1. Small non-coding RNA with Big Impact in Biology
microRNA (miRNA)
Small non-coding RNA with Big Impact in Biology
Hua-Chien Chen Ph.D

2. Type of RNA molecules RNA ncRNA mRNA snRNA snoRNA tRNA RNAi Other rRNA
Protein-coding RNA
ncRNA
Non-coding RNA. 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
Incl. 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 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

3. The Nobel Prize in Physiology or Medicine 2006
Andrew Z. Fire and Craig C. Mello
for their discovery of "RNA interference – gene silencing by double-stranded RNA"

4. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Nature (1998)391:806-11
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process

5. RNA interference pathway
Exogenous dsRNA, transposon, viral products, etc.
dsRNA
Dicer
siRNAs
RISC
Target genes
mRNA cleavage, degradation

6. Major differences between siRNA and microRNA
miRNA: microRNA, nt
Encoded by endogenous genes
ssRNA with stem-loop structure
Partial complement to the 3’UTR of target genes
Recognize multiple targets
siRNA: short-interfering RNA, nt
Mostly exogenous origin
dsRNA precursors
May be target specific

7. miRNA and RNAi pathways
microRNA pathway
RNAi pathway
Exogenous dsRNA, transposon, etc.
MicroRNA primary transcript
Drosha
precursor
Dicer
Dicer
siRNAs
miRNA
target mRNA
RISC
RISC
RISC
“translational repression”
and/or mRNA degradation
mRNA cleavage, degradation

8. C. elegans lin-4 : first identified microRNA
lin-4 precursor
lin-4 RNA
target mRNA
V. Ambros lab
“Translational repression”
lin-4 RNA
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.
1993 Victor Ambros (Dartmouth) and colleagues showed that lin-4, a gene that controls developmental timing in C. elegans encodes two small RNA molecules and not protein
lin-4 small RNA gene product showed sequence complementarity to multiple sites on 3’ UTR of lin-14
Lin-4 inhibits lin-14 protein synthesis after the initiation of translation (1999)
At the time this mechanism was believed to be exclusive to nematodes

9. 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

10. Nature (2000)

11. microRNAs at a glance miRNA 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 miRNAs

12. More than 4,000 miRNAs in public databases
Homo sapiens (541)
Mus musculus (443)
Rattus norvegicus (287)
Drosophila melanogaster (152)
Caenorhabditis elegans (137)
Arabidopsis thaliana (184)
Epstein Barr virus (23)
Human cytomegalovirus (11)
Kaposi sarcoma-associated herpesvirus (13)
Simian virus (1)
From miRBase Release 10.1 (Dec 2007)

13. MicroRNA Biogenesis and Mechanism of Action

14. Summary of microRNA biogenesis
Dicer

15. microRNA biogenesis
MicroRNA (miRNA) genes are generally transcribed by RNA Polymerase II (Pol II) in the nucleus to form large pri-miRNA transcripts, which are capped (7MGpppG) and polyadenylated (AAAAA).
These pri-miRNA transcripts are processed by the RNase III enzyme Drosha and its co-factor, Pasha, to release the ~70-nucleotide pre-miRNA precursor product.
RAN–GTP and exportin 5 transport the pre-miRNA into the cytoplasm
Subsequently, another RNase III enzyme, Dicer, processes the pre-miRNA to generate a transient ~22- nucleotide miRNA:miRNA* duplex.

16. microRNA biogenesis
This duplex is then loaded into the miRNA-associated multiprotein RNA-induced silencing complex (miRISC), which includes the Argonaute proteins, and the mature single-stranded miRNA
The mature miRNA then binds to complementary sites in the mRNA target to negatively regulate gene expression in one of two ways that depend on the degree of complementarity between the miRNA and its target.
mRNA degradation
Translational repression

17. miRNA biogenesis player: Drosha
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?

18. miRNA biogenesis player: 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?

19. 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

20. Exporting of microRNA
The pre-miRNA with its typical ~2 nucleotide overhang at its 3′end is specifically recognized by exportin‑5 and is transported to the cytoplasm, where it dissociates from its receptor after RanGTP hydrolysis.

21. microRNA mediated gene silencing
miRNA
miRNA
Translational repression
mRNA degradation

22. microRNA-mediated mRNA Degradation
Contains a member of the argonaute family
Between 130 kDa and 500 kDa
Other components are being characterized
Cleaves RNA complementary to the siRNA, in the middle of the sequence
Assembling the RISC complex requires ATP, while RNA cleavage does not.
Novina and Sharp, 2004c

23. microRNA-mediated translational repression
Imperfect match between miRNA in RISC and target mRNAs
RISC usually binds 3’ UTR
Mechanism of inhibition... ????
He and Hannon, 2004

24. Processing bodies
microRNA-mediated mRNA degradation and translational repression are converge in P-body

25. From base pairing to gene silencing

26. 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

27. Two classes of microRNA binding sites in animal

28. Biological Functions

29. 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

30. 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

31. microRNAs and cardiogenesis
microRNA-1-1 (miR-1-1) and miR-1-2 are specifically expressed in cardiac and skeletal muscle precursor cells.
miR-1 genes are direct transcriptional targets of muscle differentiation regulators including serum response factor, MyoD and Mef2.
Hand2, a transcription factor that promotes ventricular cardiomyocyte expansion, is a target of miR-1
Zhao et al. Nature 2005

33. microRNA promotes photoreceptor differentiation
miR-7 promotes photoreceptor development.

34. Genomic Localization of EBV-miRNAs
BHRF-1-1
BHRF-1-2
BHRF-1-3
BART-3, 4, 1, 15, BART-5, 16, 17, 6
BART-2
BART-18, 7, 8, 9, 10, 11
-12, 19, 20, 13, 14
EBV microRNAs were first discovered by Pfeffer et al in hairpins with 6 mature miRNAs were cloned from B95.8, including 3 BHRFs and 2 BARTs (Science 304:734, 2004). All 6 EBV miRNAs were expressed by EBV+ lymphoma cells and the expression seem to be related to the latency of EBV.
Cai et al reported at least 17 EBV microRNAs by cloning using BC-1 cells (PLos Pathogens 3:e23, 2006). These miRNAs were encoded by the B95.8-deleted region.
Grundoff et al combined a computational and microarray-based approach and reported 18 new EBV microRNAs in Jijoye cells (RNA 12:1-18, 2006). Some of these predicted sequences were confirmed by Northern. Mature region was not fully mapped.
Kim et al evaluated the expression of selected EBV microRNAs in EBV-associated gastric carcinoma (J Virol 81:1033, 2007). BART microRNAs but not BHRF miRs were expressed in EBV+ GCs.
BHRF1 cluster
- span 1.5 kb
- 3 precursor
- 4 mature miRs
BART1 cluster
- span 1.0 kb
8 precursors
12 mature miRs
BART7 cluster
- span 2.8 kb
11 precursors
15 mature miRs
96 kb
5.9 kb
3.9 kb
BART2 cluster
1 precursors
1 mature miR

35. microRNA and Cancer

36. Mechanisms that link microRNA to disease
Change in miRNA expression levels
Change in miRNA target spectrum

37. miRNA frequently located at chromosome fragile sites

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

39. miR-17-92 cluster is over-expressed in human lung cancer
miR cluster (containing miR-19a and miR-20) is markedly overexpressed in lung cancer cell lines
Figure 1. Search for miRNAs with altered expression in lung cancers.
A, Northern blot analysis of miRNAs in lung cancer cell lines. Note marked
overexpression of miR-19a, miR-20, miR-106a, and miR-106b. Normal Lung,
a mixture of RNAs from 11 normal lung tissues; BEAS-2B and HPL1D, two
immortalized human epithelial cell lines representing proximal and distal airway
cells, respectively. B, unsupervised hierarchical clustering analysis highlighting
overexpression of miR-19a, miR-20, miR-106a, and miR-106b mainly in
small-cell lung cancer cell lines (red). Blue, normal lung tissues and the two
immortalized human epithelial cell lines, BEAS-2B and HPL1D.
Cancer Research (2005) 65 : 9628

40. 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
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 : 828

41. miR-34 and p53 network
miR‑34 is a direct transcriptional target of p53, which in turn downregulates genes required for proliferation and survival.
Along with other p53 targets, such as p21 and BAX, miR‑34-family miRNAs promote growth arrest and cell death in response to cancer related stress.

42. microRNAs associated with human cancer
TS : tumor suppressor
OG : oncogene

43. microRNAs are oncogenes or tumor suppressors
microRNAs down-regulated in tumor
microRNAs up-regulated in tumor

44. Hierarchical clustering analysis of microRNA expression profiles in 59 tumor-derived cell lines
Expression levels of majority microRNAs are down-regulated in tumor cells

45. Comparison of dendrograms derived from hierarchical clustering of miRNA and mRNA expression profiles in NCI60 cell lines
miRNA
mRNA

46. Hierarchical clustering of miRNA expression
Clustering of 73 bone marrow samples from patients with acute lymphoblastic leukaemia
(ALL). Coloured bars indicate the different ALL subtypes.
Samples from colon, liver, pancreas and stomach all clustered together in 214 miRNA profiling, reflecting their common derivation from tissues of embryonic endoderm
A 16,000 mRNA profiling of the same samples failed to observe the coherence of gut derived sample in clustering
Nature (2005) 435 :

47. Global miRNA change during tumorigenesis
Human samples
K-ras mice
Figure 3 | Comparison between normal and tumour samples reveals global
changes in miRNA expression. a, Markers were selected to correlate with
the normal versus tumour distinction. A heatmap of miRNA expression is
shown, with miRNAs sorted according to the variance-thresholded t-test
score. b, miRNA markers of normal versus tumour distinction in human
tissues from a, applied to normal lungs and lung adenocarcinomas of
K-RasLA1 mice. A k-nearest neighbour (kNN) classifier based on human
sample-derived markers yielded a perfect classification of the mouse samples
(euclidean distance, k ¼ 3). Mouse tumour T_MLUNG_5 (third column
from right) was occasionally classified as normal using other kNN
parameters (see Supplementary Information).
Most of the miRNAs (129 out of 217) had lower expression levels in human tumors compared with normal tissues, irrespective of cell type
Cancer cell lines also have lower miRNA levels
A tumor/normal classifier constructed using human sample had 100% accuracy when tested in the mouse
Nature (2005) 435 :

48. A MicroRNA Signature Associated with Prognosis and Progression in Chronic Lymphocytic Leukemia
Expression profile of 13 miRNA represents the patient’s prognosis
N Engl J Med (2005) 353 : 1793

49. Detection of microRNA

50. Technologies for microRNA detection
Solution phase hybridization
RNase protection assay
Splint ligation method
Solid phase hybridization
Northern hybridization
Microarray technology
Bead-base technology
Real-time PCR amplification
Precursor detection
Primer extension
Stem-loop RT primer

51. Solution phase hybridization : Splint ligation
RNA (2007) 13: 1-7

52. Solution phase hybridization : Splint ligation
RNA (2007) 13: 1-7

53. Array-based miRNA detection
Total RNA vs purified small RNA
RNA (2007) 13:

54. Array-based miRNA detection
Specificity
Sensitivity & dynamic range
RNA (2007) 13:

55. qPCR-based miRNA detection
Nucleic Acids Research (2005) 33: e179

56. microRNA-related Database

57. miRNA target prediction programs

58. microRNA database: miRBase

59. microRNA database: miRBase
(Precursor and mature miRNA sequence)
(Transcript)
(Chromosome)
(Cluster)

61. miRBase: target prediction