In this module, the biology of various types of regulatory non-coding RNAs are described. Currently, biogenesis and functions of small interfering RNAs (siRNAs) and microRNAs are annotated.
View original pathway at Reactome.
Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, Kim VN.; ''MicroRNA genes are transcribed by RNA polymerase II.''; PubMedEurope PMCScholia
Taylor DW, Ma E, Shigematsu H, Cianfrocco MA, Noland CL, Nagayama K, Nogales E, Doudna JA, Wang HW.; ''Substrate-specific structural rearrangements of human Dicer.''; PubMedEurope PMCScholia
Bohnsack MT, Czaplinski K, Gorlich D.; ''Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs.''; PubMedEurope PMCScholia
Verdel A, Vavasseur A, Le Gorrec M, Touat-Todeschini L.; ''Common themes in siRNA-mediated epigenetic silencing pathways.''; PubMedEurope PMCScholia
Haase AD, Jaskiewicz L, Zhang H, Lainé S, Sack R, Gatignol A, Filipowicz W.; ''TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing.''; PubMedEurope PMCScholia
Kok KH, Ng MH, Ching YP, Jin DY.; ''Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA.''; PubMedEurope PMCScholia
Borchert GM, Lanier W, Davidson BL.; ''RNA polymerase III transcribes human microRNAs.''; PubMedEurope PMCScholia
Li LC, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, Dahiya R.; ''Small dsRNAs induce transcriptional activation in human cells.''; PubMedEurope PMCScholia
Weinberg MS, Villeneuve LM, Ehsani A, Amarzguioui M, Aagaard L, Chen ZX, Riggs AD, Rossi JJ, Morris KV.; ''The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells.''; PubMedEurope PMCScholia
La Rocca G, Olejniczak SH, González AJ, Briskin D, Vidigal JA, Spraggon L, DeMatteo RG, Radler MR, Lindsten T, Ventura A, Tuschl T, Leslie CS, Thompson CB.; ''In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA.''; PubMedEurope PMCScholia
Kabachinski G, Schwartz TU.; ''The nuclear pore complex--structure and function at a glance.''; PubMedEurope PMCScholia
Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD.; ''Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.''; PubMedEurope PMCScholia
Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R.; ''MicroRNA-373 induces expression of genes with complementary promoter sequences.''; PubMedEurope PMCScholia
Younger ST, Corey DR.; ''Transcriptional gene silencing in mammalian cells by miRNA mimics that target gene promoters.''; PubMedEurope PMCScholia
Peters L, Meister G.; ''Argonaute proteins: mediators of RNA silencing.''; PubMedEurope PMCScholia
Lund E, Güttinger S, Calado A, Dahlberg JE, Kutay U.; ''Nuclear export of microRNA precursors.''; PubMedEurope PMCScholia
Aravin AA, Sachidanandam R, Bourc'his D, Schaefer C, Pezic D, Toth KF, Bestor T, Hannon GJ.; ''A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice.''; PubMedEurope PMCScholia
Fontoura BM, Blobel G, Matunis MJ.; ''A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates Nup98 and the novel nucleoporin, Nup96.''; PubMedEurope PMCScholia
Wei JX, Yang J, Sun JF, Jia LT, Zhang Y, Zhang HZ, Li X, Meng YL, Yao LB, Yang AG.; ''Both strands of siRNA have potential to guide posttranscriptional gene silencing in mammalian cells.''; PubMedEurope PMCScholia
Nishi K, Nishi A, Nagasawa T, Ui-Tei K.; ''Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus.''; PubMedEurope PMCScholia
Ahlenstiel CL, Lim HG, Cooper DA, Ishida T, Kelleher AD, Suzuki K.; ''Direct evidence of nuclear Argonaute distribution during transcriptional silencing links the actin cytoskeleton to nuclear RNAi machinery in human cells.''; PubMedEurope PMCScholia
Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R.; ''Control of translation and mRNA degradation by miRNAs and siRNAs.''; PubMedEurope PMCScholia
Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, Ojo T, Hafner M, Zavolan M, Tuschl T.; ''Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs.''; PubMedEurope PMCScholia
Gould DW, Lukic S, Chen KC.; ''Selective constraint on copy number variation in human piwi-interacting RNA Loci.''; PubMedEurope PMCScholia
Baillat D, Shiekhattar R.; ''Functional dissection of the human TNRC6 (GW182-related) family of proteins.''; PubMedEurope PMCScholia
Younger ST, Corey DR.; ''Transcriptional regulation by miRNA mimics that target sequences downstream of gene termini.''; PubMedEurope PMCScholia
Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, Shiekhattar R.; ''TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing.''; PubMedEurope PMCScholia
Ye X, Huang N, Liu Y, Paroo Z, Huerta C, Li P, Chen S, Liu Q, Zhang H.; ''Structure of C3PO and mechanism of human RISC activation.''; PubMedEurope PMCScholia
Handler D, Meixner K, Pizka M, Lauss K, Schmied C, Gruber FS, Brennecke J.; ''The genetic makeup of the Drosophila piRNA pathway.''; PubMedEurope PMCScholia
Maniataki E, Mourelatos Z.; ''A human, ATP-independent, RISC assembly machine fueled by pre-miRNA.''; PubMedEurope PMCScholia
Rosenkranz D, Zischler H.; ''proTRAC--a software for probabilistic piRNA cluster detection, visualization and analysis.''; PubMedEurope PMCScholia
Gredell JA, Dittmer MJ, Wu M, Chan C, Walton SP.; ''Recognition of siRNA asymmetry by TAR RNA binding protein.''; PubMedEurope PMCScholia
Alló M, Agirre E, Bessonov S, Bertucci P, Gómez Acuña L, Buggiano V, Bellora N, Singh B, Petrillo E, Blaustein M, Miñana B, Dujardin G, Pozzi B, Pelisch F, Bechara E, Agafonov DE, Srebrow A, Lührmann R, Valcárcel J, Eyras E, Kornblihtt AR.; ''Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells.''; PubMedEurope PMCScholia
Lee Y, Hur I, Park SY, Kim YK, Suh MR, Kim VN.; ''The role of PACT in the RNA silencing pathway.''; PubMedEurope PMCScholia
Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W.; ''Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP.''; PubMedEurope PMCScholia
Gagnon KT, Li L, Chu Y, Janowski BA, Corey DR.; ''RNAi factors are present and active in human cell nuclei.''; PubMedEurope PMCScholia
Yang Q, Hua J, Wang L, Xu B, Zhang H, Ye N, Zhang Z, Yu D, Cooke HJ, Zhang Y, Shi Q.; ''MicroRNA and piRNA profiles in normal human testis detected by next generation sequencing.''; PubMedEurope PMCScholia
Ipsaro JJ, Joshua-Tor L.; ''From guide to target: molecular insights into eukaryotic RNA-interference machinery.''; PubMedEurope PMCScholia
Lazzaretti D, Tournier I, Izaurralde E.; ''The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence bound transcripts independently of Argonaute proteins.''; PubMedEurope PMCScholia
Weinmann L, Höck J, Ivacevic T, Ohrt T, Mütze J, Schwille P, Kremmer E, Benes V, Urlaub H, Meister G.; ''Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.''; PubMedEurope PMCScholia
Yi R, Qin Y, Macara IG, Cullen BR.; ''Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs.''; PubMedEurope PMCScholia
Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Rådmark O, Kim S, Kim VN.; ''The nuclear RNase III Drosha initiates microRNA processing.''; PubMedEurope PMCScholia
Ameyar-Zazoua M, Rachez C, Souidi M, Robin P, Fritsch L, Young R, Morozova N, Fenouil R, Descostes N, Andrau JC, Mathieu J, Hamiche A, Ait-Si-Ali S, Muchardt C, Batsché E, Harel-Bellan A.; ''Argonaute proteins couple chromatin silencing to alternative splicing.''; PubMedEurope PMCScholia
Höck J, Weinmann L, Ender C, Rüdel S, Kremmer E, Raabe M, Urlaub H, Meister G.; ''Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells.''; PubMedEurope PMCScholia
Sano M, Sierant M, Miyagishi M, Nakanishi M, Takagi Y, Sutou S.; ''Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference activity and strand selection.''; PubMedEurope PMCScholia
Zhang F, Wang J, Xu J, Zhang Z, Koppetsch BS, Schultz N, Vreven T, Meignin C, Davis I, Zamore PD, Weng Z, Theurkauf WE.; ''UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery.''; PubMedEurope PMCScholia
Yoda M, Kawamata T, Paroo Z, Ye X, Iwasaki S, Liu Q, Tomari Y.; ''ATP-dependent human RISC assembly pathways.''; PubMedEurope PMCScholia
Kim DH, Saetrom P, Snøve O, Rossi JJ.; ''MicroRNA-directed transcriptional gene silencing in mammalian cells.''; PubMedEurope PMCScholia
Seong Y, Lim DH, Kim A, Seo JH, Lee YS, Song H, Kwon YS.; ''Global identification of target recognition and cleavage by the Microprocessor in human ES cells.''; PubMedEurope PMCScholia
Shin C.; ''Cleavage of the star strand facilitates assembly of some microRNAs into Ago2-containing silencing complexes in mammals.''; PubMedEurope PMCScholia
Eystathioy T, Jakymiw A, Chan EK, Séraphin B, Cougot N, Fritzler MJ.; ''The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies.''; PubMedEurope PMCScholia
Francia S, Michelini F, Saxena A, Tang D, de Hoon M, Anelli V, Mione M, Carninci P, d'Adda di Fagagna F.; ''Site-specific DICER and DROSHA RNA products control the DNA-damage response.''; PubMedEurope PMCScholia
Huang V, Zheng J, Qi Z, Wang J, Place RF, Yu J, Li H, Li LC.; ''Ago1 Interacts with RNA polymerase II and binds to the promoters of actively transcribed genes in human cancer cells.''; PubMedEurope PMCScholia
Lee HY, Zhou K, Smith AM, Noland CL, Doudna JA.; ''Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing.''; PubMedEurope PMCScholia
Cai X, Hagedorn CH, Cullen BR.; ''Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs.''; PubMedEurope PMCScholia
Eulalio A, Helms S, Fritzsch C, Fauser M, Izaurralde E.; ''A C-terminal silencing domain in GW182 is essential for miRNA function.''; PubMedEurope PMCScholia
Elkayam E, Faehnle CR, Morales M, Sun J, Li H, Joshua-Tor L.; ''Multivalent Recruitment of Human Argonaute by GW182.''; PubMedEurope PMCScholia
Cronshaw JM, Krutchinsky AN, Zhang W, Chait BT, Matunis MJ.; ''Proteomic analysis of the mammalian nuclear pore complex.''; PubMedEurope PMCScholia
Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ.; ''Argonaute2 is the catalytic engine of mammalian RNAi.''; PubMedEurope PMCScholia
Muerdter F, Guzzardo PM, Gillis J, Luo Y, Yu Y, Chen C, Fekete R, Hannon GJ.; ''A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila.''; PubMedEurope PMCScholia
Zeng L, Zhang Q, Yan K, Zhou MM.; ''Structural insights into piRNA recognition by the human PIWI-like 1 PAZ domain.''; PubMedEurope PMCScholia
Rabut G, Doye V, Ellenberg J.; ''Mapping the dynamic organization of the nuclear pore complex inside single living cells.''; PubMedEurope PMCScholia
Provost P, Dishart D, Doucet J, Frendewey D, Samuelsson B, Rådmark O.; ''Ribonuclease activity and RNA binding of recombinant human Dicer.''; PubMedEurope PMCScholia
Kim DH, Villeneuve LM, Morris KV, Rossi JJ.; ''Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells.''; PubMedEurope PMCScholia
Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN.; ''The Drosha-DGCR8 complex in primary microRNA processing.''; PubMedEurope PMCScholia
Betancur JG, Tomari Y.; ''Dicer is dispensable for asymmetric RISC loading in mammals.''; PubMedEurope PMCScholia
Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R.; ''The Microprocessor complex mediates the genesis of microRNAs.''; PubMedEurope PMCScholia
Carthew RW, Sontheimer EJ.; ''Origins and Mechanisms of miRNAs and siRNAs.''; PubMedEurope PMCScholia
Bernstein E, Caudy AA, Hammond SM, Hannon GJ.; ''Role for a bidentate ribonuclease in the initiation step of RNA interference.''; PubMedEurope PMCScholia
Wei W, Ba Z, Gao M, Wu Y, Ma Y, Amiard S, White CI, Rendtlew Danielsen JM, Yang YG, Qi Y.; ''A role for small RNAs in DNA double-strand break repair.''; PubMedEurope PMCScholia
Hutvágner G, McLachlan J, Pasquinelli AE, Bálint E, Tuschl T, Zamore PD.; ''A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.''; PubMedEurope PMCScholia
Takimoto K, Wakiyama M, Yokoyama S.; ''Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression.''; PubMedEurope PMCScholia
Noland CL, Doudna JA.; ''Multiple sensors ensure guide strand selection in human RNAi pathways.''; PubMedEurope PMCScholia
Ori A, Banterle N, Iskar M, Iskar M, Andrés-Pons A, Escher C, Khanh Bui H, Sparks L, Solis-Mezarino V, Rinner O, Bork P, Lemke EA, Beck M.; ''Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines.''; PubMedEurope PMCScholia
Stalder L, Heusermann W, Sokol L, Trojer D, Wirz J, Hean J, Fritzsche A, Aeschimann F, Pfanzagl V, Basselet P, Weiler J, Hintersteiner M, Morrissey DV, Meisner-Kober NC.; ''The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.''; PubMedEurope PMCScholia
Fukunaga R, Han BW, Hung JH, Xu J, Weng Z, Zamore PD.; ''Dicer partner proteins tune the length of mature miRNAs in flies and mammals.''; PubMedEurope PMCScholia
Ohrt T, Mütze J, Staroske W, Weinmann L, Höck J, Crell K, Meister G, Schwille P.; ''Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.''; PubMedEurope PMCScholia
Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A.; ''Identification of mammalian microRNA host genes and transcription units.''; PubMedEurope PMCScholia
MacRae IJ, Ma E, Zhou M, Robinson CV, Doudna JA.; ''In vitro reconstitution of the human RISC-loading complex.''; PubMedEurope PMCScholia
Suntharalingam M, Wente SR.; ''Peering through the pore: nuclear pore complex structure, assembly, and function.''; PubMedEurope PMCScholia
Lin DH, Stuwe T, Schilbach S, Rundlet EJ, Perriches T, Mobbs G, Fan Y, Thierbach K, Huber FM, Collins LN, Davenport AM, Jeon YE, Hoelz A.; ''Architecture of the symmetric core of the nuclear pore.''; PubMedEurope PMCScholia
Koscianska E, Starega-Roslan J, Krzyzosiak WJ.; ''The role of Dicer protein partners in the processing of microRNA precursors.''; PubMedEurope PMCScholia
Jung I, Park JC, Kim S.; ''piClust: a density based piRNA clustering algorithm.''; PubMedEurope PMCScholia
Kosinski J, Mosalaganti S, von Appen A, Teimer R, DiGuilio AL, Wan W, Bui KH, Hagen WJ, Briggs JA, Glavy JS, Hurt E, Beck M.; ''Molecular architecture of the inner ring scaffold of the human nuclear pore complex.''; PubMedEurope PMCScholia
Short single-stranded RNA of 24-32 nucleotides derived from a long single-stranded precursor by a process independent of DICER. The 2' hydroxyl at the 3' end of mature piRNAs is methylated.
Short single-stranded RNA of 24-32 nucleotides derived from a long single-stranded precursor by a process independent of DICER. The 2' hydroxyl at the 3' end of mature piRNAs is methylated.
The RNA-induced silencing complex contains an Argonaute (AGO) protein, whose PAZ domain binds the 3' end of the miRNA. The PIWI domain of AGO is responsible for cleavage of target RNAs, that is, RNAs complementary to the miRNA. Only AGO2 (EIF2C2) is capable of cleavage, however. AGO1 (EIF2C1), AGO3 (EIF2C3), and AGO4 (EIF2C4) repress translation of target RNAs by binding without cleavage.
The RNA-induced silencing complex contains an Argonaute (AGO) protein, whose PAZ domain binds the 3' end of the miRNA. The PIWI domain of AGO is responsible for cleavage of target RNAs, that is, RNAs complementary to the miRNA. Only AGO2 (EIF2C2) is capable of cleavage, however. AGO1 (EIF2C1), AGO3 (EIF2C3), and AGO4 (EIF2C4) repress translation of target RNAs by binding without cleavage.
The Drosha:DGCR8 complex is also known as the MicroProcessor Complex. Drosha:DGCR8 contains DGCR8, an RNA-binding protein, and Drosha, an RNaseIII-class endonuclease that cleaves double-stranded RNA, leaving a 2-nucleotide protrusion at the 3' end.
The RNA-induced silencing complex contains an Argonaute (AGO) protein, whose PAZ domain binds the 3' end of the miRNA. The PIWI domain of AGO is responsible for cleavage of target RNAs, that is, RNAs complementary to the miRNA. Only AGO2 (EIF2C2) is capable of cleavage, however. AGO1 (EIF2C1), AGO3 (EIF2C3), and AGO4 (EIF2C4) repress translation of target RNAs by binding without cleavage.
The RNA-induced silencing complex contains an Argonaute (AGO) protein, whose PAZ domain binds the 3' end of the miRNA. The PIWI domain of AGO is responsible for cleavage of target RNAs, that is, RNAs complementary to the miRNA. Only AGO2 (EIF2C2) is capable of cleavage, however. AGO1 (EIF2C1), AGO3 (EIF2C3), and AGO4 (EIF2C4) repress translation of target RNAs by binding without cleavage.
Short single-stranded RNA of 24-32 nucleotides derived from a long single-stranded precursor by a process independent of DICER. The 2' hydroxyl at the 3' end of mature piRNAs is methylated.
Pre-miRNA binds the RISC loading complex (RLC), a complex containing DICER1, AGO2, and TARBP2 (TRBP). Alternative loading complexes contain AGO1, AGO3, or AGO4 rather than AGO2 and PRKRA (PACT) rather than TARBP2. The pre-miRNA substrate has an internal loop and a protruding 3' end created by cleavage by DROSHA:DGCR8. The DICER1:TARBP2 subcomplex or DICER1:PRKRA subcomplex recognizes this structure and the DICER1 component cleaves the pre-miRNA near the loop. The product is a double-stranded RNA of 21-25 nucleotides having 2-nucleotide protrusions at each 3' end. The products have 5' phosphates and 3' hydroxyl groups. Diffusion activity of TARBP2 and PRKRA along duplex RNA may enhance processing by DICER1.
Nuclear processing by Drosha Microprocessor complex. The primary-microRNA (pri-miRNA) is recognized by the Microprocessor complex (Drosha:DGCR8) and both strands of the pri-miRNA are cleaved by Drosha near the free 5' and 3' ends of the pri-miRNA, that is, at the ends distal from the internal loop. The product is a double-stranded RNA having 2 nucleotides protruding at the 3' end and having an internal loop.
Transcription of miRNA genes. Most miRNAs are transcribed by RNA polymerase II. The miRNAs may be autonomous transcription units or they may be located in other transcripts, including locations within introns and other untranslated regions. Of the polymerase II transcribed miRNAs, about 60% are located in introns of protein coding genes, 12 % are in introns of non-coding RNAs, 18% are in exons of non-coding RNAs, and 10% uncertain. A second class of miRNA genes are associated with Alu and other repetitive elements and are cotranscribed with these elements by RNA polymerase III. There are currently only a few proven examples of polymerase III transcribed miRNAs.
Nuclear Export by Exportin-5. The pre-microRNA is bound by the Exportin-5:RanGTP complex in the nucleus and the complex is translocated through the nuclear pore into the cytoplasm. In the process GTP is hydrolyzed to GDP.
The duplex miRNA (designated miRNA-miRNA*) is reoriented on DICER1 after cleavage and then transferred from DICER1 to an Argonaute protein (AGO2 or, by inference, AGO1, AGO3, or AGO4) within the RISC loading complex. Particular Argonaute proteins do not appear to have significantly different populations of miRNAs, however Argonaute identity can affect the resulting length of the miRNA.
The short double-stranded RNA passed from DICER1 to an Argonaute protein is rendered single-stranded by removal and loss of the passenger strand through a mechanism that is not well characterized. All Argonautes (AGO1 (EIF2C1), AGO2 (EIF2C2), AGO3 (EIF2C3), AGO4 (EIF2C4)) can remove the passenger strand without cleaving it. AGO2 (EIF2C2) possesses endonucleolytic activity and cleaves the passenger strand of siRNAs, which facilitates removal of the passenger strand but is not required (Matranga et al. 2005). RNA helicase A associated with the RISC loading complex can also facilitate removal of the passenger strand. The mechanism that selects which strand is retained as the guide RNA is not well understood in humans. Overhanging nucleotides and strength of base-pairing at each end of the input duplex are observed to influence strand selection. Argonaute proteins loaded with miRNAs or siRNAs are predominantly located in association with TARBP2 or PRKRA at the cytosolic face of the rough endoplasmic reticulum in cultured cells.
Following cleavage the duplex siRNA reoriented on DICER1 and then transferred from DICER1 to an Argonaute protein (AGO1, AGO2, AGO3, or AGO4) within the RISC loading complex (RLC).
A short double-stranded RNA is passed from DICER1 to an Argonaute protein and rendered single-stranded by removal and loss of the passenger strand. All Argonautes (AGO1 (EIF2C1), AGO2 (EIF2C2), AGO3 (EIF2C3), AGO4 (EIF2C4)) can remove the passenger strand without cleaving it and most miRNAs are processed in this way. AGO2 (EIF2C2) can cleave the passenger strand of a subset of miRNAs that have no mismatches in the central region (Shin et al. 2008). RNA helicase A associated with the RISC loading complex can facilitate removal of the passenger strand. The mechanism that selects which strand is retained as the guide RNA is not well understood in humans. Overhanging nucleotides and strength of base-pairing at each end of the input duplex are observed to influence strand selection. In cultured cells Argonaute proteins loaded with miRNAs or siRNAs are predominantly located in association with TARBP2 or PRKRA at the cytosolic face of the rough endoplasmic reticulum. In adult non-dividing cells most Argonaute-bound miRNAs are located in low molecular weight complexes but shift to larger complexes containing GW182 in response to phosphoinositide-3-kinase/mTOR signaling.
Double stranded RNA binds the RISC loading complex and DICER1, an RNase III component of the complex, cleaves double-stranded RNAs to yield short double-stranded RNAs of 21-25 nucleotides, duplex siRNAs (small interfering RNAs). SiRNAs are similar to microRNAs (miRNAs) in their final structure but differ from miRNAs in their source: siRNAs are produced from long double stranded RNAs that originate from viruses, transposable elements, centromeric repeats and other repetitive structures. The RLC as originally characterized contains DICER1, AGO2, and TARBP2 (TRBP). Alternative RLCs appear to contain other Argonaute proteins (AGO1, AGO3, AGO4) rather than AGO2 and PRKRA rather than TARBP2. Diffusion activity of TARBP2 and PRKRA along duplex RNA may enhance processing by DICER1.
RISCs can bind target RNAs that do not exactly match the guide RNA carried by an Argonaute. Binding is especially dependent on base-pairing between the target RNA and the eight 5' nucleotides of the guide RNA (miRNA or siRNA). After binding, Argonaute-1 (AGO1, EIF2C1), AGO3 (EIF2C3), and AGO4 (EIF2C4) are incapable of cleavage in all cases. AGO2 is capable of cleaving the target RNA but not if mismatches exist in the middle of the guide (centered about 10 nucloetides from the 5' end of the guide RNA). In the absence of cleavage the target RNA remains bound by the RISC, which inhibits translation of the target RNA and causes the RNA to enter the decay pathway. In vivo, inhibition of translation requires interaction of AGO with a TNRC6 protein and MOV10. The phosphoinositide-3 kinase/mTOR pathway increases expression of GW182, a TNRC6 protein, which increases the portion of AGO:miRNA in high molecular weight complexes with mRNA.
Human Argonaute-2 (AGO2, EIF2C2) possesses ribonucleolytic activity in its PIWI domain and cleaves target RNAs that are exactly complementary to the guide RNA at a location around 10 nucleotides from the 5' end of the match with the guide RNA. The products of cleavage have a 5' phosphate and a 3' hydroxyl group. Both complexes containing siRNAs and miRNAs are capable of cleavage. Although Argonaute proteins interact with many other proteins, the complex of AGO2 and the guide RNA are sufficient to direct cleavage of target RNAs in vitro. In vivo, cleavage requires interaction of AGO2 with a TNRC6 protein and MOV10.
RISCs containing Argonaute-1 (AGO1, EIF2C1), AGO3 (EIF2C3), and AGO4 (EIF2C4) bind to target RNAs by base-pairing between the target RNA and the guide RNA of the RISC. AGO1,3,4 do not possess ribonuclease activity therefore exact matches between the guide and the target do not result in cleavage of the target. Rather, the effect of binding is inhibition of translation followed by decay of the target RNA. Argonaute proteins have been shown to interact with ribosomal proteins and with components of processing bodies (P-bodies) where RNA degradation occurs. Direct interaction between AGO and a TNRC6 protein is required for inhibition of translation and targeting to P-bodies in vivo.
Importin-8 (IPO8, IMP8, RANBP8) binds AGO2:miRNA complexes in the cytosol and participates in the importation of AGO2:miRNA complexes into the nucleus (Weinmann et al. 2009, Wei et al. 2014). IPO8 is also required for recruitment of AGO2:miRNA complexes to many target mRNAs in the cytosol and their efficient silencing (Weinmann et al. 2009). Moreover, other Argonautes (AGO1, AGO3, AGO4) are also observed in the nucleus (Kim et al. 2008, Weinmann et al. 2009, Ahlenstiel et al. 2012, Gagnon et al. 2014) and may be imported by the same mechanism.
BCDIN3D transfers a methyl group from S-adenosylcysteine to the each of the 2 hydroxyl groups of the 5' phosphate of pre-miR-145 and pre-miR-23b (Xhemalce et al. 2012). The methylation eliminates the negative charges on the phosphate and thereby interferes with the recognition of pre-miRNAs by Dicer, inhibiting production of mature miR-145.
Complexes containing small RNAs and AGO1 or AGO2 are observed within the nucleus and at the inner nuclear envelope, respectively, associated with the actin cytoskeleton (Ahlenstiel et al. 2012, Huang et al. 2013). Argonaute:miRNA complexes associate with genomic regions possessing sequences that match the miRNA, possibly via RNA transcripts tethered to chromatin (Li et al. 2006, Weinber et al. 2006, Kim et al. 2008, Place et al. 2008, Younger and Corey 2011). AGO2:miRNA appears to be in complexes containing DICER and TNRC6A (Gagnon et al. 2014) and AGO1 has been shown to associate with RNA polymerase II, TARBP2, and EZH2 at transcriptionally silenced promoters (Kim et al. 2006, Huang et al. 2013). AGO1 also associates with RNA polymerase II at active promoters (Huang et al. 2013). Other AGO:miRNA complexes may form similar complexes. Association of AGO:miRNA complexes with genes may cause transcriptional activation (Li et al. 2006, Place et al. 2008), transcriptional repression (Kim et al. 2008, Younger and Corey 2011), alternative splicing (Ameyar-Zazoua et al. 2012), or DNA repair (Francia et al. 2012, Wei et al. 2012). The determinants for transcriptional activation and repression are not known. Transcriptional effects are mediated through changes in histone methylation, especially methylation of histone H3 at lysine-4, lysine-9, and lysine-27 (Li et al. 2006, Kim et al. 2006, Kim et al. 2008, Younger and Corey 2011).
The AGO2:miRNA complex is formed in the cytosol (Ohrt et al 2008) and is imported into the nucleus in a complex with Importin-8 (IPO8, Imp8, RanBP8) (Weinmann et al. 2009, Wei et al. 2014). Once in the nucleus, Imp8 in complex with the cargo interacts with RAN:GTP, causing the dissociation of Imp8 from the complex with AGO2:miRNA (Gorlich et al. 1997). Other Argonautes are also observed in the nucleus (Robb et al. 2005, Weinmann et al. 2009, Doyle et al. 2013, Ahlenstiel et al. 2012, Gagnon et al. 2014) and may be imported by the same mechanism.
TNRC6A (GW182) is a major component of miRISC and processing bodies (P bodies or GW bodies) where transcripts are degraded (Eystathioy et al. 2003). GW182 posesses several glycine-tryptophan (GW) repeats that enable interactions with Argonaute proteins (Eulalio et al. 2009, Takimoto et al. 2009). Humans express three paralogs (TNRC6A, TNRC6B, and TNRC6C) which can each silence expression of mRNAs to which they are bound (Lazzaretti et al. 2009). In the cytosol TNRC6A binds AGO2:miRNA via three GW-repeat motifs (Landthaler et al. 2008, Takimoto et al. 2009, Nishi et al. 2013).
TNRC6A (GW182) possesses both a nuclear localization signal (NLS) and a nuclear export signal (NES) that enable it to shuttle between the cytoplasm and the nucleus (Nishi et al. 2013). Thus the TNRC6A:AGO2:miRNA complex is transported into the nucleus by an unknown importation mechanism (Nishi et al. 2013). (TNRC6A is exported by Exportin 1.) The interaction between AGO2 and TNRC6A affects gene silencing activity in the nucleus (Nishi et al. 2013).
RNA cleaved by PIWIL2:piRNA is transferred to PIWIL4 (HIWI2 in human, MIWI2 in mouse). The reaction requires MAEL and is enhanced by the chaperone activity of FKBP6:HSP90. PIWIL4, TDRD9, and MAEL are located in piP bodies, a type of nuage (electron-dense perinuclear material). PIWIL4 and PIWIL2 are in separate nuages.
As inferred from homologs in Drosophila and mouse, PLD6 (MitoPLD) located on the cytoplasmic face of the mitochondrial outer membrane makes the first endonucleolytic cleavage of primary piRNA transcripts. The cleavage yields a 5' phosphate and a 3' hydroxyl. Cleavage is believed to precede loading into PIWIL1 (HIWI, MIWI) or PIWIL2 (HILI, MILI). Most mature piRNAs have uracil at the 5' end. This appears to be due to selective binding by PIWI proteins rather than selective cleavage (reviewed in Bortvin 2013).
As inferred from mouse homologs, after binding PIWIL1 (HIWI in human, Miwi in mouse) the 3' end of the pre-piRNA is trimmed by an unknown nuclease. The final size of the piRNA appears to be determined by the particular PIWI protein with which it is associated. PIWIL1 and TDRD6 are located in the chromatoid body. Both TDRD6 and TDRKH are associated with PIWIL1 in adult testes but only TDRKH is present in embryonic prospermatogonia. TDRKH is required for spermatogenesis and appears to participate in trimming of the 3' end of pre-piRNAs.
As inferred from homologs in mouse, PIWIL4 is loaded with piRNA in the cytosol and then translocates to the nucleus where it directs transcriptional silencing of cognate loci by an unknown mechanism. Most cellular PIWIL4 is translocated to the nucleus at E16.5 of mouse development and proper localization depends on PIWIL2 and TDRD1. TDRD9 and MAEL interact with PIWIL4, are observed in the nucleus, and may play a role in the translocation of PIWIL4. Knockout of TDRD9, however, does not affect nuclear localization of PIWIL4. Knockout of MAEL delays but does not prevent localization of PIWI4L to the nucleus. TDRKH is required for translocation of PIWIL4, however TDRKH is only observed in the cytosol.
As inferred from homologs in mouse, PIWIL2 (HILI in human, homolog of MILI in mouse) bound to a piRNA cleaves target RNAs complementary to the piRNA. The cleaved RNA can either be transferred to another PIWIL2 as part of the "ping pong cycle" that generates secondary piRNAs or the cleaved RNA can be transferred to PIWIL4, which then transits to the nucleus to transcriptionally silence loci complementary to the piRNA.
RNA cleaved by PIWIL2 (HILI in human, homolog of MILI in mouse) can be transferred to another molecule of PIWIL2. This is part of the "ping pong cycle" that generates further secondary piRNAs from a longer precursor.
Primary (unprocessed) transcripts of piRNAs are transported from the nucleus to the cytosol by an unknown mechanism. Studies with Drosophila indicate that Uap56, Nxt1, Nxf2, Nup154, and Nup43 may be involved in exporting piRNA precursors from the nucleus (Zhang et al. 2012, Muerdter et al. 2013, Handler et al. 2013).
As inferred from experiments with mouse homologs, primary piRNA transcripts originate from multiple copy transposable elements and unique copy non-coding RNAs and mRNAs. As male germ cells progress from fetus to adult, the composition of piRNAs shifts from transposons to unique copy sequences (reviewed in Bortvin 2013). Computational analyses have identified 161 to 242 piRNA clusters and many other smaller piRNA hotspots in the mouse genome (Aravin et al. 2008, Rosenkranz and Zischler 2012, Jung et al. 2014). About 18% of pre-pachytene piRNAs in mouse originate from mRNAs encoding proteins (Aravin et al. 2008). Likewise 235 to 368 piRNA clusters were identified in the human genome (Rosenkranz and Zischler 2012, Gould et al. 2012, Yang et al. 2013, Jung et al. 2014). As inferred from the mouse homolog, the MYBL1 (A-MYB) transcription factor drives transcription of both piRNA precursors and mRNAs encoding PIWI family proteins.
After the cleaved RNA binds PIWIL2 (HILI in human, homolog of Mili in mouse) the 3' end is trimmed by an unknown nuclease to generate a mature piRNA. The resulting PIWIL2:piRNA complex can then participate in further amplification by the "ping-pong" cycle.
After cleavage by PLD6 at the 5' end, the pre-piRNA is bound by PIWIL2 (HILI, homolog of MILI in mouse), likely in a complex with TDRD1, TDRD12, DDX4 (MVH), ASZ (GASZ), and MOV10L, all of which are required for wild-type levels of piRNA biogenesis. Binding by PIWIL2 is believed to be selective for pre-piRNAs that have uracil residues at their 5' ends.
As inferred from mouse homologs, after binding PIWIL2 (HILI in human, MILI in mouse) the 3' end of the pre-piRNA is trimmed by an unknown nuclease. The final size of the piRNA appears to be determined by the particular PIWI protein with which it is associated. MOV10L1, which has a helicase domain, associates with PIWIL2 and is required for loading PIWIL2 with piRNA. PIWIL2, TDRD1, MVH, and ASZ are located in the intermitochondrial cement, the chromatoid body, and the pi-body, a type of nuage (reviewed in Pillai and Chuma 2012). (Nuage is electron-dense perinuclear material also known as germinal granules.)
After cleavage by PLD6 at the 5' end, the pre-piRNA is bound by PIWIL1 (HIWI, homolog of MIWI in mouse), likely in a complex with other proteins such as TDRD6 and TDRKH, which interact with methylated arginine residues on PIWIL1 and are required for piRNA biogenesis. Binding by PIWIL1 is believed to be selective for pre-piRNAs that have uracil residues at their 5' ends.
As inferred from mouse homologs, HENMT1 transfers a methyl group from S-adenosylmethionine to the 2' hydroxyl group of a trimmed piRNA bound by the PIWIL1 complex in the cytosol.
As inferred from mouse homologs, HENMT1 transfers a methyl group from S-adenosylmethionine to the 2' hydroxyl group at the 3' end of piRNA bound to the PIWIL2 complex in the cytosol.
As inferred from mouse homologs, HENMT1 transfers a methyl group from S-adenosylmethionine to the 2' hydroxyl group at the 3' end of a piRNA bound by PIWIL4.
C3PO appears to act as a nuclease that hydrolyzes the passenger strand after cleavage by AGO2. C3PO could also be part of a DICER1-independent pathway for loading AGO2. AGO2 of humans may contain either miRNAs or siRNAs. The mechanism that selects which strand is retained as the guide RNA is not well understood in humans. Overhanging nucleotides and strength of base-pairing at each end of the input duplex are observed to influence strand selection.
C3PO appears to act as a nuclease that hydrolyzes the passenger strand after cleavage by AGO2. C3PO could also be part of a DICER1-independent pathway for loading AGO2. AGO2 of humans may contain either miRNAs or siRNAs. The mechanism that selects which strand is retained as the guide RNA is not well understood in humans. Overhanging nucleotides and strength of base-pairing at each end of the input duplex are observed to influence strand selection.
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transposon
RNA:TDRD1:TDRD12:DDX4:ASZ:MOV10L1Phosphate and 3'
Hydroxyltransposon
RNA:TDRD9:MAEL:TDRKHholoenzyme complex
(generic)holoenzyme complex
(unphosphorylated)Annotated Interactions
transposon
RNA:TDRD1:TDRD12:DDX4:ASZ:MOV10L1transposon
RNA:TDRD1:TDRD12:DDX4:ASZ:MOV10L1Phosphate and 3'
Hydroxyltransposon
RNA:TDRD9:MAEL:TDRKHtransposon
RNA:TDRD9:MAEL:TDRKHA second class of miRNA genes are associated with Alu and other repetitive elements and are cotranscribed with these elements by RNA polymerase III. There are currently only a few proven examples of polymerase III transcribed miRNAs.
The mechanism that selects which strand is retained as the guide RNA is not well understood in humans. Overhanging nucleotides and strength of base-pairing at each end of the input duplex are observed to influence strand selection.
Argonaute proteins loaded with miRNAs or siRNAs are predominantly located in association with TARBP2 or PRKRA at the cytosolic face of the rough endoplasmic reticulum in cultured cells.
RNA helicase A associated with the RISC loading complex can facilitate removal of the passenger strand.
The mechanism that selects which strand is retained as the guide RNA is not well understood in humans. Overhanging nucleotides and strength of base-pairing at each end of the input duplex are observed to influence strand selection.
In cultured cells Argonaute proteins loaded with miRNAs or siRNAs are predominantly located in association with TARBP2 or PRKRA at the cytosolic face of the rough endoplasmic reticulum. In adult non-dividing cells most Argonaute-bound miRNAs are located in low molecular weight complexes but shift to larger complexes containing GW182 in response to phosphoinositide-3-kinase/mTOR signaling.
The RLC as originally characterized contains DICER1, AGO2, and TARBP2 (TRBP). Alternative RLCs appear to contain other Argonaute proteins (AGO1, AGO3, AGO4) rather than AGO2 and PRKRA rather than TARBP2. Diffusion activity of TARBP2 and PRKRA along duplex RNA may enhance processing by DICER1.
Association of AGO:miRNA complexes with genes may cause transcriptional activation (Li et al. 2006, Place et al. 2008), transcriptional repression (Kim et al. 2008, Younger and Corey 2011), alternative splicing (Ameyar-Zazoua et al. 2012), or DNA repair (Francia et al. 2012, Wei et al. 2012). The determinants for transcriptional activation and repression are not known. Transcriptional effects are mediated through changes in histone methylation, especially methylation of histone H3 at lysine-4, lysine-9, and lysine-27 (Li et al. 2006, Kim et al. 2006, Kim et al. 2008, Younger and Corey 2011).
As inferred from the mouse homolog, the MYBL1 (A-MYB) transcription factor drives transcription of both piRNA precursors and mRNAs encoding PIWI family proteins.
The mechanism that selects which strand is retained as the guide RNA is not well understood in humans. Overhanging nucleotides and strength of base-pairing at each end of the input duplex are observed to influence strand selection.
The mechanism that selects which strand is retained as the guide RNA is not well understood in humans. Overhanging nucleotides and strength of base-pairing at each end of the input duplex are observed to influence strand selection.
holoenzyme complex
(generic)holoenzyme complex
(generic)holoenzyme complex
(unphosphorylated)