microRNAs Small Non-coding RNAs with Big Impact in Biology

1. microRNAsSmall 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 Warm Yeast Human • 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? Genome: 15Mb Genes: 6,000 Cell: 1 Genome: 100Mb Genes: 18,500 Cell: 1,100 Genome: 3,200Mb Genes: 25,000 Cell: 1X1012

4. The proportion of noncoding DNA broadly increases with developmental complexity Nature Rev. Genetics (2004) 5: 316-323

5. Type of RNA molecules RNA mRNA Protein-coding RNA ncRNA: non-coding RNAs Transcribed RNA with a structural, functional or catalytic role tRNA Transfer RNA Interface between mRNA & amino acids snRNA Small nuclear RNA RNA thatform 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 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. lin-4 precursor lin-4 RNA target mRNA “Translational repression” C. elegans lin-4 : first identified microRNA 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).

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: 901-906

8. Let-7 sequence and gene regulation Nature (2000) 403: 901-906

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 40 (1) From miRBase Release 13.0 (Mar 2008)

11. Gene regulation by transcription factors and microRNAs Transcription factors microRNAs

12. microRNA Biogenesis

13. microRNA biogenesis  Transcription  Processing  Maturation  Execution Nature Rev. Immunology (2008) 8: 120-130

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 • Transcribed by RNA polymerase II • 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 • 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? 1,374 aa

17. 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. Multiple mechanisms for miRNA processing

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 RIIIDa RIIIDb dsRBD PAZ • 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? 1,922 aa

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 silencingAGO2-mediated RNA degradation

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 12-17 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 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 Nature (2005) 435 : 834-838

41. C-myc induces expression of the miR-17/92 cluster Tet-off system to induce c-myc expression in P493 cells Nature (2005) 435 : 839-843

42. miR-17/92 cluster showed increase expression in B lymphoma and colon cancers Nature (2005) 435 : 828-833

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 Nature (2005) 435 : 828-833

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: 11099-11101

45. Expression of miR-378 Promotes Tumorigenesis and Angiogenesis Tumor growth Capillary formation U87 cells  transfect with miR-378 exp. Vector  tumor xenograft model PNAS (2007) 104: 20350-20355

46. microRNAs regulate tumor angiogenesis • Pro-angiogenic microRNAs • miR-17-92 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 Nature Cell Biol (2007)

48. miR-373 and miR-520c promote tumor metastasis in vivo Nature Cell Biol (2007)

49. miR-10b is highly expressed in metastatic breast cancer cells Ref: 1789_8713

50. miR-10b induced tumor metastasis in vivo