Yang-Ming University, 2009 microrna Biology and Application 3/03 microrna biogenesis and functions Woan-Yuh Tarn 3/10 micrornas and development Woan- Yuh Tarn 3/17 micrornas and nervous system Jeng-Ya Yu 3/24 Plant micrornas Tzyy-Jen Chiou 3/31 microrna bioinformatics Wen-chang Lin 4/07 micrornas in diseases Woan- Yuh Tarn 4/14 micrornas in cancer Jeng-Ya Yu 4/21 Application of Small RNA Jeng-Ya Yu 4/28 Discussion Woan-Yuh Tarn/ Jan-Ya Yu
microrna biogenesis and functions Outline 1) Introduction 2) Discovery of mirnas 3) mirna classification 4) Biogenesis of micrornas 5) Distinct pathways for assembly of Ago-miRNA complexes 6) Alternative pathways for generation of small RNAs 7) RNA editing controls mirna biogenesis 8) Specific cellular localization of mirnps 9) Viral micrornas 10) Mechanisms of mi/sirna functions
1) Introdcution Non-coding regulatory RNAs localization function snrna nucleoplasm splicing snorna nucleoli guide 2 -O-methylation and pseudouridylation scarna Cajal bodies similar to snorna Telomerase RNA Cajal bodies telomere DNA synthesis 7SK RNA nucleoplasm transcription regulation Xist and Tisx X chromosome dosage compensation Micro/siRNA cytoplasm mrna degradation and translational control chromatin transcriptional silencing micrornas are expressed in fission yeast, ciliates, fungi, flies, worms, plants and higher eukaryotes. mirnas are predicted to regulate 30% of mammalian protein-encoding genes by interactions with their 3' untranslated regions (UTRs).
2) Discovery of mirnas sirnas 1990: Transgenic introduction of a gene silenced endogenous gene expression in plant (Petunia). Mechanism: Introduced dsrna is processed by Dicer into a 21-23 nt small interfering RNA (sirna). Dicer (RNase III-like RNase) plays a role in post-transcriptional gene silencing (PTGS) or transgene quelling. micrornas regulate developmental timing (heterochronic gene pathway) in C. elegans. C. elegans lin-4 (identified in 1993) controls developmental timing. Andrew Fire and Craig Mello (2006 Nobel Prize in Physiology or Medicine) reported in 1998 that small regulatory RNAs (micrornas) interfere with target gene expression. [Potent and specific genetic interference by double-stranded RNA in C. elegans. Nature (1998) 391:806-11] Also found that double-stranded RNA mixtures caused potent and specific interference in animals. First identified microrna lin-4
3) microrna classification sirna Small interfering RNA (20-24 nt) from invasive nucleic acids (viruses or foreign genes introduced for experiemntal and clinical purposes, or from other environmental sources) perfect complementarity to their mrna targets targeted sites may disperse throughout the entire transcripts primarily cause mrna degradation mirna microrna (20-24 nt) most mammalian mirnas contain mismatches usually target untranslated regions of mrnas primarily cause translational suppression or can also facilitate RNA degradation if not perfectly match to the target rasirna Repeat-associated small interfering RNA (24-29 nt) derived from repetitive elements within the genome (heterochromatin regions including centromeres and telomeres, and rertotransposons). arise mainly from the antisense strand cause transcriptional silencing via chromatin remodeling pirna Piwi-interacting RNA (26-31 nt) in the germ line associated with Piwi predicted to have function in gametogenesis
tasirna trans-acting RNA (24-29 nt) in plant endogenous sirnas derived from noncoding transcripts that are cleaved by a microrna (mirna) facilitate protein-coding mrna degradation function in plant stress responses similar sirnas (tiny noncoding RNA) found in C. elegans mirtrons short hairpin introns provide an alternative source for animal microrna biogenesis and use the splicing machinery to bypass Drosha cleavage in initial maturation. Mol Cell (2007) 28, 328-36. LINE (L1) retrotransposons can generate dsrnas from both sense and antisense promoters in germ cells. These dsrnas can be processed into sirnas. L1-specific sirnas target to the 5 UTR of L1 transcripts and cause its degradation. Thus, sirnas suppress L1 retrotransposition in germ cells. Nature Stru. Mol. Biol. (06) 13, 758-9
4) Biogenesis of micrornas Primary mirnas (pri-mirnas) are probably transcribed by RNA polymerase II. The primary transcripts may be polycistronic (host several micrornas), and can be spliced and polyadenylated. In the nucleus, pri-mirnas are processed into ~70-nt pre-mirnas by the complex containing Drosha (RNase III-like nuclease) and Pasha/DGCR8 (dsrna-binding protein). Nature (03) 425, 415-9. Drosha/DGCR8 (microprocessor): DGCR8 binds junction with more flexible tails and appropriately posits the processing center of Drosha to creat ~65nt long pre-mirna. Ref: Nat Rev Genet (04) 5, 522-531; Nat Rev Mol Cell Biol (05) 6, 376-85
Pre-miRNAs is exported by importin-β family member exportin-5 in a RanGTP-dependent manner. Genes Dev. (03) 17, 3011-6. Pre-miRNA is subsequently processed into mirna duplex by Dicer. The RISC-loading complex (RLC) (in Drosophila and mammalian cells): Dicer/R2D2 (dsrna binding protein) binds the double-stranded-(si/mi)rna through its interaction with the 5 phosphorylated end of RNA. (R2D2: Drosophila) Note: Plant pri-mirnas and pre-mirnas are both processed by Dcl1 in the nucleus. The processed duplex mirnas are exported to the cytoplasm. The PAZ domain of Dicer (molecular ruler) binds the end of dsrna and the binding surface matches the length of 25 bp dsrna (a mirna:mirna* duplex). The RNase III domains cleave the precursor RNA into 25-26 nt from the 3 end of the opposite strand and both strands can generate ssrna. The 3 2-nt overhang facilitates proper processing. Nat. Struct. Mol. Biol. (07) 14, 934-40; Cell (09) 136, 642-655
The dsrna unwinds from the less stable end associated with Dicer. The passenger stand is ejected and degraded. As unwinding proceeds, Argonaute replaces Dicer/R2D2 (in mammalian cells) to interact with the sirna and form the functional RNA-induced silencing complexes (RISCs). [Note that: In Drosophila, an 80S holo-risc is found to contain Ago2, Dicer-2, R2D2 and other proteins.] Ago proteins Ago proteins are critical RNA silencing effectors. The Mid domain and the PAZ domain interact with the 5 end and the 3 end of the guide strand mi/sirna, respectively. The Piwi domain contains the target cleavage site. The mismatch between si/mirna and the target inhibits the cleavage activity. Ago/miRNA-associated factors: GW182, Gemin3, Gemin4, Mov10 (RNA helicase), the importin-β member imp-8. [GW182 is important for the function of mirna-mediated gene silencing.]
5) Distinct pathways for assembly of Ago-miRNA complexes Drosophila micrornas sort into distinct RISCs Nature Stru. Mol. Biol. (06) 14, 684-6 Argonaute proteins contain similar domains but may have distinct activities. e.g. The PIWI domain can cleave DNA/RNA hybrids (containing the slicer catalytic site), structurally similar to ribonuclease H. Ago2 but not other members can execute cleavage of targeted mrnas. In Drosophila, most mirnas (with more mismatches and bulges) are assembled with Dcr1/loqs (loquacious), but those with less mismatches (such as mir-277) are loaded into the Ago2-RISCs via the action of Dcr2/R2D2. Mammalian Ago complexes Separation of Ago-associated mrna complexes showed that Ago1 and Ago2 reside in distinct complexes that exhibit different activities. e.g. a large Ago-2- containing complex can process mirna precursors. EMBO report (07) 8, 1052-60
6) Alternative pathways for generation of small RNAs Drosophila pirna Drosophila Piwi-associated pirnas can be derived from (1) the sense strand of retrotransposons that bind Ago3 or from (2) the antisense strand that bind AUB (Aubergine). The Ping-Pong model for generation of Piwi-associated pirnas: AGO3-piRNA and AUB-piRNA are partially complementary; AGO3-piRNAs interact with AUB-piRNA precursors and produce AUB-piRNAs, and vice versa. Therefore, pirnas can be exponential generated. Drosophila pirnas are 3 -methylated. Nat. Rev. Mol. Cell Biol. (08) 9, 2232
Plant TasiRNA Transitive RNA silencing: Primary sirnas (21-24 nt) act as primers for secondary sirnas production by RNA-dependent RNA polymerase (RDR6). However, 5 and 3 secondary sirnas are probably generated by primer-dependent and primerindependent RDR6 activities, respectively. DCL4 produces the 21-nt sirnas (in homogenous size).
mirtrons certain debranched introns mimic the structural features of pre-mirnas to enter the mirna-processing pathway without Drosha-mediated cleavage. The hairpin exported by Exportin-5, and subsequently processed by Dicer- 1/loqs. Mirtrons are originally identified in Drosophila and C.elegans. Mol Cell (2007) 28, 328-36. LINE (L1) retrotransposon can generate dsrnas from both sense and antisense promoters in germ cells. These dsrnas can be processed into sirnas. L1-specific sirnas target to the 5 UTR of L1 transcripts and cause its degradation. Thus, sirnas suppress L1 retrotransposition in germ cells. Nature Stru. Mol. Biol. (06) 13, 758-9
7) RNA editing controls mirna biogenesis ADAR (adenosine deaminase acting on RNA) converts adenosines to inosines (A to I RNA editing) in dsrnas. RNA editing can occur on mrnas and may regulate retrotransposons and gene silencing. Editing of hematopoietic cellspecific pri-mir-142 suppresses its processing by Drosha. The edited pri-mir-142 was degraded by Tudor-SN, a component of RISC and also a ribonuclease specific to inosine-containing dsrnas. Nat Struct Mol Biol (06) 13, 13-21 RNA editing may provide mechanisms for regulation of mirnas or directing mirnas to alternative targets. Nat Rev Mol Cell Biol 7(12):919-31
8) Specific cellular localization of mirnps A part of mirna/ago complexes is concentrated in cytoplasmic Processing bodies (P bodies; PBs). [Note: P bodies contain proteins that are involved in mrna degradation, translational repression, mrna surveillance and RNA-mediated gene silencing, together with their mrna targets. e.g. the decapping enzyme (Dcp1/2) and exonuclease (such as 5 to 3 exonuclase Xrn1) for mrna destruction are located in PBs.] Imp-8 is involved in targeting of the mirna/ago complex to mrnas and moves the complex to PBs. (Cell 136, 496-507; 2009) Science (05) 309, 1519-26 Upon stress induction, mirnas are (slightly) enriched at stress granules (SGs) [SGs contain several translation initiation factors and 40S ribosomal components, implicated in translational suppression.] The majority of mirna/agos and their targets are diffusely located in the cytoplasm. [perhaps for function in translational repression, deadenylation/degradation of mrnas.]
9) Viral micrornas Viral mirnas may play a role in modulating both viral and host gene expression. Cell Microbiol (07) 9, 2784-94 HIV: SV40: EBV: potentially target host genes involved in the HIV pathogenesis and establish latency help virus evasion into T cytotoxic cells by targeting early transcripts target genes that function in apoptosis and tumor suppression Mouse CMV (cytomegalovirus): expressed at early times of infection and dominate the total microrna pool KHSV (Kaposi's sarcoma-associated herpesvirus): expressed in latently infected B cells HSV (herpes simplex virus): The non-coding long RNA latency-associated transcript (LAT) produces a mirna that confers resistance to apoptosis and promotes the survival of infected neurons by downregulation of transforming growth factor (TGF)- beta 1 and SMAD3 expression. Nature (06) 442, 82-5
10) Mechanisms of mi/sirna functions Primary functions of mi/sirna in posttranscriptional gene expression: translational repression and mrna degradation. Si/miRNA-directed target RNA destruction (A) (B) The active RISC complex can target RNAs via complete complementarity to the mirna or sirna, leading to site-specific endonuclease cleavage by Ago (PIWI domain similar to ribonuclease H; termed slicer ), followed by degradation of the resulting target RNA fragments by the 5-3 exonuclease Xrn1 and the exosome (from the 3 to 5 ). When si/mirnas are partially complementary to the targets, Ago slows down translation and induces decapping by Dcp1/Dcp2 and deadenylation by the Ccr4-Caf1-NOT deadenylase complex (RNA 15, 21-32; 2009). Note: CCR4 (deadenylase); Not1 (transcriptional repressor). (A) (B) Cell 136, 642-55; 2009).
mirnp-mediated translation suppression 1) mirnps block translation initiation steps (A, B and C) (A) mirnp competes with the cap-binding protein eif4e [the MC domain of Ago binds to the mrna 5 cap structure], preventing association of eif4e/4g with PABP. (B) The human RISC associates with the 60S ribosome subunit and Ago recruits anti-association factor eif6 to prevent productive assembly of the 80S ribosome. (C) mirnps induce deadenylation of the poly(a) tail and then prevent circularization of the mrna. [eif4e/g and PABP respectively bind to the 5 and 3 of mrna during translation, which circularizes the mrna and facilitates translation efficiency.] (A) (B) (C) (D)
2) mirnps block translation after the initiation steps The ribosome drop-off hypothesis: mirnps prevent efficient translational elongation and lead to premature termination (evidence: lin-4 mirna/lin- 14 mrna is accumulated in polyribosomes, suggestion that initiation is not impaired.) [model D] 3) Drosophila mir2 prevents 60S ribosomal subunit joining and polyribosome formation but induces the formation of dense (heavier than 80S) mirnps (termed 'pseudopolysomes'). This mechanism requires the cap structure.
sirna-directed DNA silencing RNA-directed homologous DNA silencing in plants RNA signals that direct DNA methylation are produced by RdRp (RDR2) and Dicer (DCL3). These RNA signals then target DNA methyltransferases (MET1 for CG methylation and DRM2 for non-cg methylation) along with AGO4 to methylate DNA. AGO proteins may also participate in maintenance of methylation. Silencing of centromeric heterochromatin in fission yeast dsrnas are generated from centromeric tandem repeats and then processed by Dicer into sirnas. These sirnas are associated with the RITS (RNA-induced transcriptional silencing) complex containing Ago1 and Chp1 (a chromodomain protein and located in centromeres). Heterochromatin establishment: Small RNAs may guide the histone methyltransferases (HMTs) to catalyse H3 methylation at K9. Maintenance of silenced state: Swi6 (HP1; heterochomatin protein 1) joins at the silenced chromatin region
rasirna (repeat-associated small interfering RNA)-induced heterochromatin in flies and mammals. rasirna-rits formation requires Piwi and particularly participates in silencing of repetitive elements. RNA-guided heterochomatine formation: epigenetic changes: reprogram gene expression dosage compensation: equalization of transcription level from two homologous chromosomes suppression of transposable element activity Non-CG methylation, prevailing in plants, is detected in mammalian embryonic stem cells and retrotransposons, but whether this involves in small RNAs is uncertain (rasirna?). Nat. Rev. Genet. (05) 6, 24-35 Cell (05 )122:9-12
Piwi RNA (pirna) pirnas bidirectionally transcribed specifically expressed in germ cells 26-31 nt in length produced by a Dicer-independent mechanism associated with Piwi the 3' termini of mouse Piwi-interacting RNAs are 2'-O-methylated Sense pirnas may serve a function in both the nucleus and in the cytoplasm (?). Antisense 1/3pi RNAs are enriched in repeats and may regulate transposon expression. In the nucleus, pirnas mark specific genomic loci through an epigenetic mechanism or through an RNA cleavage event. In the cytoplasm, pirnas might act at the chromatoid body to degrade (transposon) RNA. Mol Cell (07) 26:603-9
pirnas may be involved in spermatogenesis Piwi (Miwi) proteins exert a pronounced role in mammalian male germ cells and are detected in the chromatoid body (perinuclear germline granules; related to the somatic P body?), and also associates with translational machinery Murine MILI Important for meiotic-progression in early prophase of meiosis I. May have a role in suppression of retrotransposable elements. Murine MIWI binds a 29-30-nucleotide testisabundant RNAs. initiates spermiogenesis, a process that transforms round spermatids into mature sperm. is required for the expression of its target mrnas involved in spermiogenesis. Genes & Dev (06) 20, 1993-7 PNAS (06) 103, 13415-20