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Watter domeine of motiewe is in staat om aan beide DNA en RNA te heg?

Watter domeine of motiewe is in staat om aan beide DNA en RNA te heg?


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Domeine of motiewe van transkripsiefaktore is katalities aktief om aan die nukleïensuur van enige tipe te heg. Waar kan ek 'n lys van domeine of motiewe vind wat aan beide DNA en RNA kan heg? byvoorbeeld KH-domeine bind aan óf RNA óf enkelstring-DNS.


Prion

Prions is verkeerd gevoude proteïene met die vermoë om hul verkeerd gevoude vorm na normale variante van dieselfde proteïen oor te dra. Hulle kenmerk verskeie noodlottige en oordraagbare neurodegeneratiewe siektes by mense en baie ander diere. [3] Dit is nie bekend wat veroorsaak dat die normale proteïen verkeerd vou nie, maar die abnormale driedimensionele struktuur word vermoed dat dit aansteeklike eienskappe verleen, wat nabygeleë proteïenmolekules in dieselfde vorm laat ineenstort. Die woord prion afgelei van "proteïenagtige aansteeklike deeltjie". [4] [5] [6] Die veronderstelde rol van 'n proteïen as 'n aansteeklike middel staan ​​in kontras met alle ander bekende aansteeklike middels soos viroïede, virusse, bakterieë, swamme en parasiete, wat almal nukleïensure bevat (DNA, RNA, of albei).

Prionisovorme van die prionproteïen (PrP), wie se spesifieke funksie onseker is, word veronderstel as die oorsaak van oordraagbare spongiforme enkefalopatieë (TSE's), [7] insluitend skrapie by skape, chroniese vermorsingsiekte (CWD) in takbokke, bees spongiforme enkefalopatie ( BSE) in beeste (algemeen bekend as "malbeessiekte") en Creutzfeldt-Jakob-siekte (CJD) by mense. Alle bekende prion-siektes by soogdiere beïnvloed die struktuur van die brein of ander neurale weefsel almal is progressief, het geen bekende effektiewe behandeling nie, en is altyd dodelik. [8] Tot 2015 is alle bekende soogdierprionsiektes beskou as veroorsaak deur die prionproteïen (PrP), maar in 2015 is daar vermoed dat meervoudige sisteematrofie (MSA) deur 'n prionvorm van alfa-sinukleen veroorsaak is. [9]

Prions vorm abnormale aggregate van proteïene genaamd amiloïede, wat in besmette weefsel ophoop en geassosieer word met weefselskade en seldood. [10] Amiloïede is ook verantwoordelik vir verskeie ander neurodegeneratiewe siektes soos Alzheimer se siekte en Parkinson se siekte. [11] [12] Prionaggregate is stabiel, en hierdie strukturele stabiliteit beteken dat prione bestand is teen denaturasie deur chemiese en fisiese middels: hulle kan nie deur gewone ontsmetting of kook vernietig word nie. Dit maak die wegdoening en insluiting van hierdie deeltjies moeilik.

'n Prionsiekte is 'n tipe proteopatie, of siekte van struktureel abnormale proteïene. By mense word geglo dat prions die oorsaak is van Creutzfeldt-Jakob-siekte (CJD), sy variant (vCJD), Gerstmann-Sträussler-Scheinker-sindroom (GSS), fatale familiële slapeloosheid (FFI) en kuru. [4] Daar is ook bewyse wat daarop dui dat prions 'n rol kan speel in die proses van Alzheimer se siekte, Parkinson se siekte, en amiotrofiese laterale sklerose (ALS), dit word genoem. prionagtige siektes. [13] [14] [15] [16] Verskeie gisproteïene is ook geïdentifiseer as met prionogeniese eienskappe. [17] [18] Prionreplisering is onderhewig aan epimutasie en natuurlike seleksie net soos vir ander vorme van replikasie, en hul struktuur verskil effens tussen spesies. [19]


Inhoud

Virale RdRps is in die vroeë 1960's ontdek uit studies oor mengovirus en poliovirus toe daar waargeneem is dat hierdie virusse nie sensitief was vir aktinomisien D nie, 'n middel wat sellulêre DNA-gerigte RNA-sintese inhibeer. Hierdie gebrek aan sensitiwiteit het voorgestel dat daar 'n virusspesifieke ensiem is wat RNA vanaf 'n RNA-sjabloon kan kopieer en nie van 'n DNA-sjabloon nie.

RdRps is hoogs behoue ​​deur virusse en is selfs verwant aan telomerase, alhoewel die rede hiervoor 'n voortdurende vraag is vanaf 2009. [3] Die ooreenkoms het gelei tot spekulasie dat virale RdRps voorvaderlik is vir menslike telomerase.

Die bekendste voorbeeld van RdRp is dié van die poliovirus. Die virale genoom is saamgestel uit RNA, wat die sel binnedring deur reseptor-bemiddelde endositose. Van daar af is die RNA in staat om onmiddellik as 'n sjabloon vir komplementêre RNA-sintese op te tree. Die komplementêre string is dan self in staat om as 'n sjabloon op te tree vir die produksie van nuwe virale genome wat verder verpak en uit die sel vrygestel word gereed om meer gasheerselle te besmet. Die voordeel van hierdie metode van replikasie is dat daar geen DNA-stadium replikasie is vinnig en maklik. Die nadeel is dat daar geen 'rugsteun' DNA-kopie is nie.

Baie RdRps is nou geassosieer met membrane en is dus moeilik om te bestudeer. Die bekendste RdRps is poliovirale 3Dpol, vesikulêre stomatitis virus L, [4] en hepatitis C virus NS5B proteïen.

Baie eukariote het ook RdRps betrokke by RNA-interferensie, dit versterk mikroRNA's en klein temporale RNA's en produseer dubbelstring-RNA deur klein interfererende RNA's as primers te gebruik. [5] Trouens, hierdie selfde RdRps wat in die verdedigingsmeganismes gebruik word, kan tot hul voordeel deur RNA-virusse oorgeneem word. [6] Hulle evolusionêre geskiedenis is hersien. [7]

RdRp verskil van RNA-polimerase aangesien dit werk om die sintese van 'n RNA-string aanvullend tot 'n gegewe RNA-sjabloon te kataliseer, eerder as om 'n DNA-sjabloon te gebruik. Die RNA replikasie proses is 'n vier-stap meganisme, soos beskryf.

  1. Nukleotiedtrifosfaat (NTP) binding - aanvanklik presenteer die RdRp met 'n vakante aktiewe plek waarin die NTP bind. Korrekte NTP-binding veroorsaak dat die RdRp 'n konformasieverandering ondergaan. [8]
  2. Aktiewe werf sluiting - die konformasie verandering geïnisieer deur die korrekte NTP binding, lei tot die beperking van aktiewe werf toegang en produseer 'n katalisties bevoegde toestand. [8]
  3. Fosfodiesterbindingsvorming - twee Mg 2+ ione is in die katalities aktiewe toestand teenwoordig en rangskik hulself so rondom die RNA-primer dat die substraat NTP in staat is om 'n fosfatidieloordrag te ondergaan en 'n fosfodiesterbinding met die bestaande nukleotiedketting te vorm. [9] Met die gebruik van hierdie Mg 2+ ione het die aktiewe plek nie meer die katalisties stabiel nie en verander die RdRp kompleks na 'n oop konformasie. [9]
  4. Translokasie - sodra die aktiewe plek oop is, is die RNA-string in staat om deur die RdRp-proteïenkompleks te beweeg en 'n nuwe NTP te bind en kettingverlenging voort te sit, tensy anders gespesifiseer deur die sjabloon. [8]

RNA-sintese kan uitgevoer word deur middel van 'n primer-onafhanklike (De novo) of 'n primer-afhanklike meganisme wat 'n virale proteïen genoom-gekoppelde (VPg) primer gebruik. [10] Die De novo inisiasie bestaan ​​uit die byvoeging van 'n nukleosiedtrifosfaat (NTP) tot die 3'-OH van die eerste inisierende NTP. [10] Tydens die volgende sogenaamde verlengingsfase word hierdie nukleotidieloordragreaksie met daaropvolgende NTP's herhaal om die komplementêre RNA-produk te genereer. Beëindiging van die ontluikende RNA-ketting wat deur RdRp geproduseer word, is nie heeltemal bekend nie, maar dit is getoon dat RdRp-terminasie volgorde-onafhanklik is. [11]

Een groot nadeel van RNA-afhanklike RNA-polimerase-replikasie is die geweldige foutkoers tydens transkripsie. [10] Dit is bekend dat RdRps 'n gebrek aan getrouheid in die orde van 10 4 nukleotiede het, wat vermoedelik 'n direkte gevolg is van sy onvoldoende proefleesvermoë. [10] Hierdie hoë tempo van variasie word bevoordeel in virale genome aangesien dit die patogeen toelaat om verdediging te oorkom wat ontwikkel is deur gashere wat probeer om infeksie te vermy wat evolusionêre groei moontlik maak.

Virale/prokariotiese RNA-gerigte RNA-polimerases, saam met baie enkel-subeenheid DNA-gerigte polimerases, gebruik 'n vou waarvan die organisasie gekoppel is aan die vorm van 'n regterhand met drie subdomeine genaamd vingers, palm en duim. [12] Slegs die palm-subdomein, saamgestel uit 'n vierstring antiparallelle beta-vel met twee alfa-helikse, is goed bewaar onder al hierdie ensieme. In RdRp bestaan ​​die palm-subdomein uit drie goed bewaarde motiewe (A, B en C). Motief A (D-x(4,5)-D) en motief C (GDD) is ruimtelik naas mekaar geplaas. Die asparaginsuurreste van hierdie motiewe word geïmpliseer in die binding van Mg 2+ en/of Mn 2+. Die asparagienresidu van motief B is betrokke by seleksie van ribonukleosiedtrifosfate bo dNTP's en bepaal dus of RNA eerder as DNA gesintetiseer word. [13] Die domeinorganisasie [14] en die 3D-struktuur van die katalitiese sentrum van 'n wye reeks RdRps, selfs dié met 'n lae algehele volgorde homologie, word bewaar. Die katalitiese sentrum word gevorm deur verskeie motiewe wat 'n aantal bewaarde aminosuurreste bevat.

Eukariotiese RNA-interferensie vereis 'n sellulêre RNA-afhanklike RNA-polimerase (c RdRp). Anders as die "hand" polimerases, lyk hulle na vereenvoudigde multi-subeenheid DNA-afhanklike RNA polimerases (DdRPs), spesifiek in die katalitiese β/β' subeenhede, deurdat hulle twee stelle dubbel-psi β-vate in die aktiewe plek gebruik. QDE1 (Q9Y7G6) in Neurospora crassa, wat albei vate in dieselfde ketting het, [15] is 'n voorbeeld van so 'n ensiem. [16] Bakteriofage homoloë, insluitend die soortgelyke enkelketting DdRp yonO (O31945), blyk nader aan c RdRps te wees as wat DdRPs is. [5] [17]

Daar is 4 superfamilies virusse wat alle RNA-bevattende virusse met geen DNA-stadium dek nie:

  • Virusse wat positiewe string RNA of dubbelstring RNA bevat, behalwe retrovirusse en Birnaviridae
    • Alle positiewe-string RNA eukariotiese virusse met geen DNA stadium nie
    • Alle RNA-bevattende bakteriofage is daar twee families van RNA-bevattende bakteriofage: Leviviridae (positiewe ssRNA fage) en Cystoviridae (dsRNA fage)
    • dsRNA virus familie Reoviridae, Totiviridae, Hypoviridae, Partitiviridae

    RNA-transkripsie is soortgelyk aan [ hoe? ] maar nie dieselfde as DNA-replikasie nie.

    Flavivirusse produseer 'n poliproteïen vanaf die ssRNA-genoom. Die poliproteïen word aan 'n aantal produkte geklief, waarvan een NS5, 'n RNA-afhanklike RNA-polimerase, is. Hierdie RNA-gerigte RNA-polimerase besit 'n aantal kort streke en motiewe wat homoloog is aan ander RNA-gerigte RNA-polimerases. [18]

    RNA replikase gevind in positiewe-string ssRNA virusse is verwant aan mekaar, vorm drie groot superfamilies. [19] Birnavirale RNA-replikase is uniek deurdat dit nie motief C (GDD) in die handpalm het nie. [20] Mononegavirale RdRp (PDB 5A22) is outomaties geklassifiseer as soortgelyk aan (+)-ssRNA RdRps, spesifiek een van Pestivirus en een van Leviviridae. [21] Bunyavirale RdRp-monomeer (PDB 5AMQ) lyk soos die heterotrimeriese kompleks van Ortomyxovirale (Influenza PDB 4WSB) RdRp. [22]

    Aangesien dit 'n proteïen is wat universeel is vir RNA-bevattende virusse, is RdRp 'n nuttige merker om hul evolusie te verstaan. [23] Die algehele strukturele evolusie van virale RdRps is hersien. [24]

    Rekombinasie wysig

    Wanneer sy (+)ssRNA-genoom herhaal word, is die poliovirus RdRp in staat om rekombinasie uit te voer. Rekombinasie blyk te plaasvind deur 'n kopiekeusemeganisme waarin die RdRp (+)ssRNA-sjablone omskakel tydens negatiewe stringsintese. [25] Rekombinasiefrekwensie word gedeeltelik bepaal deur die getrouheid van RdRp-replikasie. [26] RdRp variante met hoë replikasie getrouheid toon verminderde rekombinasie, en lae getrouheid RdRps toon verhoogde rekombinasie. [26] Rekombinasie deur RdRp string omskakeling vind ook gereeld plaas tydens replikasie in die (+)ssRNA plant karmovirusse en tombusvirusse. [27]

    Intrageniese aanvulling Edit

    Sendai-virus (familie Paramyxoviridae) het 'n lineêre, enkelstrengige, negatiewe sin, nie-gesegmenteerde RNA-genoom. Die virale RdRp bestaan ​​uit twee virusgekodeerde subeenhede, 'n kleiner een P en 'n groter een L. Wanneer verskillende onaktiewe RdRp-mutante met defekte oor die lengte van die L-subeenheid in paarsgewyse kombinasies getoets is, is herstel van virale RNA-sintese waargeneem in sommige kombinasies. [28] Daar word na hierdie positiewe L-L-interaksie verwys as intrageniese komplementering en dui aan dat die L-proteïen 'n oligomeer in die virale RNA-polimerasekompleks is.

    RdRps kan as geneesmiddelteikens vir virale patogene gebruik word aangesien hul funksie nie nodig is vir eukariotiese oorlewing nie. Deur RNA-afhanklike RNA-polimerase-funksie te inhibeer, kan nuwe RNA's nie vanaf 'n RNA-sjabloonstring gerepliseer word nie, maar DNA-afhanklike RNA-polimerase sal funksioneel bly.

    Daar is tans antivirale middels teen Hepatitis C en COVID-19 wat spesifiek RdRp teiken. Dit sluit in Sofosbuvir en Ribavirin teen Hepatitis C [29] en Remdesivir, die enigste FDA-goedgekeurde middel teen COVID-19.

    GS-441524 trifosfaat, is 'n substraat vir RdRp, maar nie soogdierpolimerases nie. Dit lei tot voortydige kettingbeëindiging en inhibisie van virale replikasie. GS-441524 trifosfaat is die biologies aktiewe vorm van die fosfaat pro-geneesmiddel, Remdesivir. Remdesivir word geklassifiseer as 'n nukleotied-analoog waarin dit werk om die funksie van RdRp te inhibeer deur kovalent te bind aan en terminering van die ontluikende RNA te onderbreek deur vroeë of vertraagde terminering of om verdere verlenging van die RNA-polinukleotied te voorkom. [30] [31] Hierdie vroeë beëindiging lei tot niefunksionele RNA wat deur normale sellulêre prosesse afgebreek sal word.

    Die gebruik van RNA-afhanklike RNA-polimerase speel 'n groot rol in RNA-inmenging in eukariote, 'n proses wat gebruik word om geenuitdrukking stil te maak via klein interfererende RNA's (siRNA's) wat aan mRNA bind wat hulle onaktief maak. [32] Eukariotiese RdRp word aktief in die teenwoordigheid van dsRNA, maar RdRp is slegs teenwoordig in 'n geselekteerde subset van eukariote, insluitend C. elegans en P. tetraurelia. [33] Hierdie teenwoordigheid van dsRNA veroorsaak die aktivering van RdRp- en RNAi-prosesse deur die aanvang van RNA-transkripsie deur die bekendstelling van siRNA's in die sisteem te begin. [33] In C. elegans, is siRNA's geïntegreer in die RNA-geïnduseerde stilmaakkompleks, RISC, wat saam met mRNA's werk wat gerig is op inmenging om meer RdRps te werf om meer sekondêre siRNA's te sintetiseer en geenuitdrukking te onderdruk. [34]


    Metodes

    Die ssDNA–ssDBPs en ssRNA–ssRBPs sisteme bestudeer

    Vir 'n omvattende ontleding van 'n verskeidenheid interaksies tussen proteïene en enkelstrengs nukleïensure, het ons 12 komplekse bestudeer: ses ssDNA–ssDBP komplekse en ses ssRNA–ssRBP komplekse waarvan die driedimensionele strukture bekend is (opgesom in Tabel 1). Die stelle proteïen-DNA- en proteïen-RNA-komplekse sluit proteïene in wat verskillende funksies het, met voue van verskillende groottes, en met heterogene ssDNA/ssRNA met verskillende lengtes en volgordes. Die proteïene in hierdie ssDNA–ssDBP komplekse behoort aan verskillende strukturele domeine: die oligonukleotied/oligosakkaried-bindende (OB) vou, die RNA herkenningsmotief (RRM) domein en die K homologie (KH) domein. Ons let daarop dat die vier komplekse met OB-voue verskil in hul strukture (i.e., proteïenlengte) en rye. Net so is die ses ssRNA–ssRBP-komplekse ook gekies om verskillende strukturele domeine te dek, naamlik die OB-vou, RRM, PUF-domein, sink-vinger-domein, RAMP-proteïen en 'n Fab. Oor die algemeen het ons verskillende voue gedek waarin die elektrostatiese en aromatiese stapelenergiebydraes wissel van 'n baie hoë stapelenergiefraksie (die OB-vou) tot 'n hoë elektrostatiese energiefraksie (KH-domein en RAMP). Te oordeel aan die beskikbare strukture van die 12 komplekse wat hier bestudeer is en gebaseer op die beskikbare ongebonde strukture, blyk dit onwaarskynlik dat die proteïene 'n aansienlike konformasieverandering ondergaan om hul ssDNA/ssRNA ligande te bind. Die ssDNA- en ssRNA-molekules is baie meer buigsaam in oplossing as gevoude proteïene. Gevolglik kan 'n mens tot die gevolgtrekking kom dat die bindingsoppervlaktes in ssDBP's en ssRBP's vooraf gedefinieer is, en groot konformasieverandering vind slegs vir ssDNA/ssRNA plaas.


    Titel: RNA-afhanklike RNA-teikening deur CRISPR-Cas9

    Dubbelstring DNA (dsDNA) binding en splitsing deur Cas9 is 'n kenmerk van tipe II CRISPR-Cas bakteriële aanpasbare immuniteit. Daar word gemeen dat alle bekende Cas9-ensieme DNA uitsluitlik herken as 'n natuurlike substraat, wat beskerming bied teen DNA-faag en plasmiede. Hier wys ons dat Cas9-ensieme van beide subtipes II-A en II-C enkelstring-RNA (ssRNA) kan herken en klief deur 'n RNA-geleide meganisme wat onafhanklik is van 'n protospacer-aangrensende motief (PAM) volgorde in die teiken RNA. RNA-geleide RNA-splyting is programmeerbaar en plek-spesifiek, en ons vind dat hierdie aktiwiteit uitgebuit kan word om infeksie deur enkelstrengige RNA-faag in vivo te verminder. Ons demonstreer ook dat Cas9 PAM-onafhanklike onderdrukking van geenuitdrukking in bakterieë kan rig. Ten slotte dui hierdie resultate aan dat 'n subset van Cas9-ensieme die vermoë het om op beide DNA- en RNA-teikenvolgordes op te tree, en stel die potensiaal voor vir gebruik in programmeerbare RNA-teikentoepassings.


    RG-ryke herhalings - Die Switserse-weermagmes van proteïen-RNA-interaksies

    'n Gewone versteurde RNA-bindende motief in RBP's bestaan ​​uit herhalings van arginien en glisien, genoem RGG-bokse of GAR-herhalings. Hierdie rye is heterogeen, beide in aantal herhalings en in hul spasiëring. 'n Onlangse ontleding het hierdie RG-ryke streke in di- en tri-RG- en -RGG-bokse verdeel, en gevalle van sulke herhalings in volgorde van tiene (di- en tri-RGG) tot honderde (tri-RG) en byna tweeduisend geïdentifiseer (di-RG) proteïene [47]. Proteïene wat sulke herhalings bevat, is verryk in RNA metaboliese funksies [47]. Dit is egter nie tans duidelik of die verskillende herhalende argitekture afsonderlike funksionele handtekeninge verskaf nie.

    Die RGG-boks is die eerste keer in die heterogene kernribonukleoproteïenproteïen U (hnRNP-U, ook bekend as SAF-A) geïdentifiseer as 'n gebied wat voldoende is en benodig word vir RNA-binding (Tabel 1, Fig. 1). hnRNP-U het nie kanoniese RBD's nie, maar het semi-gestruktureerde SAP-domein betrokke by DNA-binding [48-50]. Daar is gevind dat hnRNP-U honderde nie-koderende RNA's teiken, insluitend klein kern (sn)RNA's betrokke by RNA-splyting, en 'n aantal lang nie-koderende (lnc)RNA's, op 'n RGG-boks-afhanklike wyse [51 ]. RGG-gemedieerde interaksie van hnRNP-U met die lncRNAs Xist [52] en PANDA [53] is geïmpliseer in epigenetiese regulering.

    RG[G]-gemedieerde RNA-binding speel ook 'n rol in kern-RNA-uitvoer, soos geïllustreer deur die kern-RNA-uitvoerfaktor 1 (NXF1). Terwyl NXF1 'n RRM huisves wat in staat is om RNA te bind [54], word die meeste van die in vivo RNA-bindingskapasiteit toegeskryf aan die RGG-bevattende, N-terminale streek [55] (Tabel 1). Die arginiene in hierdie motief speel 'n sleutelrol in die interaksie met RNA, wat bewys is dat dit volgorde-onafhanklik is, maar nodig is vir RNA-uitvoer [55]. NXF1 algehele affiniteit vir RNA is laag [55, 56], en vereis samewerking met die uitvoeradapter ALY/REF [57]. ALY/REF dra ook 'n N-terminale versteurde arginienryke streek wat lyk soos 'n RGG-boks [57] en bemiddel beide RNA-binding [54, 58, 59] en die interaksie met NXF1 [60]. Die aktivering van NXF1 word voorgestel om geaktiveer te word deur die vorming van 'n drieledige kompleks tussen ALY/REF en NXF1, waarin hul RG-ryke wanordelike streke 'n sentrale rol speel. Analoge volgordes is in virale proteïene geïdentifiseer en fasiliteer ook virale RNA-uitvoer deur kanoniese kernuitvoerpaaie te omseil (Tabel 1).

    Brose X verstandelike gestremde proteïen (FMRP) is nog 'n RBP met 'n goed gekarakteriseerde, RNA-bindende RGG-boks (Fig. 1). Betrokke by vertaling onderdrukking in die brein [61], verlies van FMRP aktiwiteit lei tot veranderinge in sinaptiese konnektiwiteit [62], verstandelike gestremdheid [63-65], en kan ook die aanvang van neurodegeneratiewe siektes bevorder [66]. Benewens sy RGG-boks, bevat FMRP twee KH-domeine wat bydra tot RNA-binding. Daar is getoon dat die RGG-boks van FMRP met hoë affiniteit met G-quadruplex RNA-strukture [67-77] interaksie het. Die RGG-boks is ongestruktureerd in sy ongebonde toestand [70, 78], maar vou by binding aan 'n guanienryke, gestruktureerde G-kwadrupleks in teiken-RNA [78] (Fig. 2). Beide arginiene en glisiene speel 'n sleutelrol in die funksie van die RGG-boks en vervanging van hierdie aminosure benadeel RNA-binding [78]. Die arginienresidu wat gebruik word om met RNA te reageer, wissel na gelang van die teiken-RNA [70, 76, 78]. Die FMRP RGG-boks teiken sy eie mRNA op 'n G-quadruplex-struktuur wat die RGG-boks kodeer [69]. Hierdie binding reguleer alternatiewe splitsing van FMRP mRNA proksimaal tot die G-kwartet, wat daarop dui dat dit die balans van FRMP isovorme outo-reguleer [74]. Verbasend genoeg het 'n onlangse transkriptoomwye studie van polisoom-geassosieerde FMRP geen verryking vir voorspelde G-quadruplex strukture in die 842 hoë-vertroue teiken mRNAs gevind nie [79]. Nog 'n studie het FMRP-bindingsplekke geïdentifiseer wat in spesifieke volgordemotiewe verryk is, waar die KH2-domeine na vore gekom het as die belangrikste spesifisiteitsdeterminante [80]. Hierdie resultate dui daarop dat die rol van RGG-boks in hierdie RBP beperk kan word om die algehele bindingsaffiniteit van die proteïen te verhoog, wat die volgorde-spesifieke interaksies ondersteun wat deur die KH2-domeine bemiddel word. Ons kan egter nie die moontlikheid van differensiële UV-kruisbindingsdoeltreffendheid van die KH2-domeine en die RGG-boks uitsluit nie, wat kan lei tot bevooroordeelde bindende handtekeninge in CLIP-studies.

    Strukturele voorbeelde RNA-gebonde wanordelike streke. a Die RGG-peptied van die menslike FMRP gebind aan a in vitro-geselekteerde guanienryke sc1 RNA bepaal deur KMR (PDB 2LA5) [78] b Basiese pleister van wanordelike bees-immuungebrekvirus (BIV) Tat vorm 'n β–draai wanneer dit met sy teiken-RNA, TAR, in wisselwerking tree. Struktuur bepaal deur KMR (PDB 1MNB) [91] c Dimer van die basiese pleister wat Rev-proteïen van die menslike immuniteitsgebrekvirus (MIV) bevat in kompleks met teiken-RNA, RRE, bepaal deur kristallografie [102] (PDB 4PMI). Rooi, peptiedgeel, RNA. Illustrasies is met PyMol geskep

    'n Aantal ander RBP's gebruik 'n RGG-herhalingsgebied om G-ryke en gestruktureerde RNA-teikens te teiken en is betrokke by neurologiese siektes sowel as kanker (Tabel 1). Hierdie RG-ryke streke kan beide onselektiewe en spesifieke interaksies met RNA bemiddel en kan betrokke wees by gevarieerde RNA metaboliese prosesse.


    Watter domeine of motiewe is in staat om aan beide DNA en RNA te heg? - Biologie

    CRISPR/Cas-stelsels voorsien bakterieë en archaea van aanpasbare immuniteit teen virusse en plasmiede deur crRNA's te gebruik om die stilswye van indringende nukleïensure te lei. Ons wys hier dat in 'n subset van hierdie stelsels, die volwasse crRNA basis-gepaard aan trans-aktiverende tracrRNA vorm 'n twee-RNA-struktuur wat die CRISPR-geassosieerde proteïen Cas9 rig om dubbelstrengs (ds) breek in teiken-DNS in te voer. Op plekke komplementêr tot die crRNA-gids volgorde, klief die Cas9 HNH nuklease domein die komplementêre string terwyl die Cas9 RuvC-agtige domein die nie-komplementêre string klief. Die dubbel-tracrRNA:crRNA, wanneer gemanipuleer as 'n enkele RNA chimera, rig ook volgorde-spesifieke Cas9 dsDNA-splyting. Ons studie onthul 'n familie van endonukleases wat dubbele RNA's gebruik vir plekspesifieke DNA-splyting en beklemtoon die potensiaal om die stelsel te ontgin vir RNA-programmeerbare genoomredigering.

    Bakterieë en archaea het RNA-gemedieerde adaptiewe verdedigingstelsels genaamd CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-geassosieerd) ontwikkel wat organismes teen indringer virusse en plasmiede beskerm (13). Hierdie verdedigingstelsels maak staat op klein RNA's vir volgorde-spesifieke opsporing en stilmaak van vreemde nukleïensure. CRISPR/Cas-stelsels is saamgestel uit cas gene georganiseer in operon(s) en 'n CRISPR-skikking wat bestaan ​​uit unieke genoom-teikenvolgorde (genoem spasieerders) afgewissel met identiese herhalings (13). CRISPR/Cas-bemiddelde immuniteit vind in drie stappe plaas. In die adaptiewe fase reageer bakterieë en archaea wat een of meer CRISPR-lokusse huisves op virale en plasmied-uitdaging deur kort fragmente van vreemde volgorde (protospacers) in die gasheerchromosoom aan die proksimale einde van die CRISPR-skikking te integreer (13). In die uitdrukkings- en interferensiefases, lewer transkripsie van die herhaal-spasieerder-element in voorloper CRISPR RNA (pre-crRNA) molekules gevolg deur ensiematiese splitsing die kort crRNA's wat kan basispaar met komplementêre protospacer-volgordes van indringer virale of plasmied teikens (411). Teikenherkenning deur crRNAs rig die stilmaak van die vreemde rye deur middel van Cas-proteïene wat in kompleks met die crRNAs funksioneer (10, 1220).

    Daar is drie tipes CRISPR/Cas-stelsels (2123). Die Tipe I- en III-stelsels deel 'n paar oorkoepelende kenmerke: gespesialiseerde Cas-endonukleases verwerk die pre-crRNA's, en sodra dit volwasse is, versamel elke crRNA in 'n groot multi-Cas-proteïenkompleks wat in staat is om nukleïensure aanvullend tot die crRNA te herken en te splits. In teenstelling hiermee verwerk tipe II-stelsels pre-crRNA's deur 'n ander meganisme waarin 'n transaktiverende crRNA (tracrRNA) komplementêr tot die herhalende volgordes in pre-crRNA verwerking deur die dubbelstring-RNA-spesifieke ribonuklease RNase III in die teenwoordigheid van die Cas9 (voorheen Csn1) proteïen (4, 24) (fig. S1). Daar word vermoed dat Cas9 die enigste proteïen is wat verantwoordelik is vir crRNA-geleide stilmaak van vreemde DNA (2527).

    Ons wys hier dat in Tipe II-stelsels, Cas9-proteïene 'n familie van ensieme uitmaak wat 'n basisgepaarde struktuur benodig wat tussen die aktiveer-tracrRNA en die teiken-crRNA gevorm word om teiken-dubbelstring (ds) DNA te klief. Plek-spesifieke splitsing vind plaas op plekke wat bepaal word deur beide basisparing komplementariteit tussen die crRNA en die teiken protospacer DNA en 'n kort motief (na verwys as die protospacer aangrensende motief, of PAM) naas die komplementêre streek in die teiken DNA. Ons studie demonstreer verder dat die Cas9-endonuklease-familie met enkele RNA-molekules geprogrammeer kan word om spesifieke DNS-plekke te splyt, en sodoende die opwindende moontlikheid verhoog om 'n eenvoudige en veelsydige RNA-gerigte stelsel te ontwikkel om dsDNA-breuke (DSB's) te genereer vir genoom-teikening en redigering.

    Cas9 is 'n DNA-endonuklease wat deur twee RNA's gelei word. Daar word vermoed dat Cas9, die kenmerkende proteïen van tipe II-stelsels, betrokke is by beide crRNA-rypwording en crRNA-geleide DNA-interferensie (fig. S1) (4, 2527). Cas9 is betrokke by crRNA rypwording (4), maar die direkte deelname daarvan aan teiken-DNS-vernietiging is nie ondersoek nie. Om te toets of en hoe Cas9 moontlik in staat is tot teiken-DNA-splyting, het ons 'n ooruitdrukkingstelsel gebruik om Cas9-proteïen wat van die patogeen afkomstig is, te suiwer Streptococcus pyogenes (fig. S2) en het sy vermoë getoets om 'n plasmied-DNS of 'n oligonukleotieddupleks wat 'n protospasieerdervolgorde aanvullend tot 'n volwasse crRNA, en 'n bona fide PAM dra, te kloof. Ons het gevind dat volwasse crRNA alleen nie in staat was om Cas9-gekataliseerde plasmied DNA-splyting te rig nie (Fig. 1A en Fig. S3A). Byvoeging van tracrRNA, wat kan basispaar met die herhalende volgorde van crRNA en noodsaaklik is vir crRNA rypwording in hierdie sisteem, het Cas9 egter geaktiveer om plasmied DNA te klief (Fig. 1A en Fig. S3A). Die splitsingsreaksie het beide magnesium en die teenwoordigheid van 'n crRNA-volgorde aanvullend tot die DNA vereis. 'n crRNA wat in staat was tot tracrRNA-basisparing, maar wat 'n nie-verwante teiken-DNA-bindende volgorde bevat het nie Cas9-gekataliseerde plasmiedsplyting ondersteun nie (Fig. 1A en fig. S3A, vergelyk crRNA-sp2 met crRNA-sp1 en fig. S4A). Soortgelyke resultate is verkry met 'n kort lineêre dsDNA-substraat (Fig. 1B en Fig. S3, B en C). Die trans-aktiverende tracrRNA is dus 'n klein nie-koderende RNA met twee kritieke funksies: sneller pre-crRNA verwerking deur die ensiem RNase III (4) en aktiveer daarna crRNA-geleide DNA-splyting deur Cas9.

    Cas9 is 'n DNA-endonuklease wat deur twee RNA-molekules gelei word. (A) Cas9 is geprogrammeer met 'n 42-nukleotied crRNA-sp2 (crRNA wat spasieerder 2 volgorde bevat) in die teenwoordigheid of afwesigheid van 75-nukleotied tracrRNA. Die kompleks is by sirkelvormige of XhoI-gelineariseerde plasmied DNA gevoeg wat 'n volgorde aanvullend tot spasieerder 2 en 'n funksionele PAM dra. crRNA-sp1, spesifisiteitskontrole M, DNA merker verwys na fig. S3A. (B) Cas9 is geprogrammeer met crRNA-sp2 en tracrRNA (nukleotiede 4-89). Die kompleks is geïnkubeer met dubbel- of enkelstring DNA's wat 'n volgorde aanvullend tot spasieerder 2 en 'n funksionele PAM (4). Die komplementêre of nie-komplementêre stringe van die DNA was 5'-radio-gemerk en uitgegloei met 'n nie-gemerkte vennoot string. Verwys na fig. S3, B en C. (C) Volgorde-analise van splitsingsprodukte van Fig. 1A. Beëindiging van primer-verlenging in die volgordebepalingsreaksie dui die posisie van die splitsingsplek aan. Die 3′ terminale A-oorhang (sterretjie) is 'n artefak van die volgordebepalingsreaksie. Verwys na fig. S5, A en C. (D) Die splitsingsprodukte van Fig. 1B is ontleed langs 5'-eind-gemerkte grootte merkers afgelei van die komplementêre en nie-komplementêre stringe van die teiken DNA dupleks. M, merker P, splitsingsproduk. Verwys na fig. S5, B en C. (E) Skematiese voorstelling van tracrRNA, crRNA-sp2 en protospacer 2 DNA-volgorde streke van crRNA komplementariteit tot tracrRNA (oranje) en die protospacer DNA (geel) word PAM-volgorde voorgestel, grys splitsingsplekke gekarteer in (C) en (D) word voorgestel deur blou pyle (C), 'n rooi pyl (D, komplementêre string) en 'n rooi lyn (D, nie-komplementêre string).

    Splyting van beide plasmied en kort lineêre dsDNA deur tracrRNA:crRNA-geleide Cas9 is plek-spesifiek (Fig. 1, C tot E, en Fig. S5, A en B). Plasmied DNA-splyting het stomp punte op 'n posisie drie basispare stroomop van die PAM-volgorde geproduseer (Fig. 1, C en E, en Fig. S5, A en C) (26). Net so, binne kort dsDNA-duplekse, word die DNA-string wat komplementêr is tot die teikenbindende volgorde in die crRNA (die komplementêre string) by 'n plek drie basispare stroomop van die PAM (Fig. 1, D en E, en Fig. S5, B en C). Die nie-komplementêre DNA-string word op een of meer plekke binne 3 tot 8 basispare stroomop van die PAM geklief. Verdere ondersoek het aan die lig gebring dat die nie-komplementêre string eers endonukleolities gekloof word en daarna deur 'n 3'-5' eksonuklease-aktiwiteit geknip word (fig. S4B). Die splitsingstempo's deur Cas9 onder enkelomsettoestande het gewissel van 0.3 tot 1 min -1, vergelykbaar met dié van restriksie-endonukleases (fig. S6A), terwyl inkubasie van wildtipe Cas9-tracrRNA:crRNA-kompleks met 'n 5-voudige molêre oormaat van substraat-DNS het bewys gelewer dat die dubbel-RNA-geleide Cas9 'n meervoudige-omset-ensiem is (fig. S6B). In teenstelling met die CRISPR Tipe I Cascade kompleks (18), Cas9 sny beide gelineariseerde en supergedraaide plasmiede (Fig. 1A en 2A). 'n Indringende plasmied kan dus in beginsel verskeie kere deur Cas9-proteïene wat met verskillende crRNA's geprogrammeer is, gesplit word.

    Cas9 gebruik twee nuklease-domeine om die twee stringe in die teiken-DNS te splits. (A) Bo: Skematiese voorstelling van Cas9-domeinstruktuur wat die posisies van domeinmutasies toon. Onder: Komplekse van wilde-tipe of nuklease-mutante Cas9-proteïene met tracrRNA:crRNA-sp2 is getoets vir endonuklease-aktiwiteit soos in Fig. 1A. (B) Komplekse van wilde-tipe Cas9 of nuklease domein mutante met tracrRNA en crRNA-sp2 is getoets vir aktiwiteit soos in Fig. 1B.

    Elke Cas9 nuklease-domein split een DNA-string. Cas9 bevat domeine wat homoloog is aan beide HNH en RuvC endonukleases (Fig. 2A en Fig. S7) (2123, 27, 28). Ons het Cas9-variante ontwerp en gesuiwer wat inaktiverende puntmutasies in die katalitiese residue van óf die HNH- óf RuvC-agtige domeine bevat (Fig. 2A en Fig. S7) (23, 27). Incubation of these variant Cas9 proteins with native plasmid DNA showed that dual-RNA–guided mutant Cas9 proteins yielded nicked open circular plasmids, while the wild-type Cas9 protein-tracrRNA:crRNA complex produced a linear DNA product (Figs. 1A and 2A and figs. S3A and S8A). This result indicates that the Cas9 HNH and RuvC-like domains each cleave one plasmid DNA strand. To determine which strand of the target DNA is cleaved by each Cas9 catalytic domain, we incubated the mutant Cas9-tracrRNA:crRNA complexes with short dsDNA substrates in which either the complementary or the noncomplementary strand was radiolabeled at its 5′ end. The resulting cleavage products indicated that the Cas9 HNH domain cleaves the complementary DNA strand, while the Cas9 RuvC-like domain cleaves the noncomplementary DNA strand (Fig. 2B and fig. S8B).

    Dual-RNA requirements for target DNA binding and cleavage. tracrRNA might be required for target DNA binding and/or to stimulate the nuclease activity of Cas9 downstream of target recognition. To distinguish between these possibilities, we used an electrophoretic mobility shift assay to monitor target DNA binding by catalytically inactive Cas9 in the presence or absence of crRNA and/or tracrRNA. Addition of tracrRNA substantially enhanced target DNA binding by Cas9, whereas little specific DNA binding was observed with Cas9 alone or Cas9-crRNA (fig. S9). This indicates that tracrRNA is required for target DNA recognition, possibly by properly orienting the crRNA for interaction with the complementary strand of target DNA. The predicted tracrRNA:crRNA secondary structure includes base-pairing between the 3′-terminal 22-nucleotides of the crRNA and a segment near the 5′ end of the mature tracrRNA (Fig. 1E). This interaction creates a structure in which the 5′-terminal 20 nucleotides of the crRNA, which vary in sequence in different crRNAs, are available for target DNA binding. The bulk of the tracrRNA downstream of the crRNA base-pairing region is free to form additional RNA structure(s) and/or to interact with Cas9 or the target DNA site. To determine whether the entire length of the tracrRNA is necessary for site-specific Cas9-catalyzed DNA cleavage, we tested Cas9-tracrRNA:crRNA complexes reconstituted using full-length mature (42-nt) crRNA and various truncated forms of tracrRNA lacking sequences at their 5′ or 3′ ends. These complexes were tested for cleavage using a short target dsDNA. A substantially truncated version of the tracrRNA retaining nucleotides 23-48 of the native sequence was capable of supporting robust dual-RNA–guided Cas9-catalyzed DNA cleavage (Fig. 3, A and C, and fig. S10, A and B). Truncation of the crRNA from either end showed that Cas9-catalyzed cleavage in the presence of tracrRNA could be triggered with crRNAs missing the 3′-terminal 10 nucleotides (Fig. 3, B and C). In contrast, a 10-nucleotide deletion from the 5′ end of crRNA abolished DNA cleavage by Cas9 (Fig. 3B). We also analyzed Cas9 orthologs from various bacterial species for their ability to support S. pyogenes tracrRNA:crRNA-guided DNA cleavage. In contrast to closely related S. pyogenes Cas9 orthologs, more distantly related orthologs were not functional in the cleavage reaction (fig. S11). Net so, S. pyogenes Cas9 guided by tracrRNA:crRNA pairs originating from more distant systems were unable to cleave DNA efficiently (fig. S11). Species specificity of dual-RNA–guided cleavage of DNA indicates co-evolution of Cas9, tracrRNA and the crRNA repeat, as well as the existence of a still unknown structure and/or sequence in the dual-RNA that is critical for the formation of the ternary complex with specific Cas9 orthologs.

    Cas9-catalyzed cleavage of target DNA requires an activating domain in tracrRNA and is governed by a seed sequence in the crRNA. (A) Cas9-tracrRNA:crRNA complexes were reconstituted using 42-nucleotide crRNA-sp2 and truncated tracrRNA constructs and assayed for cleavage activity as in Fig. 1B. (B) Cas9 programmed with full-length tracrRNA and crRNA-sp2 truncations was assayed for activity as in (A). (C) Minimal regions of tracrRNA and crRNA capable of guiding Cas9-mediated DNA cleavage (blue box). (D) Plasmids containing wild-type or mutant protospacer 2 sequences with indicated point mutations (right) were cleaved in vitro by programmed Cas9 as in Fig. 1A (left top) and used for transformation assays of wild-type or pre-crRNA-deficient S. pyogenes (left bottom). The transformation efficiency was calculated as CFU per μg of plasmid DNA error bars represent standard deviations for three biological replicates. (E) Plasmids containing wild-type and mutant protospacer 2 inserts with varying extent of crRNA-target DNA mismatches (right) were cleaved in vitro by programmed Cas9 (left). The cleavage reactions were further digested with XmnI. The 1880 bp and 800 bp fragments are Cas9-generated cleavage products.

    To investigate the protospacer sequence requirements for Type II CRISPR/Cas immunity in bacterial cells, a series of protospacer-containing plasmid DNAs harboring single-nucleotide mutations were analyzed for their maintenance following transformation in S. pyogenes and their ability to be cleaved by Cas9 in vitro. In contrast to point mutations introduced at the 5′ end of the protospacer, mutations in the region close to the PAM and the Cas9 cleavage sites were not tolerated in vivo and resulted in decreased plasmid cleavage efficiency in vitro (Fig. 3D). Our results are in agreement with a previous report of protospacer escape mutants selected in the Type II CRISPR system from S. thermophilus in vivo (27, 29). Furthermore, the plasmid maintenance and cleavage results hint at the existence of a “seed” region located at the 3′ end of the protospacer sequence that is crucial for the interaction with crRNA and subsequent cleavage by Cas9. In support of this notion, Cas9 enhanced complementary DNA strand hybridization to the crRNA and this enhancement was the strongest in the 3′-terminal region of the crRNA targeting sequence (fig. S12). Corroborating this, a contiguous stretch of at least 13 base pairs between the crRNA and the target DNA site proximal to the PAM is required for efficient target cleavage, while up to six contiguous mismatches in the 5′-terminal region of the protospacer are tolerated (Fig. 3E). These findings are reminiscent of the previously observed seed sequence requirements for target nucleic acid recognition in Argonaute proteins (30, 31) and the Cascade and Csy CRISPR complexes (13, 14).

    A short sequence motif dictates R-loop formation. In multiple CRISPR/Cas systems, recognition of self versus non-self has been shown to involve a short sequence motif that is preserved in the foreign genome, referred to as the PAM (27, 29, 3234). PAM motifs are only a few base pairs in length, and their precise sequence and position vary according to the CRISPR/Cas system type (32). In die S. pyogenes Type II system, the PAM conforms to an NGG consensus sequence, containing two G:C base pairs that occur one base pair downstream of the crRNA binding sequence, within the target DNA (4). Transformation assays demonstrated that the GG motif is essential for protospacer plasmid DNA elimination by CRISPR/Cas in bacterial cells (fig. S13A), consistent with previous observations in S. thermophilus (27). The motif is also essential for in vitro protospacer plasmid cleavage by tracrRNA:crRNA-guided Cas9 (fig. S13B). To determine the role of the PAM in target DNA cleavage by the Cas9-tracrRNA:crRNA complex, we tested a series of dsDNA duplexes containing mutations in the PAM sequence on the complementary or noncomplementary strands, or both (Fig. 4A). Cleavage assays using these substrates showed that Cas9-catalyzed DNA cleavage was particularly sensitive to mutations in the PAM sequence on the noncomplementary strand of the DNA, in contrast to complementary strand PAM recognition by Type I CRISPR/Cas systems (18, 34). Cleavage of target single-stranded DNAs was unaffected by mutations of the PAM motif. This observation suggests that the PAM motif is required only in the context of target dsDNA and may thus be required to license duplex unwinding, strand invasion, and the formation of an R-loop structure. Using a different crRNA-target DNA pair (crRNA-sp4 and protospacer 4 DNA), selected due to the presence of a canonical PAM not present in the protospacer 2 target DNA, we found that both G nucleotides of the PAM were required for efficient Cas9-catalyzed DNA cleavage (Fig. 4B and fig. S13C). To determine whether the PAM plays a direct role in recruiting the Cas9-tracrRNA:crRNA complex to the correct target DNA site, binding affinities of the complex for target DNA sequences were analyzed by native gel mobility shift assays (Fig. 4C). Mutation of either G in the PAM sequence substantially reduced the affinity of Cas9-tracrRNA:crRNA for the target DNA. This finding argues for specific recognition of the PAM sequence by Cas9 as a prerequisite for target DNA binding and possibly strand separation to allow strand invasion and R-loop formation, which would be analogous to the PAM sequence recognition by CasA/Cse1 implicated in a Type I CRISPR/Cas system (34).

    A PAM is required to license target DNA cleavage by the Cas9-tracrRNA:crRNA complex. (A) Dual RNA-programmed Cas9 was tested for activity as in Fig. 1B. Wild-type and mutant PAM sequences in target DNAs are indicated (right). (B) Protospacer 4 target DNA duplexes (labeled at both 5′ ends) containing wild-type and mutant PAM motifs were incubated with Cas9 programmed with tracrRNA (nt 23-89):crRNA-sp4. At indicated time points (min), aliquots of the cleavage reaction were taken and analyzed as in Fig. 1B. (C) Electrophoretic mobility shift assays were performed using RNA-programmed Cas9 (D10A/H840A) and protospacer 4 target DNA duplexes [same as in (B)] containing wild-type and mutated PAM motifs. Cas9 (D10A/H840A)-RNA complex was titrated from 100 pM to 1 μM.

    Cas9 can be programmed with a single chimeric RNA. Examination of the likely secondary structure of the tracrRNA:crRNA duplex (Figs. 1E and 3C) suggested the possibility that the features required for site-specific Cas9-catalyzed DNA cleavage could be captured in a single chimeric RNA. Although the tracrRNA:crRNA target selection mechanism works efficiently in nature, the possibility of a single RNA-guided Cas9 is appealing due to its potential utility for programmed DNA cleavage and genome editing (Fig. 5A). We designed two versions of a chimeric RNA containing a target recognition sequence at the 5′ end followed by a hairpin structure retaining the base-pairing interactions that occur between the tracrRNA and the crRNA (Fig. 5B). This single transcript effectively fuses the 3′end of crRNA to the 5′ end of tracrRNA, thereby mimicking the dual-RNA structure required to guide site-specific DNA cleavage by Cas9. In cleavage assays using plasmid DNA, we observed that the longer chimeric RNA was able to guide Cas9-catalyzed DNA cleavage in a manner similar to that observed for the truncated tracrRNA:crRNA duplex (Fig. 5B and fig. S14, A and C). The shorter chimeric RNA did not work efficiently in this assay, confirming that nucleotides 5-12 positions beyond the tracrRNA:crRNA base-pairing interaction are important for efficient Cas9 binding and/or target recognition. Similar results were observed in cleavage assays using short dsDNA as a substrate, which further indicate that the position of the cleavage site in target DNA is identical to that observed using the dual tracrRNA:crRNA as a guide (Fig. 5C and fig. S14, B and C). Finally, to establish whether the design of chimeric RNA might be universally applicable, we engineered five different chimeric guide RNAs to target a portion of the gene encoding the green-fluorescent protein (GFP) (fig. S15, A to C), and tested their efficacy against a plasmid carrying the GFP coding sequence in vitro. In all five cases, Cas9 programmed with these chimeric RNAs efficiently cleaved the plasmid at the correct target site (Fig. 5D and fig. S15D), indicating that rational design of chimeric RNAs is robust and could in principle enable targeting of any DNA sequence of interest with few constraints beyond the presence of a GG dinucleotide adjacent to the targeted sequence.

    Cas9 can be programmed using a single engineered RNA molecule combining tracrRNA and crRNA features. (A) Top: In Type II CRISPR/Cas systems, Cas9 is guided by a two-RNA structure formed by activating tracrRNA and targeting crRNA to cleave site-specifically target dsDNA (refer to fig. S1). Bottom: A chimeric RNA generated by fusing the 3′ end of crRNA to the 5′ end of tracrRNA. (B) A plasmid harboring protospacer 4 target sequence and a wild-type PAM was subjected to cleavage by Cas9 programmed with tracrRNA(4-89):crRNA-sp4 duplex or in vitro-transcribed chimeric RNAs constructed by joining the 3′ end of crRNA to the 5′ end of tracrRNA with a GAAA tetraloop. Cleavage reactions were analyzed by restriction mapping with XmnI. Sequences of chimeric RNAs A and B are shown with DNA-targeting (yellow), crRNA repeat-derived (orange) and tracrRNA-derived (light blue) sequences. (C) Protospacer 4 DNA duplex cleavage reactions were performed as in Fig. 1B. (D) Five chimeric RNAs designed to target the GFP gene were used to program Cas9 to cleave a GFP gene-containing plasmid. Plasmid cleavage reactions were performed as in Fig. 3E, except that the plasmid DNA was restriction mapped with AvrII following Cas9 cleavage.

    Conclusions. In summary, we identify a DNA interference mechanism involving a dual-RNA structure that directs a Cas9 endonuclease to introduce site-specific double-stranded breaks in target DNA. The tracrRNA:crRNA-guided Cas9 protein utilizes distinct endonuclease domains, HNH and RuvC-like, to cleave the two strands in the target DNA. Target recognition by Cas9 requires both a seed sequence in the crRNA and a GG dinucleotide-containing PAM sequence adjacent to the crRNA-binding region in the DNA target. We further show that the Cas9 endonuclease can be programmed with guide RNA engineered as a single transcript to target and cleave any dsDNA sequence of interest. The system is efficient, versatile and programmable by changing the DNA target-binding sequence in the guide chimeric RNA. Zinc-Finger Nucleases (ZFNs) and Transcription-Activator Like Effector Nucleases (TALENs) have attracted considerable interest as artificial enzymes engineered to manipulate genomes (3538). We propose an alternative methodology based on RNA-programmed Cas9 that could offer considerable potential for gene targeting and genome editing applications.


    Kink-turns in RNA

    The kink-turn (usually abbreviated to k-turn) is a widespread structural motif in RNA, first noted as a repeated structural element in the large ribosomal subunit by the Steitz lab (1). It introduces a very tight kink into the axis of helical RNA. It clearly plays an important structural role in RNA, and is significant in many aspects of RNA function including translation, modification and splicing, as well as genetic regulation.

    The sequence of Kt-7, an almost canonical k-turn found in the 50S subunit of the Haloarcula marismortui ribosome.

    The standard k-turn comprises a three-nucleotide bulge flanked on its 3 side by A G and G A basepairs (N-C stem), and on its 5 side by a section of regular basepairing (C stem) (1).

    The structure of a generic k-turn. A schematic of the structure is shown on the left, and the structure of Kt-7 in the H. marismortui ribosome is shown on the right.

    Kt-7 of the 23S rRNA of the H. marismortui ribosome is close to the consensus sequence for k-turns. The structure introduces a pronounced kink in the RNA (from whence its name), with an included angle between the axes of

    50 . The minor grooves of the two helices are juxtaposed, and the conformation is stabilized by interactions between the stacked adenosines of the A G basepairs and the C stem, and by stacking of the 5 and central bases of the bulge on the ends of the C and N-C stems respectively.

    The two A G basepairs at the center of the k-turn. The A G pairs of Kt-7 are shown in the molecular graphics.

    The A G basepairs are highly conserved. Both are trans sugar edge (G) to Hoogsteen edge (A) pairs, linked by potential hydrogen bonds G-N2 to A-N7 and A-N6 to G-N3. The 1b 1n pair is strongly buckled, while the bases of the 2n 2b pair are much closer to coplanarity. In some k-turns, the A-N6 to G-N3 hydrogen bond is not formed in the 2b 2n pair (see below). In the folded structure the minor groove edges of the two A G pairs are juxtaposed with the minor groove of the opposing C helix to participate in A-minor interactions (2). The structural principles of k-turn geometry have been analyzed using isostericity matrices (3).

    Many k-turns are bound by proteins in their natural state, at least some of which stabilize the kinked geometry. This is discussed further below.

    A general nucleotide numbering system for k-turns

    We have devised a universal nomenclature for the nucleotide positions in k-turns (4), thus avoiding the confusion that could arise from the many different numbering systems used in various crystal structures.

    A systematic nomenclature for the nucleotides of a k-turn.

    In this system, the unpaired bulge nucleotides are labelled Lj, where the index j is numbered sequentially from the 5 side. Unpaired nucleotides on the opposite strand (found in a small fraction of k-turns) are designated Lnj, with j increasing 5 to 3 . For all the remaining nucleotides, the two strands are differentiated by the suffix of b for the bulge-containing strand or n for the non-bulged strand. The nucleotides of the non-canonical stem are positively numbered from the first G A mismatch in the 5 to 3 direction relative to the bulged strand, while those of the canonical stem are negatively numbered in the 3 to 5 direction. The numbering system can accommodate loops of different sizes.

    Where are k-turns found ?

    Some k-turns have been identified unequivocally, from their structure in situ within crystal structures of larger RNA structures or complexes. Others are assumed by virtue of their sequence, and perhaps their homology with known k-turns in related species. The pronounced kink of the k-turn can also be observed using a variety of biophysical methods, such as FRET (5).

    The k-turn was first identified as a novel motif occurring multiple times in the ribosome (1,6,7). Further examples have been found in mRNA (8-11), and in riboswitches (12-16). They are very commonly found in C/D and H/ACA guide snoRNAs, and U3 snoRNA species (17-25). There is also a near-canonical k-turn in a stem-loop of the human U4 snRNA (26,27).

    Metal ion-induced folding of k-turns

    In order to adopt the tightly-kinked geometry, k-turn motifs require the presence of metal ions (5,27,28), or the binding of proteins (29). In the absence of either of these factors the RNA adopts a conformation that is more extended, like that of any three-nucleotide bulge in a duplex (30). The kinked conformation can be stabilized by addition of either divalent or monovalent metal ions (5), and folding can be treated as a two-state process.

    Ion-induced folding of Kt-7 observed by the increase in fluorescence resonance energy transfer between terminally-attached fluorophores that become closer when the RNA adopts the kink turn conformation.

    Folding is induced by micromolar concentrations of divalent ions, or millimolar concentrations of monovalent ions. For example, FRET analysis reveals that the folding of Kt-7 is achieved with a [Mg 2+ ]1/2 = 80 M, and a [Na + ]1/2 = 30 mM (4).

    There is no evidence for site-specific binding of metal ions to k-turns. It is likely that electrostatic interactions must be screened to allow the folded structure to achieve stability, and this is achieved by an ion atmosphere of loosely bound metal ions.

    This suggests a dynamic character for k-turn structures, where they sample both the kinked and a more extended geometry, consistent with fluorescent lifetime measurements (31). Computer modelling studies have suggested that k-turns undergo hinge-like motions on a fast timescale (32-34).

    Protein binding by k-turn RNA

    The majority of k-turns are known to bind one or more proteins. A few are not, including that found in the SAM riboswitch (12), although it remains possible that in the cellular environment even that too is bound by proteins.

    The archetypal k-turn binding protein is the ribosomal L7Ae and related proteins. These form a family of RNA-binding proteins including the eukaryotic and archaeal proteins L7Ae, L30e and S12e (35), the yeast Nhp2 and Snu13p proteins, and the human 15.5 kDa protein (36). Each of these proteins binds k-turn motifs in RNA, and some functional substitutions are tolerated (21). The assembly of the RNA methylation (box C/D) and pseudouridylation (box H/ACA) nucleoproteins are initiated by the binding of L7Ae-type proteins to k-turns contained within the guide RNA (37,38). The 15.5 kDa protein binds a k-turn of the U4 stem-loop in the U4-U6.U5 tri-snRNP (36). Crystal structures are available for the complexes of Archaeoglobus fulgidus L7Ae and box C/D RNA (22), Methanococcus jannaschii L7Ae and box H/ACA RNA (23) and the human 15.5 kDa protein and the U4 snRNA (26). In each case, the k-turn adopts the tightly-kinked conformation in the complex with the protein.

    Crystal structure of the L7Ae protein complexed with the box C/D k-turn (22).

    Even in the absence of added metal ions, the kinked conformation is induced by the binding of the protein. Moreover, the binding occurs with extremely high affinity. We have measured a dissociation constant of Kd = 10 pM for Archeoglobus fulgidus L7Ae binding to Kt-7 (39).

    The binding of L7Ae to Kt-7 observed by FRET. The k-turn becomes folded into the kinked geometry on binding L7Ae, and thus adopting the structure that exhibits high FRET efficiency. Thus the position of equilibrium can be followed in this manner. The data are fitted to a two-state model. However, the binding is so tight that only an upper limit can be measured for the Kd. An accurate value was measured using kinetic measurements of association and dissociation rates (39).

    In principle it could be imagined that k-turn folding could be promoted by protein binding in one of two ways, by passive selection of the kinked structure (conformational selection), or a more active process in which the protein manipulates the RNA structure (induced fit). The former requires an equilibrium between extended and folded k-turn structure in solution, as indicated in our measurement of fluorescent lifetimes (40). Real-time binding single-molecule experiments provide no evidence for a more extended intermediate down to a time resolution of 16 ms, supporting the conformational selection model (41).

    Stabilization of k-turn structure by tertiary interactions

    There is a third mechanism of stabilization, arising from longer-range tertiary interactions within the RNA. The k-turn introduces an abrupt transition in helical trajectory in the RNA helix that can be an important element in building the large-scale architecture of large RNA species. This is exemplified by the SAM-I riboswitch, where a long helix (P2) is kinked by a standard k-turn to allow the terminal loop to dock into a receptor in helix P4 (12,14).

    Schematic of the secondary structure of the SAM-I riboswitch. The k-turn is colored in our conventional manner. Note the loop-receptor interaction with the terminal loop of helix P2B.

    This stabilizes the global fold of the riboswitch that creates the binding pocket for its ligand S-adenosyl methionine (SAM - shown in red above), and removal of the k-turn completely prevents SAM binding. Conversely though, the tertiary structure of the RNA stabilizes the local structure of the k-turn. We observed that a G2nA substitution prevented the k-turn from folding as an isolated duplex, yet allowed the riboswitch to retain full function indicative of unperturbed folding (42). Solving the crystal structure of the modified SAM-I riboswitch showed that the k-turn was folded completely normally, despite the presence of an A A pair at the 2b 2n position.

    Crystal structure of the SAM-I riboswitch containing a k-turn that has been modified by a G2nA substitution. A. The 3D structure of the complete riboswitch. B. The structure of the k-turn (colored) superimposed with that of the unmodified sequence (grey).

    We conclude that the free energy of the tertiary interactions in the riboswitch is coupled to the folding of the k-turn so that an intrinsically unstable k-turn can fold normally.

    Thus we see that the folded structure of the k-turn can be stabilized in three ways : 1. By neutralization of charge on addition of metal ions. 2. By the binding of proteins, including members of the L7Ae family. 3. By tertiary interactions within larger RNA species. There is likely to be an interplay between these different processes in larger RNA-protein assemblies.

    Hydrogen bonding interactions that stabilize the kinked geometry

    The majority of k-turns have a G A pair at the 1b 1n position, and an A G pair at the 2b 2n position. Sequence substitution of any of the four nucleotides is usually highly detrimental to folding (5). Both are trans sugar edge (G) to Hoogsteen edge (A) pairs, linked by potential hydrogen bonds G-N2 to A-N7 and A-N6 to G-N3. We have found experimentally that all four hydrogen bonds are important to the stability of the kinked form of the RNA. But the G-N2 to A-N7 hydrogen bonds of the two G A pairs are the most critical to the stability of kinked form of Kt-7 in Mg 2+ ions, so that folding is completely prevented by G to inosine substitutions at either position (39).

    A cartoon summarizing the important hydrogen-bonding interactions involving O2 groups that stabilize the kinked geometry of the k-turn. Hydrogen bonds are indicated by the cyan arrows, with a thickness that reflects their conservation and importance in stabilizing the structure.

    In addition to the bonds linking the G A pairs, a number of critical hydrogen bonds involving O2 groups play a key role in stabilizing the kinked structure. These are summarized in the figure above. The most important are those in the core of the turn, and ribose-phosphate interactions around the bulge. These are strongly conserved in all k-turns, and critical to folding. Of these the single-most important hydrogen bond is one donated from the O2 of L1 ribose to the N1 of the A1n in the kink-proximal A G pair.

    The critical hydrogen bond formed between the O2 of L1 ribose to the N1 of the A1n.

    This is present in all known k-turn structures, and removal of the O2 from L1 completely prevents metal ion-induced folding (4). Another important hydrogen bond stabilizes the tight turn made at the loop of the k-turn. An interaction between the O2 of L3 and the proS non-bridging O of the phosphate between L1 and L2 bridges the neck of the turn, and is observed in most k-turn crystal structures. Removal of the O2 from L3 ribose in Kt-7 led to marked impairment of ion-induced folding (4).

    Two important hydrogen bonds in the loop, seen here in Kt-7. One is donated from the O2 of L3 to the proS non-bridging O of the phosphate between L1 and L2. The other is between the O2 of L2 and the proS O of the L2/L3 phosphate group.

    There is a further key hydrogen bond in the A-minor interaction between the C and NC helices, from the O2' of -1n to the nucleobase of the conserved adenine 2b. However, the acceptor can be N3 or N1, and this divides most of the known k-turn structures into two structural classes (31) . This is also true for the k-turns with an A A pair at the 2b 2n position. The rotation of the adenine nucleobase required to present either N3 or N1 as acceptor affects the basepairing with the base at the 2n position. For example, where this is guanine, the G A pair is bonded by two hydrogen bonds for the N3 class however in the N1 class the A2b N6 to G2n N3 is too long to be considered hydrogen bonded.

    Examples of the N3 and N1 classes of k-turn, showing the A-minor interaction between A2b and the O2' at the -1n position, and the hydrogen bonding between A2b and G2n. The red broken line in Kt-38 indicates an N-N distance of 4.7 , too long to be bonded.

    K-turns that depart from the consensus sequence

    Some k-turns break the rules of the standard motif. We divide these into two classes :
    1. The simple k-turns. These retain the same ordering of key nucleotides with respect to the primary sequence.
    2. The complex k-turns. In these k-turns the key nucleotides do not map onto the primary sequence in a linear way.

    Some of the simple k-turns have sequence changes that alter the seemingly essential G A pairs. This is well exemplified by Kt-23 of the small ribosomal subunit, that exhibits frequent changes from the consensus in the 2n position.

    WebLogo (43) summary of Kt-23 sequences

    In Kt-23 of Thermus thermophilus (6), the 2n nucleotide is uridine, forming a non-Watson-Crick A U pair with A2b. Although making the same change in Kt-7 totally prevents folding from occurring, Kt-23 folds efficiently on addition of magnesium ions, and its structure in the 30S ribosomal subunit is superimposable with that of Kt-7 (44).

    Overlay of the crystal structures of Kt-7 (blue) and Kt-23 (red), showing the closely similar positions of the adenine nucleobases at the 1n and 2b positions.

    A relatively rare subset of Kt-23 sequences have adenine at the 2n position, giving a potential A A pair at the 2b 2n position. One such example is found in Thelohania solenopsae. This can be folded into the k-turn conformation by the binding of L7Ae, or by tertiary interactions in the SAM-I riboswitch (45). The crystal structure of the latter construct shows that the RNA adopts standard k-turn geometry, with the 1n and 2b adenine nucleobases directed into the C helix minor groove as normal, and preservation of the standard hydrogen bonding pattern (45). This is an N1 structure, and consequently there is no hydrogen bond between A2b N6 and A2n N3 (N-N distance 4.7 ).

    The structure of the A2b A2n pair observed in the crystal structure of T. solenopsae Kt-23 (45). The electron density is the calculated composite omit map.

    The complex k-turns exhibit more radical departure from the simple k-turns such as H. marismortui Kt-7, where the key nucleotides appear to be out of order (as in T. thermophilus Kt-11), and can even pass between strands (as in H. marismortui Kt-15).

    The connectivity of the nucleotides in the structures of H. marismortui Kt-15, and T. thermophilus Kt-11.

    Despite the major departures of the sequences from the conventional k-turn, both form standard k-turn structures in the ribosome. We recommend naming the nucleotides according to their location in the structure, rather than their position in the linear sequence. Thus the adenine paired with G2n in Kt-15 is best termed 2b, even though it is covalently located on the non-bulged strand.

    In some species k-turns are functionally replaced by structures that are strongly kinked, yet not k-turns (if we define k-turns by the A G pairs and the juxtaposition of the N and NC helix minor grooves). Byvoorbeeld, die B. subtilis lysine riboswitch contains a probable k-turn that introduces a kink into the structure required for the tertiary structure (13), yet in Thermatoga maritima this is replaced by a sequence that is plainly not a k-turn (40). The latter may well be a point of flexibility rather than an intrinsic kink, but it functions in a similar manner in situ. In another example, RNase P of T. maritima contains a sequence (termed a pk turn) that is kinked similarly to a k-turn (47) , but lacks the G A pairs and cross-strand hydrogen bonding. However the pk-turn and the k-turn are functionally exchangeable between the SAM-I riboswitch and RNase P (40). In the crystal structure of the group I intron ribozyme of Azoarcus, Strobel and colleagues found a sequence that they termed the reverse k-turn (48). This motif has an A A pair at the putative 1b 1n position, and bends in the opposite direction compared to conventional k-turns, so that the major grooves become juxtaposed. We feel that this should not be classified as a k-turn because it lacks all the key elements that define a k-turn.

    A role for k-turns in building complex RNA structures ?

    Kink turns could play a key role in RNA architecture and the biogenesis of large assemblies. This includes the formation of functional RNA-protein complexes such as the box C/D in guided methylation, where the first even is the binding of an L7Ae-related protein to a k-turn. On a larger scale, the ribosome has many k-turns that are bound by a variety of different proteins. The dynamic character of the k-turn should provide opportunity for RNA to explore conformational space, but once folded the tight kink can provide long-range organization of the structure in a delicate interplay between the local structure and tertiary interactions. This may provide flexibility during the assembly of the structure, with subsequent fixation by the high-affinity binding of specific proteins to k-turns.


    Erkennings

    We thank K. Makarova for assistance with the PSIBLAST search and S. Gottesman, S. Melamed, M. Raina, T. Updegrove, J. Vogel, and Z. Wroblewska for comments. Research in the M.O. lab is supported by the National Science Centre in Poland (grant no. 2014/15/B/NZ1/03330), KNOW RNA Research Centre in Poznań (grant no. 01/KNOW2/2014), and the Foundation for Polish Science (grant no. TEAM/2011-8/5), co-financed by the European Union Regional Development Fund. Research in the G.S. lab is supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Die skrywers verklaar geen botsing van belange nie.

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