MicroRNA Biogenesis Dr. V. Narry Kim

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1 MicroRNA Biogenesis V. Narry Kim Seoul National University 1 What is a microrna (mirna)? A mirna is defined as a single-stranded RNA of ~22 nt, which is generated by the RNase III-type enzyme from an endogenous transcript that contains a local hairpin structure 2 What do mirnas do? mirnas acts as guide molecules in post-transcriptional gene silencing Each mirna directly regulates hundreds of target mrnas, sometimes in combinati on with other mirnas. In humans, over 30% of protein-coding genes may be regulated directly by mirnas mirnas control diverse pathways: devel opm ent, cell differentiati on, cell proliferati on, apoptosis, hormone secretion, stem cell maintenance, and tumorigenesis 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Function of animal micrornas Kim et al., MicroRNA biogenesis: coordinated cropping and dicing. Nature Reviews Molecular Cell Biology, Key protein factors in small RNA pathways There are two RNase III subfamilies involved in small RNA pathways: Drosha subfamily (class II) and Dicer subfamily (class III) Drosha initiates mirna maturation by cleaving the primary transcript of mirna. Drosha is conserved only among metazoans and only one Drosha homologue is found in each animal species Dicer cleaves dsrna precursors into nt RNA duplexes. Dicer is a highly conserved protein with one Dicer homologue in fission yeast (Dcr), one in human (Dicer), one in nematode worm (DCR-1), two in Drosophila (DCR-1 and DCR-2), and four in Arabidopsis (DCL1, DCL2, DCL3, DCL4) 5 Key protein factors in small RNA pathways Argonaute (Ago) proteins interact directly with small RNAs and form the effector complexes, known as the RISC (RNA-induced silencing complex) The PAZ domain consisting of ~130 amino acids interacts with the 3 overhang of dsrna The PIWI domain containing ~300 amino acids binds to the 5 end of mirna and exhibits structural homology to RNase H. Human Ago2 has the slicer activity that cleaves target mrna There exsit one Ago in S. pombe (Ago1), more than 20 in C. elegans, five in Drosophila and eight in humans, and at least ten in Arabidopsis 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Key protein factors in small RNA pathways Proteins containing dsrna-bindi ng domains (dsrbd) interact with other essential factors such as RNase III and Ago to assist their functions DGCR8, for exampl e, interacts with Drosha and functions as an essential cofactor in the initiation of mirna processing R2D2, forms a tight complex with DCR-2 and functions in strand selection and RISC assembly 7 Key protein factors in small RNA pathways A number of proteins containing the DEAD-box helicase motif have been identified as factors required for small RNA-mediated gene silencing. The biochemical roles of these helicase proteins remain unclear Many additional factors are known to be involved in small RNA pathways, including FMR, VIG, Gemin3, Gemin4, and HEN1 8 mirna vs. sirna microrna (mirna) Small interfering RNA (sirna) 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 mirna vs. sirna mirnas differ from small interfering RNAs (sirnas) in the origin. mirnas originate from hairpin-shaped precursors whereas sirnas are produced from long dsrnas Both mirnas and sirnas induce translational repression and mrna cleavag e Similar set of protein factors are involved in mirna pathway and sirna pathway In humans, the mirisc is indistinguishabl e from the sirisc. In flies and plants, different homologues are often assigned to take on different roles. For instance, fly AGO1 functions in the mirna pathway while fly AGO2 is critical in sirna pathway In mammalian cells, most of the known endogenous small RNAs belong to the class of microrna 10 Stats of mirna (as of Nov. 2005) 326 mirnas are currently annotated in humans Bioinformatic estimates of human mirnas are over 800 Over 30% of worm mirnas are conserved in mammals microrna database: mirbase er.ac.uk/ 11 How to find a new mirna? 1. Forward genetic screening Random mutagenesis and screening for mutants with a particular phenotype is followed by the search for the mutated gene locus that is responsi bl e for the altered phenotype The first mirnas, lin-4 and let-7, were discover ed by this approach 2. cdna cloning Size fractionated RNAs are ligated to the adapters at the 5 and 3 ends before they are amplified by RT-PCR and sequenced Hundreds of mirnas were discovered by this method 3. Computational prediction Conserved sequences that can form a stem-loop are potential gene loci for mirnas Experimental validation (usually by Northern blotting) is required 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Genomic location and gene structure Some mirna loci are found in non-protei n coding transcription units whereas some are located in protein coding genes More than half of the human mirna loci are found in introns 13 Gene structure and transcription mirna genes are transcribed by RNA polymerase II (pol II) Pol II transcription factors participate in mirna gene regulation, allowing elaborate control. For instance, c-myc transacti vates the mir-17 gene Approximatel y half of mirna genes are found in clusters. Clustered mirna genes constitute a polycistronic transcripti on unit mir-37 mir-38 mir-39 mir-40 mir-35 mir mirna gene microrna maturation in animals Transcription RNA polymerase II pri-mirna Cropping pre-mirna Drosha-DGCR8 (Microprocessor) Export Exportin 5 Dicing Dicer Strand selection & RISC assembly Ago2 TRBP Mature mirna in mirisc Nucleus Cytoplasm 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Nuclear processing (cropping) of primary microrna (pri-mirna) The primary transcript for mirna is abbreviated as pri-mirna Pri-miRNAs are usually over several kilobases long and contain a 7-methyl guanosine cap and a poly(a) tail 16 Nuclear processing (cropping) by Drosha The hairpin embedded in pri-mirna is released to generate the intermediate, pre-mirna, by the nuclear RNase III protein, Drosha Drosha interacts with a dsrna-bindi ng protein, DGCR8, to constitute a complex called Microprocesso r Homologues of Drosha and DGCR8 are found in animals but not in plants or fungi. DGCR8 homologues are called Pasha in worms and flies (Lee et al., 2002) (Deli et al., 2004; Gregory et al., 2004; Han et al., 2002) 17 Substrate recognition and cleavage site selection by Microprocessor Microprocessor (Drosha-DGCR8 complex) Pri-miRNAs are typically consists of a stem of 3 helical turns and Drosha cleaves at ~2 helical turns from the terminal loop The mechanism of substrate recognition and cleavag e site selection remains unclear (Han et al., Genes and Dev, 2004; Zeng et al., EMBO J. 2005) 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Nuclear export by exportin 5 (Exp5) Pre-miRNA is recognized and exported to the cytoplasm by exportin 5 (Exp5) Exp5 belongs to the Ran-dependent nuclear transport receptor family. Exp5 forms an export complex together with pre-mirna and a small GTPase, Ran (Lund et al., 2003; Yi et al., 2003) 19 Exportin 5 recognition motif Exp5 recognizes the minihelix motif which comprises of a stem of ~14 bp and a short 3 overhang Minihelix motif = ~14-nt stem + short 3 overhang Pre-miRNAs are the major natural cargos for Exp5 (Gwizdek et al., 2001) (Lund et al., 2003; Lee et al., 2003) Artificial small hairpin RNAs (shrnas) can be transported by Exp5 (Yi et al., 2003; Brummelkamp et al., 2002) 20 Cytoplasmic processing (dicing) by Dicer Pre-miRNA is processed into the ~22 nt mirna duplex in the cytoplasm The cleavag e reaction is carried out by the cytoplasmic RNase III protein, Dicer Homologues of Dicer are found in all eukaryotic species. Some species have multiple Dicer homologues that play differentiated roles. For instance, Drosophil a DCR1 cleaves pre-mirna while DCR2 dices long dsrnas (Kim, Nature Reviews Molecular Cell Biology, 2005) 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 Partnership between RNase III and dsrbd proteins Like Drosha, Dicer homologues interact with dsrna-binding proteins Function of Dicer cofactors are highly variabl e Human Dicer TRBP Drosophila Dicer-2 R2D2 Strand selection, sirna pathway Dicer-1 Loquacious/R3D1 dsrbd dsrbd dsrbd Pre-miRNA processing C. elegans dsrbd dsrbd dsrbd dsrbd dsrbd mirna accumulation, RNAi process Dicer RDE4 dsrbd dsrbd sirna accumulation Plant DCL-1 HYL1 dsrbd dsrbd mirna accumulation 22 Cleavage site selection by Dicer Dicer cleaves at ~22 nt from the terminus The PAZ domain of Dicer interacts with the 3 overhang. So it is believed that the PAZ domain holds the terminus and helps the processing center locate at ~22 nt from the terminus (Zhang and Filipowicz, Cell, 2004) 23 RNase III type proteins RNase III introduces a staggered cut into dsrna, leaving a ~2 nt 3 overhang in the product. The products have a 5 phosphate group and a 3 hydroxyl group 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 RNase III type proteins RNase III contains at least one conser ved catalytic domain (RIIID) RNase III types proteins can be grouped into three classes based on the domain organization Classes Class I Class II (Drosha) Class III (Dicer) Domain organization RIIID dsrbd RIIIDa RIIIDb dsrbd Helicase PAZ RIIIDa RIIIDb dsrbd Dimerization Intermolecular Intramolecular Intramolecular 25 Tandem RIIIDs form intramolecular dimer The RIIIDa cleaves the 3 strand while the RIIIDb cuts the 5 strand 26 Coordinated events in mirna maturation The 2 nt 3 overhang serves as a signature motif for RNAs involved in small RNA pathway Cleavage at ~22-nt from the bottom end Control of microrna biogenesis may be possible at multiple steps 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 Usually only one strand of the mirna hairpin is selected for mature mirna while the other strand is discarded The strand whose 5 end is less stable is selected over the other stand In flies, the dsrna-bindi ng protein R2D2 senses the relative stability of the ends of sirna duplex Ago2 binds to the guide strand and destroys the passenger strand Strand selection Mechanism for mirna strand selection is unclear. The slicer activity of Ago2 is unlikely involved in the removal of the passenger strand 28 mirisc assembly A number of proteins have been identified as components of the RISC or RISC loading complex (RLC). But the key components are Ago, Dicer, and dsrbd proteins In flies, Loqs/R3D1 participates in both pre-mirna cleavage and mirisc assembly In humans, TRBP facilitates RISC assembly. Dicer, TRBP and hago2 constitute a complex that is responsibl e for both dicing and RISC assembly (Zamore and Haley, Science, 2005) 29 Regulation of mirna expression Transcriptional control Post-transcripti onal control 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 How to determine mirna levels in cells Method Detection target Sensitivity Note Northern blotting RT-PCR Primer extension Microarray Mature mirna and pre-mirna Mature mirna, pre-mirna, pri-mirna (depending on primer design) Mature mirna and pre-mirna (depending on primer design) Mature mirna, some pre-mirna Good for most purposes (quantitation, validation, routine analysis) Recommendable for pri-mirna detection; Also suitable if the sample is limited Suitable for the quantitation of mature mirna Recommendable for high throughput experiments Bead-based flow cytometry Mature mirna, some pre-mirna + Adequate for high throughput experiments 31 mirna expression profiling - 1 A number of mirna genes are expressed in a spatially and/or temporally regulated manner Liver-specific expression Brain-specific expression Genomics of MicroRNA (Kim et al., Trends in Genetics 2005) 32 mirna expression profiling - 2 Strong correlations between mirna expressi on and diseased status of the cells have been observed MicroRNA expression profiles classify human cancers. (Golubet al., Nature. 2005) 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 mirna gene mirna biogenesis in plants Transcription RNA polymerase II pri-mirna Dicer-like 1 (DCL1) HYL1 Processing Export Hasty (HST) Strand selection & RISC assembly Mature mirna AGO1 in mirisc HEN1 Nucleus Cytoplasm 34 Generation of shrnas mirna gene shrna cassette (pol II) shrna cassette (pol III) Understanding mirna biogenesis is important not only to dissect the biology of mirnas but also to improve RNAi technology mirna biogenesis factors are required for producti on of sirnas from small hairpin RNAs (shrnas) Drosha-DGCR8 Mature mirna (mirisc) sirna (sirisc) The screen versions of these slides have full details of copyright and acknowledgements 12