (30,000).
Most of the proteins are orthologous and have similar functions from nematodes to humans, and many are common with yeast.
Where is the information that programs our complexity?

4. The proportion of noncoding DNA broadly increases with developmental complexity
Nature Rev. Genetics (2004) 5:

5. ncRNA: non-coding RNAs
Type of RNA molecules
RNA
mRNA
Protein-coding RNA
ncRNA: non-coding RNAs
Transcribed RNA with a structural,
functional or catalytic role
rRNA
Ribosomal RNA
Participate in
protein synthesis
tRNA
Transfer RNA
Interface between
mRNA &
amino acids
snRNA
Small nuclear
RNA
RNA that form
part of the
spliceosome
snoRNA
Small nucleolar
RNA
Found in nucleolus,
involved in
modification
of rRNA
RNAi
RNA interference
Small non-coding
RNA involved
in regulation
of gene 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 protein-coding gene
Modified from Dr Morten Lindow slide

6. C. elegans lin-4 : first identified microRNA
lin-4 precursor
lin-4 RNA
“Translational repression”
target mRNA
lin-4 RNA
V. Ambros lab
lin-4 encodes two small RNA molecules, a more abundant 22 nt that are processed from a rare 61 nt pre-lin-4 . These hairpin precursor is a characteristic feature of the miRNA class of regulatory RNAs.
One of lin-4’s target genes, lin-14, encodes a novel nuclear protein and is a putative transcription factor. The lin-4 microRNA regulates lin-14 through specific sequences in the 3’ UTR of the lin-14 mRNA
Upon lin-4 expression, lin-14 protein levels are reduced. Although transcription from the lin-14 gene still occurs, it is of no consequence. (Posttranscriptional control).
The story of microRNAs begins almost ten years ago when the Ambros lab made the remarkable discovery that the lin-4 gene, important for controlling development in the nematode C. elegans, did not code for protein, but instead, produced a pair of small RNAs.
The longer ~61-nt RNA was predicted to fold into a stem-loop precursor which gave rise to a ~22 nt RNA that was found to function by triggering repression of the translation of the target gene lin-14. The specificity of interaction was thought to derive from complementarity between the lin-4 RNA and the 3’ untranslated region of the target mRNA as shown in this diagram.
Not until 2000 did this question begin to be answered when a second gene of this type called let-7 was characterized by the Ruvkun lab. Let-7 also had a strong developmental phenotype when mutated and turned out to encode a 21 nucleotide untranslated RNA. But unlike lin-4… let-7 was found to be conserved in a wide range of other animals including molluscs, worms, flies and vertebrates including human.

7. lin-4 and let-7 are funding members of microRNA
Seven years later, let-7 (another non-coding gene) was shown to regulate development in worms
A homolog of let-7 was identified in humans and Drosophila
Lin-4 and let-7 became founding members of a group of endogenous small RNA molecules with regulatory functions
Lin-4: regulates heterochronic development at L1 to L2 stage
Let-7: regulates heterochronic development at L4 to adult stage
Nature (2000) 403:

8. Let-7 sequence and gene regulation
Nature (2000) 403:

9. microRNAs at a glance microRNA precursor
Small, single-stranded forms of RNA (~22 nucleotides in length)
generated from endogenous hairpin-shaped transcripts encoded in the genomes
Negatively regulate protein-coding genes through translational repression or targeting mRNA for degradation
More than 500 microRNAs encoded in human genenome constitute a largest gene family
It has been estimate that more than 30% of protein-coding genes can be regulated by microRNAs

10. More than 5,000 miRNAs in public databases
Homo sapiens (706)
Mus musculus (547)
Rattus norvegicus (286)
Drosophila melanogaster (152)
Caenorhabditis elegans (155)
Arabidopsis thaliana (187)
Epstein Barr virus (25)
Human cytomegalovirus (11)
Kaposi sarcoma-associated herpesvirus (13)
Simian virus (1)
From miRBase Release 13.0 (Mar 2008)

11. Gene regulation by transcription factors and microRNAs

12. microRNA Biogenesis

13. microRNA biogenesis  Transcription  Processing  Maturation
 Execution
Nature Rev. Immunology (2008) 8:

14. Majority of miRNAs are transcribed by RNA polymerase II
Intronic miRNAs
Located in the intron region of protein coding or non-coding transcripts
Transcribed by RNA polymerase II
Extronic miRNAs
Located in the exon region of protein-coding or non-coding transcripts
Intergenin miRNAs
Located in the intergenic region of chromosome
Transcribed by RNA polymerase II or polymerase III

15. Genomic location of microRNAs

16. Pri-miRNAs are processed by Drosha RNase III enzyme
Pro-rich
RS-rich
RIIIDa
RIIIDb
dsRBD
1,374 aa
Processes pri-miRNA into pre-miRNA
Leaves 2 bp 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?

17. Multiple mechanisms for miRNA processing
The microprocessor complex Drosha–DGCR8 cleaves the pri-miRNA, releasing the pre-miRNA
Some miRNAs require additional specificity factors (for example p68 and p72) for efficient cleavage
Interaction of pri-miR-18a with hnRNP A1 facilitates cleavage of this specific miRNA by Drosha
TGF-β signalling induces SMAD binding to the miR-21 precursor and enhances its efficient processing by Drosha.
Splicing can replace Drosha processing if the released and debranched intron (mirtron) has the length and hairpin structure of a pre-miRNA.
Figure 2 Regulation of pri-miRNA processing. (a) The microprocessor
complex Drosha–DGCR8 cleaves the pri-miRNA, releasing the pre-miRNA.
(b) Some miRNAs require additional specificity factors (for example
p68 and p72) for efficient cleavage. (c) Interaction of pri-miR-18a with
hnRNP A1 facilitates cleavage of this specific miRNA by Drosha. (d) TGF-β
signalling induces SMAD binding to the miR-21 precursor and enhances its
efficient processing by Drosha. (e) Splicing can replace Drosha processing
if the released and debranched intron (mirtron) has the length and hairpin
structure of a pre-miRNA.

18. Ran/Expotin 5 system for pre-miRNA export
Export cargo
NES
Export cargo
Exportin
Exportin
NES
Ran
GTP
Ran
GDP
RanGAP PI
Cytoplasm
Nucleoplasm
Export cargo
NES
Ran-GTP: predominantly in the nucleoplasm
Ran-GDP: predominantly in the cytoplasm
Exportin-5: transport pre-miRNA
Export cargo
Exportin
Exportin
NES
Ran
GTP
Ran
GTP

19. Mature miRNAs are generated by Dicer
DEAD
Helicase
PAZ
RIIIDa
RIIIDb
dsRBD
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?

20. Working hypothesis of Dicer
First contact of dsRNA
2 nt overhang on the 3’ end of dsRNA
Binds to the PAZ binding domain at an oligonucleotide (OB) fold
Second contact at Platform Domain
Anti-parallel-beta sheet
Positive charged residues
Residues interact with negative charge of RNA backbone
A connector helix forms 65 Angstrom (24nt) distance between the PAZ holding and the RNase III cleaving domains – “ruler”
Third contact at the 2 RNase III domains
2 Mn cation binding sites per RNase domain
RNase III domains positioned via bridging domain
Bind to scissile phosphates of dsRNA backbone
A cluster of Acidic residues near the Mn cation binding sites in the RNase III domains is responsible for the hydrolytic cleavage of dsRNA
The small guide RNA is then released and incorporated into the RISC complex by the PAZ-like Argonaut protein

21. Mechanisms of miRNA-mediated gene silencing AGO2-mediated RNA degradation
Gene silencing by double-stranded RNA triggers. Long
dsRNAs or shRNAs are recognized by the Dicer-TRBP complex to
generate 21- to 22-nt siRNA duplexes with 2-nt overhangs at the 3(
ends and phosphorylated at the 5( ends. Similarly, synthetic 21-nt
siRNAs can be introduced into cells, phosphorylated by cellular
kinase, and incorporated with the RISC loading complex. During the
formation of RISC, the guide strand of the siRNA duplex is loaded
onto Ago2 while the passenger strand is removed. Active siRISC
recognizes and cleaves its target mRNA. RISC is a catalytic enzyme
complex that is recycled for multiple rounds of mRNA cleavage.

22. From base pairing to gene silencing
Plant miRNA
Animal miRNA

23. Current model for miRNA-mediated translational repression

24. Majority of miRNAs are binding to the 3’UTR of mRNA genes
3’UTR: regulates mRNA stability and translational efficacy

27. Prediction of miRNA targets

28. Target Prediction by TargetScan
Seed region : TargetScan defines a seed as positions 2-7 of a mature miRNA.
miRNA family : A miRNA family is comprised of miRNAs with the same seed region (positions 2-8 of the mature miRNA, also called seed+m8).
8mer : An exact match to positions 2-8 of the mature miRNA (the seed + position 8) with a downstream 'A' across from position 1 of the miRNA
7mer-m8 : An exact match to positions 2-8 of the mature miRNA (the seed + position 8)
7mer-1A : An exact match to positions 2-7 of the mature miRNA (the seed) with a downstream 'A' across from position 1 of the miRNA

29. Additional factors impact miRNA efficacy
Number of miRNA binding sites in 3’UTR
Closely Spaced Sites Often Act Synergistically
Additional Watson-Crick Pairing at Nt Enhances miRNA Targeting
Effective Sites Preferentially Reside within a Locally AU-rich Context
Effective Sites Preferentially Reside in the 3’UTR, but Not too close to the stop codon
Effective Sites preferentially reside near both ends of the 3’UTR

30. Pathophysiological Function of microRNA

31. Physiological Roles of microRNA
Organ (or tissues) development
Stem cell differentiation and maturation
Cell growth and survival
Metabolic homeostasis
Oncogenic malignancies and tumor formation
Viral infection
Epigenetic modification

32. Tissue specific expression of microRNA
Brain and spine code
Muscle
The expression of miR-124a is restricted to the brain and the spinal cord in fish and mouse or to the ventral nerve cord in the fly.
The expression of miR-1 is restricted to the muscles and the heart in the mouse.
The conserved sequence and expression of miR-1 and miR-124a suggests ancient roles in muscle and brain development.
Dev Cell (2006) 11:441

33. Control of skeletal muscle proliferation and differentiation by miR-1 and miR-133
MEF2: myocyte enhancer factor 2
HDAC4: histone deacetylase 4
SRF: serum response factor
Control of skeletal muscle proliferation and differentiation by miR-1 and miR-133. In skeletal muscle cells, the process of proliferation is antagonistic to the process
of myoblast differentiation. miR-1 and miR-133 function at the center of a network of transcription factors to regulate skeletal myoblast proliferation and differentiation. (a)
Myocyte enhancer factor-2 (MEF2) and myogenic basic helix loop helix (bHLH) proteins, including MyoD (green), regulate their own expression, as well as the expression of
downstream muscle structural genes. Additionally, these transcription factors use upstream and intragenic enhancers to activate transcription of bicistronic pri-miRNAs
encoding miR-1 (blue rectangle) and miR-133 (black rectangle) in differentiated skeletal muscle. miR-1 represses expression of HDAC4 (purple), a signal-dependent
repressor of MEF2 (red) activity, thereby establishing a negative feedback loop to modulate miR-1 and miR-133 expression and promoting myoblast differentiation. miR-133
represses expression of serum response factor (SRF; yellow), a positive regulator of miR-1/133 expression and repressor of myoblast proliferation. (b) E11.5 transgenic
mouse embryos, which harbor a lacZ transgene controlled by the miR-1–2/133a-1 intragenic enhancer, are shown. The wildtype (WT) enhancer directs transgene expression
in heart and skeletal muscle (note the blue color), whereas an enhancer with a mutation in the MEF2 site is not expressed in skeletal muscle or ventricular myocardium.
No miR-1/miR-133a expression in MEF2 knockout mice
E11.5 transgenic mouse embryos

34. Muscle-specific microRNAs and their targets
Trend in Genetics (2008)

35. Tissue specific expression of miRNA
Nature Rev Genetics 2004)

36. microRNA networks and diseases
The number of microRNA in human genome may over 1,000 genes (currently 570 miRNAs in miRBase database)
Tens to hundreds of protein-coding genes are regulated by single miRNA
Estimated that around 30% of genes are regulated by microRNA
Almost every cellular processes are regulated by microRNA
Mutation or dysregulation of microRNA  Diseases formation

37. Several evidences suggest that microRNAs may play an important role in tumor development
More than 50% of microRNAs are located within the chromosome fragile sites
Expression levels of microRNA in tumor biopsies are commonly altered
Several microRNAs have been shown to regulate the proliferation and differentiation of cells
micorRNAs also control the pathways of cell death (apoptosis)

38. miRNA frequently located at chromosome fragile sites

39. Examples of miRNAs located in chromosome fragile sites
D : deleted region
A : amplified region

40. microRNAs are commonly down regulated in tumor biopsies
Hierarchical clustering of miRNA expression. a, miRNA profiles
of 218 samples from several different tissues were clustered (average linkage,
correlation similarity). Samples are in columns, miRNAs in rows. Samples of
epithelial (EP) origin or derived from the gastrointestinal tract (GI) are
indicated.More detail is shown in Supplementary Fig. 4. b, Clustering of 73
bone marrow samples from patients with acute lymphoblastic leukaemia
(ALL). Coloured bars indicate the different ALL subtypes. c, Comparison of
miRNA data and mRNA data. For 89 epithelial samples from a that had
mRNA expression data, hierarchical clustering was performed. Samples of
GI origin are shown in blue. GI-derived samples largely cluster together in
Nature (2005) 435 :

41. C-myc induces expression of the miR-17/92 cluster
c-Myc induces expression of the miR-17 cluster. a, Schematic
representation of the mir-17, mir-106a and mir-106b clusters. mir-18b and
mir-20b are predicted on the basis of homology to mir-18a and mir-20a,
respectively30. b, Northern blot analysis of miRNAs in P493-6 cells.
Duplicate samples are shown, and miR-30 served as a loading control. Blots
were also probed for miR-16 and miR-29 as loading controls, and similar
results were obtained (data not shown). c, Northern blot analysis of total
RNA from P493-6 cells with a probe specific for the mir-17 cluster. 7SK RNA
served as a loading control
Tet-off system to induce c-myc expression in P493 cells
Nature (2005) 435 :

42. miR-17/92 cluster showed increase expression in B lymphoma and colon cancers
Nature (2005) 435 :

43. miR-17/92 clusters function as oncogenes
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
Figure 2 | Overexpression of the mir-17–19b cluster accelerates
c-myc-induced lymphomagenesis in mice. a, Schematic representation of
the adoptive transfer protocol using Em-myc HSCs. b, Mice reconstituted
with HSCs expressing mir-17–19b in an MSCV retroviral vector (MSCV
mir-17–19b) or infected with a control MSCV virus were monitored by
blood smear analysis starting 5 weeks after transplantation. The Kaplan-
Meier curves represent the percentage of leukaemia-free survival or overall
survival. c, External GFP imaging of tumour-bearing mice with Em-myc/mir-
17–19b or Em-myc/MSCV shows the overall distribution of tumour cells.
Em-myc/mir-17–19b tumours show a more disseminated phenotype
compared with control tumours. These animals are representative of their
genotype.
Nature (2005) 435 :

44. miR-34 family function as tumor suppressors
miR-34 family members are highly conserved during evolution
miR-34a is located within chromosome 1p36 region, which is commonly deleted in human neuroblastoma
Primary neuroblastomas and cell lines often showed low levels of miR-34a expression
Forced expression of miR-34a in these cells inhibited proliferation and activated cell death pathways
Cancer Research (2007) 67:

45. Expression of miR-378 Promotes Tumorigenesis and Angiogenesis
Tumor growth
Capillary formation
Tumor formation and angiogenesis affected by miR-378 over expression. (A) U87 cells transfected with miR-378 or a control vector were injected s.c. into nude mice. (Left) Four weeks after the injection, mice were photographed and killed. (Right) Tumor sizes were measured and tumor growth curves were obtained. Asterisks indicate significant differences. *, P Error bars indicate SD (n 3). (B) Tumors formed by cells transfected with the miR-378 construct or a control vector were subjected to immunohistochemistry probed with anti-CD34 antibody. Expression of the miR-378 construct elevated sizes of blood vessels. (C) Expression of miR-378 was analyzed by real-time PCR in RNAs isolated from papillary thyroid tumor and adjacent normal tissue.
U87 cells  transfect with miR-378 exp. Vector  tumor xenograft model
PNAS (2007) 104:

46. microRNAs regulate tumor angiogenesis
Pro-angiogenic microRNAs
miR cluster: TSP-1, CTGF
miR-378: Sufu (suppressor of fused)
Let-7f
Anti-angiogenic microRNAs
miR-221 and miR-222: c-Kit and eNOS
miR-15 and miR-16: VEGF and Bcl-2
miR-20a and -20b: VEGF and Bcl-2

47. Identification of miRNA involved in cell migration and invasion
Migration and invasion assays
Identification of human miRNAs that induce cell migration and
invasion. (a) General scheme of a genetic screen for miRNAs that promote
cell migration. (b) Identification of miRNA-expressing vectors enriched in the
migrating population. Left, a barcode array experiment comparing migrating cells
and total population. Right, direct sequencing of 24 miR-Vec inserts obtained
by PCR from genomic DNA isolated from migrating cells. (c, d) MCF-7 cells
stably expressing miR-373, mutant miR-373, miR-520c and control miRNAs,
or knockdown constructs targeting p53, LATS2 or non-target control, were
subjected to migration (c) and invasion (d) assays. (e) The expression level of
miR-373 in human cancer cell lines was determined by quantitative RT-PCR.
(f, g) MDA-MB-435 and HCT15 were subjected to cell migration assay following
treatment with the indicated antagomiRs
Nature Cell Biol (2007)

48. miR-373 and miR-520c promote tumor metastasis in vivo
Human miR-373 and miR-520c promote tumour metastasis in vivo.
(a) MCF-7 cells stably expressing luciferase and either miR-373, miR-520c
or a vector control were transplanted into SCID mice via tail vein injection.
Mice were imaged 6–8 weeks after transplantation. Bone metastases in skull
and pulmonary metastases were observed in MCF-7 cells expressing miR-373
or miR-520c. Dissection of mice that were transplanted with control MCF-7
cells expressing luciferase did not show metastases in secondary organs
Nature Cell Biol (2007)

49. miR-10b is highly expressed in metastatic breast cancer cells
miR-10b is highly expressed in metastatic breast cancer cells and
positively regulates cell migration and invasion. a, RT–PCR of miR-155,
miR-9 and miR-10b in a series of human mammary epithelial cells. b, c, Realtime
RT–PCR of miR-9 (b) and miR-10b (c) in four different human
mammary epithelial cells. d, e, Transwell migration assay (d) and Matrigel
invasion assay (e) of MDA-MB-231 cells transfected with the inhibitor for
miR-9 (anti-miR-9) or miR-10b (anti-miR-10b) (quantified below).
Magnification in e, f, RT–PCR of miR-10b in HMECs and SUM149
cells infected with the miR-10b-expressing or empty vector. g, h, Transwell
migration assay and Matrigel invasion assay of HMECs (g) and SUM149
cells (h) infected with the miR-10b-expressing or empty vector.
Ref: 1789_8713

50. miR-10b induced tumor metastasis in vivo
MECA-32- and Ki-67-
stained sections of a primary mammary tumour formed by miR-10btransduced
SUM149 cells, at week 6 after orthotopic transplantation. Red
arrows in panel A indicate tumour cells within a vessel, and black arrows in
panel D indicate endothelial cells. Magnification: panels A and C, 3100;
panels B and D: b, H&E- and AE1/AE3-stained sections of lungs
isolated from mice that received orthotopic injection of miR-10btransduced
or mock-infected SUM149 cells, at week 9 after transplantation.
Circles indicate clusters of metastatic cells. The arrow indicates normal
bronchial epithelium. Inset, AE1/AE3 staining of a SUM149 primary
tumour. Magnification, c, Numbers of lung micrometastases
(micromets) per section in individual mice that received orthotopic

51. HOXD10 transcription factor is a down-stream target of miR-10b
Ref: 1789_8713

52. Potential mechanisms that link microRNA to diseases
Genomic alteration of microRNA
Chromosome deletion, amplification, and translocation
Single nucleotide polymorphism of miRNA or miRNA targets
Alteration on the expression of levels of miRNA
Transcriptional control: transcription factor, enhancer, repressor
Epigenetic modification: DNA methylation, histone acetylation
Alteration on the processes of microRNA biogenesis

53. Mechanisms that link microRNA to diseases
Change in miRNA expression levels
Change in miRNA target spectrum

54. Regulatory mechanism in miRNA expression
Regulate by transcription factors
P53, c-myc, AP1, NFkB pathway ---
Regulate epigenetic mechanisms
DNA methylation
Histone modification
Regulate at miRNA processing

55. Myc regulates the expression of miR-17-92 cluster
Cancer Research (2008) 68:

56. Nature Rev Cancer (2007)

57. Regulation of miRNA expression: DNA methylation in CpG Islands
Coding region
Promoter
Exon 1
Exon 2
= CpG
CpG Island
Definition
At least 200 bases long
G+C content: > 55%
observed CpG/expected CpG ratio: >= 0.65
There are about 29,000 such regions in the human genome
65% protein-coding genes contain CpG islands in promoter region

58. microRNAs regulated by promoter methylation
Expression of miRNA also regulated by other epigenetic mechanisms