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Kan verskillende proteïene geproduseer word tydens translasie van 'n enkele mRNA in eukariote?

Kan verskillende proteïene geproduseer word tydens translasie van 'n enkele mRNA in eukariote?


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Is daar 'n translasiemeganisme wat eukariote kan gebruik om verskillende proteïene van 'n enkele getranskribeerde mRNA te produseer?


Daar is verskeie meganismes wat bekend is om te lei tot vertaling van wesenlik (of heeltemal) verskillende proteïene vanaf 'n enkele mRNA. Terwyl hierdie meganismes meer tipies in virusse gesien word, fokus ek op voorbeelde wat binne die endogene transkripsie van eukariote gedokumenteer is.

Alternatiewe vertaling inisiëring

Een proses wat daartoe kan lei dat verskillende proteïene vanaf dieselfde mRNA in eukariote vertaal word, is die gebruik van alternatiewe translasie-inisiasieplekke.1,2 Translasie begin tipies met 'n pre-inisiasie kompleks wat die 5' cap herken en op die mRNA laai. Hierdie kompleks skandeer totdat dit 'n gepaste beginplek vind. Die keuse van beginplek hang af van hoe goed die ribonukleotiedvolgorde ooreenstem met die Kozak-konsensusvolgorde.3 As die streek rondom die eerste AUG nie 'n goeie pasmaat vir daardie konsensus is nie, vind 'n proses bekend as lekkende skandering plaas en die voorinisiasiekompleks kan langs die mRNA voortgaan totdat 'n "goeie" beginplek gevind word.

Alhoewel dit kan lei tot proteïene met verskillende funksies, sal hulle tipies steeds groot streke van aminosuurvolgorde in gemeen hê. Een voorbeeld hiervan is in 'n kinase bekend as MK2, wat "'n sleutelreguleerder van transkripsie, migrasie, doodsein en post-transkripsionele geenregulering" is.4

Polisistroniese mRNA's

Alhoewel dit meer algemeen in prokariote voorkom, het eukariote in sommige gevalle ook polisistroniese transkripsies5,6. Hierdie transkripsies kodeer verskeie afsonderlike proteïene (d.w.s. van onafhanklike oop leesrame). Die voorbeelde wat ek vir soogdiere gevind het, is almal bisistronies (operone met twee gene): LASS1-GDF1, SNRPN-SNURF, MTPN-LUZP6 en MFRP-C1QTNF5. Jy kan vir daardie geenpare soek, maar dit lyk of daar nie 'n groot hoeveelheid inligting beskikbaar is nie en in baie gevalle is een van die gene amper heeltemal ongekarakteriseerd. Eukariotiese operone (ook bekend as polisistroniese mRNA's) is alomteenwoordig in tripanosome, blyk baie algemeen in aalwurms (ronde wurms) te wees en word ook gereeld in Drosophila ('n vlieg) gesien.6

Vertaling deurlees

Vertaling deurlees aka. stopkodononderdrukking vind plaas wanneer 'n in-raam stopkodon óf stogasties óf onder spesifieke toestande geïgnoreer word en translasie verder as daardie punt voortduur. Dit lei tot 'n C-terminale verlenging van die proteïen wat in sommige gevalle getoon is dat dit funksionele implikasies het7,8. 'n Voorbeeld hiervan is die [PSI+] prion in gis, wat translasielees deur die gistranskriptoom bevorder deur 'n faktor wat betrokke is by translasieterminasie te inaktiveer.7

Translasieraamverskuiwings

Dit word goed gedek in die antwoord deur @Dirigible.

Verwysings:

1: Kochetov, A. V. (2008). Alternatiewe translasie-beginplekke en verborge koderingspotensiaal van eukariotiese mRNA's. Bioessays, 30(7), 683-691.

2: Wan, J., & Qian, S. B. (2013). TISdb: 'n databasis vir alternatiewe translasie-inisiasie in soogdierselle. Nukleïensure-navorsing, 42(D1), D845-D850.

3: Acevedo, J. M., Hoermann, B., Schlimbach, T., & Teleman, A. A. (2018). Veranderinge in globale translasie-verlenging of inisiasietempo's vorm die proteoom via die Kozak-volgorde. Wetenskaplike verslae, 8(1), 4018. 4: Trulley, P., Snieckute, G., Bekker-Jensen, D., Menon, MB, Freund, R., Kotlyarov, A.,... & Gaestel, M. ( 2019). Alternatiewe vertaling inisiëring genereer 'n funksioneel duidelike isovorm van die stres-geaktiveerde proteïen kinase MK2. Sel verslae, 27(10), 2859-2870.

5: Tautz, D. (2008). Policistroniese peptiedkoderende gene in eukariote - hoe wydverspreid is dit? Briefings in Functional Genomics and Proteomics, 8(1), 68-74.

6: Blumenthal, T. (2004). Operone in eukariote. Briefings in Functional Genomics, 3(3), 199-211.

7: Schueren, F., & Thoms, S. (2016). Funksionele translasielees: 'n sisteembiologie-perspektief. PLoS genetika, 12(8), e1006196.

8: Loughran, G., Jungreis, I., Tzani, I., Power, M., Dmitriev, R.I., Ivanov, I.P.,... & Atkins, J.F. (2018). Stop kodon deurlees genereer 'n C-terminaal verlengde variant van die menslike vitamien D reseptor met verminderde kalsitriol reaksie. Tydskrif vir Biologiese Chemie, 293(12), 4434-4444.


Afsonderlik van die alternatiewe vertaling begin webwerf meganisme wat tyersome beskryf is geprogrammeerde translasie raamverskuiwing. Dit is onafhanklik van alternatiewe splitsing of post-translasionele wysigings, en gebeur wanneer 'n ribosoom leesrame verander terwyl 'n proteïen reeds vertaal word. Tipies word hierdie verskynsel geassosieer met virale vertaling, en laat virusse toe om baie proteïene op relatief kort genome te enkodeer. Neem MIV as 'n voorbeeld: die poliproteïen gag-pol vereis doeltreffende -1 raamverskuiwing vir uitdrukking van die individuele gag- en pol-geenprodukte.

In eukariote is voorbeelde meer yl. Kyk na hierdie resensie van 2012 --

… voorbeelde van soogdiergene wat −1 raamverskuiwing gebruik, is die muis embrioniese karsinoom differensiasie gereguleerde (EDR) geen en sy menslike ortoloog PEG10. 'n Gladde volgorde van G GGA AAC, in kombinasie met 'n pseudoknot, bemiddel hoogs doeltreffende −1 raamverskuiwing, soortgelyk aan virale raamverskuiwingsmotiewe (Clark et al., 2007). Onlangs is 'n geprogrammeerde ribosomale -1 raamverskuiwing in die adenomatous polyposis coli (APC) mRNA in Caenorhabditis elegans geïdentifiseer wat bemiddel word deur 'n gladde volgorde A AAA AAA of A AAA AAC (Baranov et al., 2011). Die funksionele relevansie van hierdie raamverskuiwing is onseker.

Raamverskuiwingsgebeurtenisse word dikwels bemiddel deur bewaarde RNA sekondêre strukture, soos pseudoknote en stamlusse. Sien die volgende publikasies vir 'n paar spesifieke voorbeelde van die tipes strukture wat by raamverskuiwing betrokke is:

Identifikasie van 'n nuwe antisiem mRNA +1 Raamverskuiwing Stimulerende Pseudoknot in 'n subset van diverse ongewerwelde diere en die oënskynlike afwesigheid daarvan in intermediêre spesies

Strukturele ondersoek en mutageniese analise van die stam-lus benodig vir Escherichia coli dnaX ribosomale raamverskuiwing: geprogrammeerde doeltreffendheid van 50%

−1 Raamverskuiwing by 'n CGA AAG-heksanukleotiedperseel word vereis vir transposisie van invoegingsvolgorde IS1222


Enkele meer onlangse voorbeelde van (potensiële) geprogrammeerde translasieraamverskuiwings in eukariote:

Soek vir potensiële leesraamverskuiwings in cd's van Arabidopsis thaliana en ander genome

Voordruk: Uitgebreide geprogrammeerde ribosomale raamverskuiwing in die mens soos onthul deur 'n massief parallelle verslaggewertoets


KOPPELING TRANSKRIPSIE EN VERTALING IN EUKARIOTES [O’SHEA LAB]

Die sentrale dogma van molekulêre biologie stel voor dat inligting van DNA na RNA vloei deur die proses van transkripsie en van RNA na proteïen deur translasie. In eukariote word aan hierdie twee prosesse gedink as ontkoppel: kernfaktore beheer transkripsie, en 'n ander stel faktore beheer translasie in die sitoplasma. Gedurende tye van stres, vertoon selle groot transkripsionele veranderinge, insluitend die opregulering van baie gene wat belangrik is vir oorlewing. Gelyktydig met hierdie groot transkripsieveranderinge, verminder selle die algehele proteïensintese dramaties, waardeur die stresreaksie op die vlak van proteïenuitdrukking gedemp word. Brian Zid en Erin O’Shea het gewonder of daar dalk voorkeurvertaling van transkripsies onder stres is. Om uit te vind, het hulle glukosehongersnood in gis bestudeer, 'n toestand waarin vertaling vinnig onderdruk word terwyl groot transkripsieveranderinge plaasvind.

Deur gebruik te maak van ribosoom-profilering, 'n tegniek wat die aantal ribosome op mRNA's meet met behulp van volgende generasie volgordebepaling, het die skrywers gevind dat 'n subset van transkripsie-opgereguleerde mRNA's verkieslik vertaal is tydens glukosehonger. 'n Bewaarde verskynsel by stres is die vorming van mRNA-proteïenaggregate, wat stresgranules genoem word. Deur gebruik te maak van fluoresserende mikroskopie, het hulle gevind dat mRNA's wat verkieslik vertaal is, diffuus deur die sitoplasma gebly het, terwyl daar gevind is dat transkripsie-opgereguleerde maar swak vertaalde mRNA's in streskorrels saamvoeg.

Verbasend genoeg word die inligting wat differensiële lokalisering en proteïenproduksie van hierdie twee klasse mRNA's spesifiseer, nie direk in die mRNA-volgorde gevind nie, maar word eerder gekodeer in die promotorvolgorde wat mRNA-produksie aandryf. Die skrywers het bevind dat promotorreaksie op die transkripsiefaktor hitteskokfaktor (Hsf1) diffuse sitoplasmiese lokalisering en hoër proteïenproduksie na glukosehonger spesifiseer, terwyl promotorelemente stroomop van swak vertaalde mRNA's hierdie mRNA's rig na stresgranules onder glukosehonger.

Dit verander die huidige paradigma dat transkripsie en vertaling in eukariote ontkoppel word, maar die skrywers vind dat hierdie ruimtelik afsonderlike prosesse gekoppel word tydens voedingsbeperking. 'n Koppeling tussen transkripsionele regulering en sitoplasmiese lokalisering kan 'n algemene aanpassing wees gedurende tye van stres, wat die sel in staat stel om die produksie van hele klasse proteïene te koördineer. Onder nie-stres toestande sal opregulering van 'n klas transkripsies deur 'n transkripsiefaktor soortgelyke hoeveelhede proteïen van elk van die mRNA's produseer, aangesien translasie teen 'n algemeen hoë tempo sal voortgaan. Onder stresvolle toestande wanneer algehele translasie verminder word, kan selektiewe translasie nodig wees om proteïene te produseer wat nodig is vir aanpassing by die nuwe toestand. Dit dui daarop dat die vertaling van geenstelle gekoördineer kan word deur 'n enkele kernfaktor te gebruik, sonder dat dit nodig is om die volgorde van elke mRNA te moduleer.

Hierdie werk, wat in die 7de Augustus-uitgawe van Nature sal verskyn, bied 'n nuwe rigting om te ondersoek of promotorafhanklike mRNA-lokalisering proteïenuitdrukking in 'n verskeidenheid seltoestande reguleer. Die O'Shea-laboratorium werk tans daaraan om faktore te identifiseer wat mede-transkripsie op mRNA's gelaai kan word om 'n mRNA se lot te bepaal.


Biochemie. 5de uitgawe.

Die basiese plan van proteïensintese in eukariote en archaea is soortgelyk aan dié in bakterieë. Die belangrikste strukturele en meganistiese temas kom terug in alle lewensdomeine. Eukariotiese proteïensintese behels egter meer proteïensintese as prokariotiese proteïensintese, en sommige stappe is meer ingewikkeld. Enkele noemenswaardige ooreenkomste en verskille is soos volg:

Ribosome. Eukariotiese ribosome is groter. Hulle bestaan ​​uit 'n 60S groot subeenheid en 'n 40S klein subeenheid, wat bymekaar kom om 'n 80S deeltjie te vorm met 'n massa van 4200 kd, vergeleke met 2700 kd vir die prokariotiese 70S ribosoom. Die 40S subeenheid bevat 'n 18S RNA wat homoloog is aan die prokariotiese 16S RNA. Die 60S-subeenheid bevat drie RNA's: die 5S- en 28S-RNA's is die eweknieë van die prokariotiese 5S- en 23S-molekules, sy 5.8S-RNA is uniek aan eukariote.

Inisieerder tRNA. In eukariote is die aanvangsaminosuur eerder metionien as N-formielmetionien. Soos in prokariote, neem 'n spesiale tRNA egter deel aan inisiasie. Hierdie aminoasiel-tRNA word Met-tRNA genoemi of Met-tRNAf (die subskripsie “i” staan ​​vir inisiasie, en 𠇏” dui aan dat dit in vitro geformileer kan word).

Inisiasie. Die aanvangskodon in eukariote is altyd AUG. Eukariote, in teenstelling met prokariote, gebruik nie 'n spesifieke purienryke volgorde aan die 5′ kant om inisieerder AUG's van interne te onderskei nie. In plaas daarvan word die AUG naaste aan die 5′ einde van mRNA gewoonlik gekies as die beginplek. 'n 40S-ribosoom heg aan die doppie aan die 5′-punt van eukariotiese mRNA (Afdeling 28.3.1) en soek na 'n AUG-kodon deur stap-vir-stap in die 3′-rigting te beweeg (Figuur 29.33). Hierdie skanderingsproses in eukariotiese proteïensintese word aangedryf deur helikases wat ATP hidroliseer. Paring van die antikodon van Met-tRNAi met die AUG-kodon van mRNA aandui dat die teiken gevind is. In byna alle gevalle het eukariotiese mRNA net een beginplek en is dus die sjabloon vir 'n enkele proteïen. Daarteenoor kan 'n prokariotiese mRNA veelvuldige Shine-Dalgarno-volgordes hê en dus beginplekke, en dit kan dien as 'n sjabloon vir die sintese van verskeie proteïene. Eukariote gebruik baie meer inisiasiefaktore as prokariote, en hul wisselwerking is baie meer ingewikkeld. Die voorvoegsel eIF dui 'n eukariotiese inisiasiefaktor aan. Byvoorbeeld, eIF-4E is 'n proteïen wat direk aan die 7-metielguanosienkap bind (Afdeling 28.3.1), terwyl eIF-4A 'n helikase is. Die verskil in inisiasiemeganisme tussen prokariote en eukariote is deels 'n gevolg van die verskil in RNA-prosessering. Die 5′ einde van mRNA is onmiddellik na transkripsie in prokariote vir ribosome beskikbaar. Daarteenoor moet pre-mRNA verwerk en na die sitoplasma in eukariote vervoer word voordat translasie begin word. Daar is dus ruim geleentheid vir die vorming van komplekse sekondêre strukture wat verwyder moet word om seine in die volwasse mRNA bloot te lê. Die 5′ pet bied 'n maklik herkenbare beginpunt. Daarbenewens bied die kompleksiteit van eukariotiese translasie-inisiasie nog 'n meganisme vir geenuitdrukking wat ons verder sal ondersoek in Hoofstuk 31.

Verlenging en beëindiging. Eukariotiese verlengingsfaktore EF1α en EF1βγ is die eweknieë van prokariotiese EF-Tu en EF-Ts. Die GTP-vorm van EF1α lewer aminoasiel-tRNA aan die A-plek van die ribosoom, en EF1βγ kataliseer die uitruil van GTP vir gebonde BBP. Eukariotiese EF2 bemiddel GTP-gedrewe translokasie op baie dieselfde manier as prokariotiese EF-G. Beëindiging in eukariote word uitgevoer deur 'n enkele vrystellingsfaktor, eRF1, in vergelyking met twee in prokariote. Ten slotte verhoed eIF3, soos sy prokariotiese eweknie IF3, die herassosiasie van ribosomale subeenhede in die afwesigheid van 'n inisiasiekompleks.

Figuur 29.33

Eukariotiese Vertaling Inisiasie. In eukariote begin translasie-inisiasie met die samestelling van 'n kompleks op die 5′ pet wat die 40S subeenheid en Met-tRNA insluiti. Gedryf deur ATP-hidrolise, skandeer hierdie kompleks die mRNA tot die eerste AUG (meer. )


Messenger RNA wysigings

Translasie vind in die sitoplasma plaas. Nadat mRNA die kern verlaat het, moet dit verskeie modifikasies ondergaan voordat dit vertaal word. Gedeeltes van die mRNA wat nie vir aminosure kodeer nie, genoem introne, word verwyder. 'n Poli-A-stert, wat uit verskeie adenienbasisse bestaan, word aan die een kant van die mRNA gevoeg, terwyl 'n guanosientrifosfaatdop aan die ander kant gevoeg word. Hierdie wysigings verwyder onnodige afdelings en beskerm die punte van die mRNA-molekule. Sodra alle modifikasies voltooi is, is mRNA gereed vir vertaling.


Eienskappe van die mRNA van Prokariote en Eukariote

mRNA wat deur die transkripsieproses geproduseer word, staan ​​ook bekend as mRNA-transkripsies. Alhoewel hulle 'n aantal soortgelyke eienskappe het, het hulle ook verskeie verskille. Die prokariotiese mRNA-transkripsie kan in 'n aantal dele/afdelings verdeel word wat insluit: die nie-koderende streek (geleë aan die 5'-punt van die transkripsie), die Shine-Dalgarno-volgorde, 'n tweede nie-koderende streek, die beginkodon , die koderende streek, stopkodon en 'n ander nie-koderende gebied aan die 3'-kant.

Die eukariotiese mRNA, aan die ander kant, begin met 'n 5'-dop en bestaan ​​uit 'n guaniennukleotied. Hierdie nukleotied is aan 'n metielgroep geheg en aan die naburige nukleotied gebind. Die guaniennukleotied is aan die nie-koderende gebied geheg, soortgelyk aan die een in prokariotiese mRNA. Die volgende afdeling is die beginkodon waaruit die koderinggebied strek.

Die koderinggebied eindig by die stopkodon. Dit word gevolg deur 'n nie-koderende streek en laastens die poli-A-stert (wat uit adeniene bestaan ​​en kan uit soveel as 2200 nukleotiede bestaan) aan die 3'-punt. In eukariote verhoed die 5'-dop en die poli-A-stert dat die mRNA afgebreek word.

Hier is dit belangrik om te onthou dat in eukariote die mRNA vrygestel moet word in die sitoplasma waar translasie plaasvind. Daarom speel die twee afdelings 'n belangrike rol in die handhawing van die integriteit van die mRNA. By prokariote kan transkripsie en translasie gelyktydig plaasvind en dus is hierdie afdelings nie nodig nie.

Anders as die eukariote transkripsie hoef hierdie mRNA nie 'n lang afstand vervoer te word nie en kom dus nie verskeie ensieme teë wat dit waarskynlik sal afbreek nie. As gevolg hiervan het die mRNA in prokariote nie bykomende beskerming nodig om skade te voorkom nie.

Soos genoem, vertaling is die proses waardeur die boustene van proteïene (polipeptiede/aminosuurkettings) gebou word deur gebruik te maak van die inligting vervat in die mRNA. Dit is 'n belangrike proses aangesien dit proteïene produseer wat benodig word vir verskeie selfunksies.

Om die proses te verstaan, is dit belangrik om sommige van die komponente en terminologieë wat in vertaling gebruik word, te ken.

Afgesien van mRNA (boodskapper-RNA), sluit hulle in:

· Polipeptiede - Kettings van aminosure en is die molekules waaruit proteïene bestaan.

· Nukleotiede - Strukturele komponente van DNA en RNA. Hulle bestaan ​​self uit nukleosied en fosfaat en sluit adenien, timien, sitosien en guanien (sowel as Uracil) in.

· Kodons - 'n Groep wat uit drie nukleotiede bestaan ​​- AUG is byvoorbeeld 'n goeie voorbeeld van 'n kodon - Terwyl kodons dien as die boustene van aminosure, stop ander die proses sodra die polipeptied voltooi is.

· tRNA (oordrag RNA) - Tree op as die brug tussen mRNA-kodons en aminosure.

· Ribosoom - Ribosoom bestaan ​​uit rRNA, en proteïen en is die strukture waarin polipeptiede vervaardig word.


Die begin van mRNA word nie vertaal nie

Interessant genoeg stem nie alle streke van 'n mRNA-molekule met spesifieke aminosure ooreen nie. Daar is veral 'n area naby die 5'-punt van die molekule wat bekend staan ​​as die onvertaalde streek (UTR) of leiervolgorde. Hierdie gedeelte van mRNA is geleë tussen die eerste nukleotied wat getranskribeer word en die beginkodon (AUG) van die koderende streek, en dit beïnvloed nie die volgorde van aminosure in 'n proteïen nie (Figuur 3).

So, wat is die doel van die UTR? Dit blyk dat die leiervolgorde belangrik is omdat dit 'n ribosoombindingsplek bevat. By bakterieë staan ​​hierdie terrein bekend as die Shine-Dalgarno-boks (AGGAGG), na wetenskaplikes John Shine en Lynn Dalgarno, wat dit die eerste keer gekenmerk het. ’n Soortgelyke terrein in gewerwelde diere is deur Marilyn Kozak gekenmerk en staan ​​dus bekend as die Kozak-boks. In bakteriële mRNA is die 5' UTR gewoonlik kort in menslike mRNA, die mediaan lengte van die 5' UTR is ongeveer 170 nukleotiede. As die leier lank is, kan dit regulatoriese volgordes bevat, insluitend bindingsplekke vir proteïene, wat die stabiliteit van die mRNA of die doeltreffendheid van sy translasie kan beïnvloed.



Bykomende verwerking

Voordat die mRNA die kern verlaat, word dit twee beskermende "pette" gegee wat verhoed dat die punte van die string afbreek tydens sy reis. Daar word na die twee punte van 'n DNS-string verwys as 3' en 5', wat verwys na die posisie van suikermolekules in die DNS. Die 5′-dop word op die 5′-punt van die mRNA geplaas. Die poli-A stert, wat aan die 3'-punt geheg is, is gewoonlik saamgestel uit 'n lang ketting van adenien (A) nukleotiede. Hierdie veranderinge beskerm die twee punte van die RNA om deur ander ensieme in die sel afgebreek te word.

'n Nukleotiedsuiker is saamgestel uit 5 koolstofstowwe wat elkeen genommer is (die apostrof " ' " word "prima" genoem). Fosfate bind aan die 3′ koolstof en die 5′ koolstof van elke suiker. Die een kant van 'n DNA- of RNA-molekule eindig met die 3'-koolstof wat blootgestel is en die ander kant eindig met 'n fosfaat wat aan die laaste suiker se 5'-punt geheg is. Dit is hoekom daar na die een kant verwys word as 3' en die ander kant word na verwys as 5'.


Kan verskillende proteïene geproduseer word tydens translasie van 'n enkele mRNA in eukariote? - Biologie

Die sentrale dogma hang af van die bestaan ​​en eienskappe van 'n leër van mRNA-molekules wat kortstondig in die proses van transkripsie tot stand gebring word en dikwels, kort daarna, weggedegradeer word. Gedurende die kort tyd wat hulle in 'n sel gevind word, dien hierdie mRNA's as 'n templaat vir die skepping van 'n nuwe generasie proteïene. Die vraag wat in hierdie vignet gestel word, is die volgende: Wat is gemiddeld die verhouding van vertaalde boodskap tot die boodskap self?

Alhoewel daar baie faktore is wat die proteïen-mRNA-verhouding beheer, dui die eenvoudigste model op 'n skatting in terme van slegs 'n paar sleutelkoerse. Om dit te sien, moet ons 'n eenvoudige "koersvergelyking" skryf wat ons vertel hoe die proteïeninhoud in 'n baie klein tydjie sal verander. Meer presies, ons soek die funksionele afhanklikheid tussen die aantal proteïenkopieë van 'n geen (p) en die aantal mRNA-molekules (m) wat dit voortbring. Die tempo van vorming van p is gelyk aan die tempo van translasie keer die aantal boodskappe, m, aangesien elke mRNA-molekule self as 'n proteïenbron beskou kan word. Terselfdertyd word nuwe proteïene egter gesintetiseer, en proteïenafbraak neem proteïene geleidelik uit sirkulasie. Verder is die aantal proteïene wat afgebreek word gelyk aan die tempo van degradasie keer die totale aantal proteïene. Hierdie omslagtige woorde kan baie meer elegant ingekapsuleer word in 'n vergelyking wat ons vertel hoe in 'n klein oomblik van tyd die aantal proteïene verander, nl.

waar α die degradasietempo is en β die vertaaltempo is (alhoewel die literatuur ongelukkig verskeur is tussen diegene wat die notasie op hierdie manier definieer en diegene wat die letters met presies die teenoorgestelde betekenis gebruik).

Figuur 1: Ribosome op mRNA as krale op 'n tou (van: http://bass.bio.uci.edu/

Ons stel belang in die bestendige toestand-oplossing, dit wil sê wat gebeur nadat 'n voldoende lang tyd verloop het en die stelsel nie meer verander nie. In daardie geval dp/dt=0=βm-αp. Dit sê weer vir ons dat die proteïen tot mRNA verhouding gegee word deur p/m = β/α. Ons let daarop dat dit nie dieselfde is as die aantal proteïene wat uit elke mRNA geproduseer word nie, hierdie waarde vereis dat ons ook die mRNA-omsettempo ken wat ons aan die einde van die vignet opneem. Wat is die waarde van b ? 'n Vinnig vertaalde mRNA sal ribosome hê wat dit soos krale aan 'n tou versier soos vasgevang in die klassieke elektronmikrograaf wat in Figuur 1 getoon word. Hul afstand van mekaar langs die mRNA is ten minste die grootte van die fisiese voetspoor van 'n ribosoom (≈20 nm) , BNID 102320, 105000) wat die lengte is van ongeveer 60 basispare (lengte van nukleotied ≈0.3 nm, BNID 103777), gelykstaande aan ≈20 aa. Die tempo van vertaling is ongeveer 20 aa/sek. Dit neem dus ten minste een sekonde vir 'n ribosoom om langs sy eie fisiese grootte voetspoor oor die mRNA te beweeg, wat 'n maksimum algehele translasietempo van b=1 s -1 per transkripsie impliseer.

Die effektiewe afbraaktempo spruit nie net uit afbraak van proteïene nie, maar ook uit 'n verdunningseffek soos die sel groei. Inderdaad, van die twee effekte is die seldeling-verdunningseffek dikwels dominant en dus is die algehele effektiewe degradasietyd, wat die verdunning in ag neem, omtrent die tydinterval van 'n selsiklus, τ. Ons het dus α = 1/τ.

In die lig van hierdie getalle is die verhouding p/m dus 1 s -1 /(1/τ)= τ. Vir E coli, τ is ongeveer 1000 s en dus p/m

1000. Natuurlik as mRNA nie teen die maksimum tempo getranskribeer word nie, sal die verhouding kleiner wees. Kom ons doen 'n gesonde verstandkontrole op hierdie resultaat. Onder eksponensiële groei teen medium groeikoers E coli Dit is bekend dat dit ongeveer 3 miljoen proteïene en 3000 mRNA bevat (BNID 100088, 100064). Hierdie konstantes impliseer dat die proteïen tot mRNA verhouding ≈1000 is, presies in lyn met die skatting hierbo gegee. Ons kan 'n tweede gesonde verstandkontrole uitvoer op grond van inligting van vorige vignette. In die vignet oor "Wat is swaarder 'n mRNA of die proteïen waarvoor dit kodeer?" ons het 'n massaverhouding van ongeveer 10:1 afgelei vir mRNA tot die proteïene waarvoor hulle kodeer. In die vignet oor "Wat is die makromolekulêre samestelling van die sel?" ons het genoem dat proteïen ongeveer 50% van die droë massa in is E coli selle terwyl mRNA slegs sowat 5% van die totale RNA in die sel is, wat self ongeveer 20% van die droë massa is. Dit impliseer dat mRNA dus ongeveer 1% van die totale droë massa is. Die verhouding van mRNA tot proteïen behoort dus ongeveer 50 keer 10 te wees, of 500 tot 1. Vanuit ons oogpunt hou al hierdie gesonde verstandskontroles baie mooi bymekaar.

Figuur 2: Gelyktydige meting van mRNA en proteïen in E. coli. (A) Mikroskopiebeelde van mRNA-vlak in E. coli-selle. (B) Mikroskopiebeelde van proteïen in E. coli-selle. (C) Proteïenkopiegetal vs mRNA-vlakke soos verkry met beide mikroskopiemetodes soos dié wat in deel (A) getoon word en met behulp van volgordebepalinggebaseerde metodes. Van Taniguchi et al. Wetenskap. 329, 533 (2010).

Eksperimenteel, hoe word hierdie getalle op proteïen tot mRNA verhoudings bepaal? Een elegante metode is om fluoressensiemikroskopie te gebruik om mRNA's gelyktydig waar te neem deur gebruik te maak van fluoressensie in-situ hibridisasie (FISH) en hul proteïenprodukte wat aan 'n fluoresserende proteïen saamgesmelt is. Figuur 2 toon mikroskopiebeelde van beide die mRNA en die ooreenstemmende vertaalde samesmeltingsproteïen vir een spesifieke geen in E coli. Figuur 2C toon resultate met behulp van hierdie metodes vir veelvuldige gene en bevestig 'n 100- tot 1000-voudige oormaat proteïenkopiegetalle oor hul ooreenstemmende mRNA's. Soos gesien in daardie figuur, is nie net direkte visualisering deur mikroskopie nuttig nie, maar volgorde-gebaseerde metodes is ook opgeroep.

Vir stadiger groeiende organismes soos gis- of soogdierselle verwag ons 'n groter verhouding met die voorbehoud dat ons aannames oor maksimum translasietempo al hoe skraaler word en daarmee saam ons vertroue in die skatting. Vir gis onder medium tot vinnige groeitempo, is die aantal mRNA gerapporteer om in die reeks van 10,000-60,000 per sel te wees (BNID 104312, 102988, 103023, 106226, 106763). Aangesien gisselle ≈50 keer groter in volume is as E coli, kan die aantal proteïene met daardie verhouding groter geskat word, of 200 miljoen. Die verhouding p/m is dan ≈2吆 8 /2吆 4 ≈10 4, in lyn met eksperimentele waarde van ongeveer 5 000 (BNID 104185, 104745). Vir die verdeling van gis elke 100 minute is dit in die orde van die aantal sekondes in sy generasietyd, in ooreenstemming met ons kru skatting hierbo.

Figuur 3: Proteïen tot mRNA verhouding in splitsingsgis. (A) Histogram wat die aantal mRNA- en proteïenkopieë illustreer soos bepaal deur onderskeidelik volgordebepalingsmetodes en massaspektrometrie. (B) Plot van proteïen oorvloed en mRNA oorvloed op 'n geen-vir-geen basis. Aangepas uit S. Marguerat et al., Cell, 151:671, 2012. Onlangse ontleding (R. Milo, Bioessays, 35:1050, 2014) dui daarop dat die proteïenvlakke onderskat is en 'n regstellingsfaktor van ongeveer 5-voudige toename toegepas moet word, om sodoende die verhouding van proteïen tot mRNA nader aan 104 te maak.

Soos met baie van die hoeveelhede wat regdeur die boek beskryf word, het die hoë-deurset, genoomwye gier ook die onderwerp van hierdie vignet getref. Spesifiek, deur 'n kombinasie van RNA-Seq te gebruik om die mRNA-kopiegetalle en massaspektrometrie-metodes en ribosomale profilering te bepaal om die proteïeninhoud van selle af te lei, is dit moontlik om verder te gaan as die spesifieke geen-vir-geen skattings en metings hierbo beskryf. Soos getoon in Figuur 3 vir splitsingsgis, bevestig die genoomwye verspreiding van mRNA en proteïen die skattings wat hierbo verskaf is, wat in die meeste gevalle meer as duisendvoudige oormaat proteïen tot mRNA toon. Net so word in soogdiersellyne 'n proteïen tot mRNA-verhouding van ongeveer 10 4 afgelei (BNID 110236).

Figuur 4: Dinamika van proteïenproduksie. (A) Uitbarstings in proteïenproduksie as gevolg van veelvuldige rondtes van translasie op dieselfde mRNA-molekule voordat dit verval. (B) Verspreiding van barsgroottes vir die proteïen beta-galaktosidase in E. coli. (Aangepas uit L. Cai et al., Nature, 440:358, 2006.)

Tot dusver het ons gefokus op die totale aantal proteïenkopieë per mRNA en nie die aantal proteïene wat per produksiebars geproduseer word wat vanaf 'n gegewe mRNA voorkom nie. Hierdie sogenaamde barstgroottemeting word in Figuur 4 uitgebeeld, wat vir die proteïen beta-galaktosidase in E coli die verspreiding van waargenome sarsiegroottes, wat vinnig afneem van die gewone handvol tot baie minder gevalle van meer as 10.

Ten slotte merk ons ​​op dat daar 'n derde betekenis is aan die vraag wat hierdie vignet gee, waar ons kan vra hoeveel proteïene van elke individuele mRNA gemaak word voordat dit afgebreek word. Byvoorbeeld, in vinnig groeiende E coli, word mRNA's rofweg elke 3 minute afgebreek soos bespreek in die vignet oor "Wat is die degradasietempo's van mRNA en proteïene?". Hierdie tydskaal is sowat 10-100 keer korter as die selsiklustyd. As gevolg hiervan, om te beweeg van die stelling dat die proteïen tot mRNA-verhouding tipies 1000 is na die aantal proteïene wat uit 'n mRNA geproduseer word voordat dit afgebreek word, moet ons die aantal mRNA-leeftye per selsiklus verdeel. Ons vind dit in hierdie vinnige verdeling E coli scenario gee elke mRNA aanleiding tot ongeveer 10-100 proteïene voordat dit afgebreek word.

'n Onlangse studie (G. Csardi et al., PLOS genetics, 2015) stel voor dat die basiese vraag van hierdie vignet weer ondersoek word. Noukeurige ontleding van tientalle studies oor mRNA en proteïenvlakke in ontluikende gis, die mees algemene modelorganisme vir sulke studies, dui op 'n nie-lineêre verband waar gene met hoë mRNA vlakke 'n hoër proteïen tot mRNA verhouding sal hê as lae uitgedrukte mRNAs. Dit dui daarop dat die korrelasie tussen mRNA en proteïen nie 'n helling van 1 in log-log skaal het nie, maar eerder 'n helling van ongeveer 1.6 wat ook verduidelik hoekom die dinamiese reeks proteïene aansienlik groter is as dié van mRNA.


Spliceosome, saamgestel uit snRNP's en 'n pre-mRNA, voer splitsing uit

Selfs voordat splitsing in vitro bewerkstellig is, het verskeie waarnemings gelei tot die voorstel dat klein kern-RNA's (snRNA's) help met die splitsingsreaksie. Eerstens is gevind dat die kort konsensusvolgorde aan die 5′ einde van introne komplementêr is tot 'n volgorde naby die 5′ einde van die snRNA genoem U1. Tweedens is snRNA's gevind wat verband hou met hnRNP's in kernekstrakte. Vyf U-ryke snRNA's (U1, U2, U4, U5 en U6), wat in lengte wissel van 107 tot 210 nukleotiede, neem deel aan RNA-splyting.

In die kern van eukariotiese selle word snRNA's geassosieer met ses tot tien proteïene in klein kernribonukleoproteïendeeltjies (snRNP's). Sommige van hierdie proteïene is algemeen vir alle snRNP's, en sommige is spesifiek vir individuele snRNP's. Eksperimente met 'n sintetiese oligonukleotied wat met die 5′-eindgebied van U1 snRNA hibridiseer en latere studies met pre-mRNA's wat in die 5′ splitsplek konsensusvolgorde gemuteer is, het sterk bewyse gelewer dat basisparing tussen die 5′ splitsing plek van 'n pre-mRNA en die 5′-gebied van U1 snRNA word benodig vir RNA-splyting.

Betrokkenheid van U2 snRNA by splitsing is aanvanklik vermoed toe gevind is dat dit 'n interne volgorde het wat grootliks komplementêr is tot die konsensusvolgorde wat die vertakkingspunt in pre-mRNA's flankeer (sien Figuur 11-14). Mutation experiments, similar to those conducted with U1 snRNA and 5′ splice sites, demonstrated that base pairing between U2 snRNA and the branch-point sequence in pre-mRNA is critical to splicing. These studies with U1 and U2 snRNAs indicate that during splicing they base-pair with pre-mRNA as shown in Figure 11-17. Significantly, the branch- point A itself, which is not base-paired to U2 snRNA, 𠇋ulges out,” allowing its 2′ hydroxyl to participate in the first transesterification reaction of RNA splicing (see Figure 11-16).

Figure 11-17

Diagram of interactions between pre-mRNA, U1 snRNA, and U2 snRNA early in the splicing process. The 5′ region of U1 snRNA initially base-pairs with nucleotides at the 5′ end of the intron (blue) and 3′ end of the 5′ exon (more. )

Similar studies with other snRNAs demonstrated that RNA-RNA interactions involving them also occur during splicing. For example, an internal region of U6 snRNA initially base-pairs with the 5′ end of U4 snRNA. Rearrangements later in the splicing process result in U6 snRNA base pairing with the 5′ end of U2 snRNA, which remains base-paired to the branch-point sequence in the intron. Later in the splicing process, base pairing of U5 snRNA with four exon nucleotides adjacent to the splice sites displaces U1 snRNA from the pre-mRNA.

Based on the results of these experiments, identification of reaction intermediates, and other biochemical analyses, the five splicing snRNPs are thought to sequentially assemble on the pre-mRNA forming a large ribonucleoprotein complex called a spliceosome, which is roughly the size of a ribosome (Figure 11-18). According to the model depicted in Figure 11-19, assembly of a spliceosome begins with the base pairing of U1 and U2 snRNAs, as part of the U1 and U2 snRNPs, to the pre-mRNA (see Figure 11-17). Extensive base pairing between the snRNAs in the U4 and U6 snRNPs forms a complex that associates with U5 snRNP. The U4/U6/U5 complex then associates, presumably via protein-protein interactions, with the previously formed complex consisting of a pre-mRNA base-paired to U1 and U2 snRNPs to yield a spliceosome.

Figure 11-18

Electron micrograph of a spliceosome. Extracts of HeLa cells were mixed with a β-globin pre-mRNA the reaction was interrupted before splicing was completed, so that the spliceosomes, containing snRNPs and the pre-mRNA substrate, could be purified. (more. )

Figure 11-19

The spliceosomal splicing cycle. The splicing snRNPs (U1, U2, U4, U5, and U6) associate with the pre-mRNA and with each other in an ordered sequence to form the spliceosome. This large ribonucleoprotein complex then catalyzes the two transesterification (more. )

After formation of the spliceosome, extensive rearrangements occur in the pairing of snRNAs and the pre-mRNA, as noted previously. The rearranged spliceosome then catalyzes the two transesterification reactions that result in RNA splicing. After the second transesterification reaction, the ligated exons are released from the spliceosome while the lariat intron remains associated with the snRNPs. This final intron-snRNP complex is unstable and dissociates. The individual snRNPs released participate in a new cycle of splicing. The excised intron is rapidly degraded by a �ranching enzyme,” which hydrolyzes the 5′,2′-phosphodiester bond at the branch point, and other nuclear RNases.

It is estimated that at least one hundred proteins are involved in RNA splicing, making this process comparable in complexity to protein synthesis and initiation of transcription. Some of these splicing factors are associated with snRNPs, but others are not. Sequencing of yeast genes encoding splicing factors has revealed that they contain domains with the RNP motif, which interacts with RNA, and the SR motif, which interacts with other proteins and may contribute to RNA binding. Some splicing factors also exhibit sequence homologies to known RNA helicases these may be necessary for the base-pairing rearrangements that occur in snRNAs during the spliceosomal splicing cycle.

Introns whose splice sites do not conform to the standard consensus sequence recently were identified in some pre-mRNAs. This class of introns begins with AU and ends with AC rather than following the usual “GU –𠁚G rule” (see Figure 11-14). Research on the biochemistry of splicing for this special class of introns soon identified four novel snRNPs. Together with the standard U5 snRNP, these snRNPs appear to participate in a splicing cycle analogous to that discussed above.


Recent advances in mRNA vaccine technology

Various mRNA vaccine platforms have been developed in recent years and validated in studies of immunogenicity and efficacy 18,19,20 . Engineering of the RNA sequence has rendered synthetic mRNA more translatable than ever before. Highly efficient and non-toxic RNA carriers have been developed that in some cases 21,22 allow prolonged antigen expression in vivo (Tabel 1). Some vaccine formulations contain novel adjuvants, while others elicit potent responses in the absence of known adjuvants. The following section summarizes the key advances in these areas of mRNA engineering and their impact on vaccine efficacy.

Optimization of mRNA translation and stability

This topic has been extensively discussed in previous reviews 14,15 thus, we briefly summarize the key findings (Box 1). The 5′ and 3′ UTR elements flanking the coding sequence profoundly influence the stability and translation of mRNA, both of which are critical concerns for vaccines. These regulatory sequences can be derived from viral or eukaryotic genes and greatly increase the half-life and expression of therapeutic mRNAs 23,24 . A 5′ cap structure is required for efficient protein production from mRNA 25 . Various versions of 5′ caps can be added during or after the transcription reaction using a vaccinia virus capping enzyme 26 or by incorporating synthetic cap or anti-reverse cap analogues 27,28 . The poly(A) tail also plays an important regulatory role in mRNA translation and stability 25 thus, an optimal length of poly(A) 24 must be added to mRNA either directly from the encoding DNA template or by using poly(A) polymerase. The codon usage additionally has an impact on protein translation. Replacing rare codons with frequently used synonymous codons that have abundant cognate tRNA in the cytosol is a common practice to increase protein production from mRNA 29 , although the accuracy of this model has been questioned 30 . Enrichment of G:C content constitutes another form of sequence optimization that has been shown to increase steady-state mRNA levels in vitro 31 and protein expression in vivo 12 .

Although protein expression may be positively modulated by altering the codon composition or by introducing modified nucleosides (discussed below), it is also possible that these forms of sequence engineering could affect mRNA secondary structure 32 , the kinetics and accuracy of translation and simultaneous protein folding 33,34 , and the expression of cryptic T cell epitopes present in alternative reading frames 30 . All these factors could potentially influence the magnitude or specificity of the immune response.

Box 1: Strategies for optimizing mRNA pharmacology

A number of technologies are currently used to improve the pharmacological aspects of mRNA. The various mRNA modifications used and their impact are summarized below.

• Synthetic cap analogues and capping enzymes 26,27 stabilize mRNA and increase protein translation via binding to eukaryotic translation initiation factor 4E (EIF4E)

• Regulatory elements in the 5′-untranslated region (UTR) and the 3′-UTR 23 stabilize mRNA and increase protein translation

• Poly(A) tail 25 stabilizes mRNA and increases protein translation

• Modified nucleosides 9,48 decrease innate immune activation and increase translation

• Separation and/or purification techniques: RNase III treatment (N.P. and D.W., unpublished observations) and fast protein liquid chromatography (FPLC) purification 13 decrease immune activation and increase translation

• Sequence and/or codon optimization 29 increase translation

• Modulation of target cells: co-delivery of translation initiation factors and other methods alters translation and immunogenicity

Modulation of immunogenicity

Exogenous mRNA is inherently immunostimulatory, as it is recognized by a variety of cell surface, endosomal and cytosolic innate immune receptors (Fig. 1) (reviewed in Ref. 35). Depending on the therapeutic application, this feature of mRNA could be beneficial or detrimental. It is potentially advantageous for vaccination because in some cases it may provide adjuvant activity to drive dendritic cell (DC) maturation and thus elicit robust T and B cell immune responses. However, innate immune sensing of mRNA has also been associated with the inhibition of antigen expression and may negatively affect the immune response 9,13 . Although the paradoxical effects of innate immune sensing on different formats of mRNA vaccines are incompletely understood, some progress has been made in recent years in elucidating these phenomena.

Innate immune sensing of two types of mRNA vaccine by a dendritic cell (DC), with RNA sensors shown in yellow, antigen in red, DC maturation factors in green, and peptide−major histocompatibility complex (MHC) complexes in light blue and red an example lipid nanoparticle carrier is shown at the top right. A non-exhaustive list of the major known RNA sensors that contribute to the recognition of double-stranded and unmodified single-stranded RNAs is shown. Unmodified, unpurified (part a) and nucleoside-modified, fast protein liquid chromatography (FPLC)-purified (part b) mRNAs were selected for illustration of two formats of mRNA vaccines where known forms of mRNA sensing are present and absent, respectively. The dashed arrow represents reduced antigen expression. Ag, antigen PKR, interferon-induced, double-stranded RNA-activated protein kinase MDA5, interferon-induced helicase C domain-containing protein 1 (also known as IFIH1) IFN, interferon m1Ψ, 1-methylpseudouridine OAS, 2′-5′-oligoadenylate synthetase TLR, Toll-like receptor.

Studies over the past decade have shown that the immunostimulatory profile of mRNA can be shaped by the purification of IVT mRNA and the introduction of modified nucleosides as well as by complexing the mRNA with various carrier molecules 9,13,36,37 . Enzymatically synthesized mRNA preparations contain double-stranded RNA (dsRNA) contaminants as aberrant products of the IVT reaction 13 . As a mimic of viral genomes and replication intermediates, dsRNA is a potent pathogen-associated molecular pattern (PAMP) that is sensed by pattern recognition receptors in multiple cellular compartments (Fig. 1). Recognition of IVT mRNA contaminated with dsRNA results in robust type I interferon production 13 , which upregulates the expression and activation of protein kinase R (PKR also known as EIF2AK2) and 2′-5′-oligoadenylate synthetase (OAS), leading to the inhibition of translation 38 and the degradation of cellular mRNA and ribosomal RNA 39 , respectively. Karikó and colleagues 13 have demonstrated that contaminating dsRNA can be efficiently removed from IVT mRNA by chromatographic methods such as reverse-phase fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC). Strikingly, purification by FPLC has been shown to increase protein production from IVT mRNA by up to 1,000-fold in primary human DCs 13 . Thus, appropriate purification of IVT mRNA seems to be critical for maximizing protein (immunogen) production in DCs and for avoiding unwanted innate immune activation.

Besides dsRNA contaminants, single-stranded mRNA molecules are themselves a PAMP when delivered to cells exogenously. Single-stranded oligoribonucleotides and their degradative products are detected by the endosomal sensors Toll-like receptor 7 (TLR7) and TLR8 (Refs 40,41), resulting in type I interferon production 42 . Crucially, it was discovered that the incorporation of naturally occurring chemically modified nucleosides, including but not limited to pseudouridine 9,43,44 and 1-methylpseudouridine 45 , prevents activation of TLR7, TLR8 and other innate immune sensors 46,47 , thus reducing type I interferon signalling 48 . Nucleoside modification also partially suppresses the recognition of dsRNA species 46,47,48 . As a result, Karikó and others have shown that nucleoside-modified mRNA is translated more efficiently than unmodified mRNA in vitro 9 , particularly in primary DCs, and in vivo in mice 45 . Notably, the highest level of protein production in DCs was observed when mRNA was both FPLC-purified and nucleoside-modified 13 . These advances in understanding the sources of innate immune sensing and how to avoid their adverse effects have substantially contributed to the current interest in mRNA-based vaccines and protein replacement therapies.

In contrast to the findings described above, a study by Thess and colleagues found that sequence-optimized, HPLC-purified, unmodified mRNA produced higher levels of protein in HeLa cells and in mice than its nucleoside-modified counterpart 12 . Additionally, Kauffman and co-workers demonstrated that unmodified, non-HPLC-purified mRNA yielded more robust protein production in HeLa cells than nucleoside-modified mRNA, and resulted in similar levels of protein production in mice 49 . Although not fully clear, the discrepancies between the findings of Karikó 9,13 and these authors 12,49 may have arisen from variations in RNA sequence optimization, the stringency of mRNA purification to remove dsRNA contaminants and the level of innate immune sensing in the targeted cell types.

The immunostimulatory properties of mRNA can conversely be increased by the inclusion of an adjuvant to increase the potency of some mRNA vaccine formats. These include traditional adjuvants as well as novel approaches that take advantage of the intrinsic immunogenicity of mRNA or its ability to encode immune-modulatory proteins. Self-replicating RNA vaccines have displayed increased immunogenicity and effectiveness after formulating the RNA in a cationic nanoemulsion based on the licensed MF59 (Novartis) adjuvant 50 . Another effective adjuvant strategy is TriMix, a combination of mRNAs encoding three immune activator proteins: CD70, CD40 ligand (CD40L) and constitutively active TLR4. TriMix mRNA augmented the immunogenicity of naked, unmodified, unpurified mRNA in multiple cancer vaccine studies and was particularly associated with increased DC maturation and cytotoxic T lymphocyte (CTL) responses (reviewed in Ref. 51). The type of mRNA carrier and the size of the mRNA–carrier complex have also been shown to modulate the cytokine profile induced by mRNA delivery. For example, the RNActive (CureVac AG) vaccine platform 52,53 depends on its carrier to provide adjuvant activity. In this case, the antigen is expressed from a naked, unmodified, sequence-optimized mRNA, while the adjuvant activity is provided by co-delivered RNA complexed with protamine (a polycationic peptide), which acts via TLR7 signalling 52,54 . This vaccine format has elicited favourable immune responses in multiple preclinical animal studies for vaccination against cancer and infectious diseases 18,36,55,56 . A recent study provided mechanistic information on the adjuvanticity of RNActive vaccines in mice in vivo and human cells in vitro 54 . Potent activation of TLR7 (mouse and human) and TLR8 (human) and production of type I interferon, pro-inflammatory cytokines and chemokines after intradermal immunization was shown 54 . A similar adjuvant activity was also demonstrated in the context of non-mRNA-based vaccines using RNAdjuvant (CureVac AG), an unmodified, single-stranded RNA stabilized by a cationic carrier peptide 57 .

Progress in mRNA vaccine delivery

Doeltreffend in vivo mRNA delivery is critical to achieving therapeutic relevance. Exogenous mRNA must penetrate the barrier of the lipid membrane in order to reach the cytoplasm to be translated to functional protein. mRNA uptake mechanisms seem to be cell type dependent, and the physicochemical properties of the mRNA complexes can profoundly influence cellular delivery and organ distribution. There are two basic approaches for the delivery of mRNA vaccines that have been described to date. First, loading of mRNA into DCs ex vivo, followed by re-infusion of the transfected cells 58 and second, direct parenteral injection of mRNA with or without a carrier. Ex vivo DC loading allows precise control of the cellular target, transfection efficiency and other cellular conditions, but as a form of cell therapy, it is an expensive and labour-intensive approach to vaccination. Direct injection of mRNA is comparatively rapid and cost-effective, but it does not yet allow precise and efficient cell-type-specific delivery, although there has been recent progress in this regard 59 . Both of these approaches have been explored in a variety of forms (Fig. 2 Table 1).

Commonly used delivery methods and carrier molecules for mRNA vaccines along with typical diameters for particulate complexes are shown: naked mRNA (part a) naked mRNA with in vivo electroporation (part b) protamine (cationic peptide)-complexed mRNA (part c) mRNA associated with a positively charged oil-in-water cationic nanoemulsion (part d) mRNA associated with a chemically modified dendrimer and complexed with polyethylene glycol (PEG)-lipid (part e) protamine-complexed mRNA in a PEG-lipid nanoparticle (part f) mRNA associated with a cationic polymer such as polyethylenimine (PEI) (part g) mRNA associated with a cationic polymer such as PEI and a lipid component (part h) mRNA associated with a polysaccharide (for example, chitosan) particle or gel (part i) mRNA in a cationic lipid nanoparticle (for example, 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids) (part j) mRNA complexed with cationic lipids and cholesterol (part k) and mRNA complexed with cationic lipids, cholesterol and PEG-lipid (part l).

Ex vivo loading of DCs. DCs are the most potent antigen-presenting cells of the immune system. They initiate the adaptive immune response by internalizing and proteolytically processing antigens and presenting them to CD8 + and CD4 + T cells on major histocompatibility complexes (MHCs), namely, MHC class I and MHC class II, respectively. Additionally, DCs may present intact antigen to B cells to provoke an antibody response 60 . DCs are also highly amenable to mRNA transfection. For these reasons, DCs represent an attractive target for transfection by mRNA vaccines, both in vivo en ex vivo.

Although DCs have been shown to internalize naked mRNA through a variety of endocytic pathways 61,62,63 , ex vivo transfection efficiency is commonly increased using electroporation in this case, mRNA molecules pass through membrane pores formed by a high-voltage pulse and directly enter the cytoplasm (reviewed in Ref. 64). This mRNA delivery approach has been favoured for its ability to generate high transfection efficiency without the need for a carrier molecule. DCs that are loaded with mRNA ex vivo are then re-infused into the autologous vaccine recipient to initiate the immune response. Die meeste ex vivo-loaded DC vaccines elicit a predominantly cell-mediated immune response thus, they have been used primarily to treat cancer (reviewed in Ref. 58).

Injection of naked mRNA in vivo. Naked mRNA has been used successfully for in vivo immunizations, particularly in formats that preferentially target antigen-presenting cells, as in intradermal 61,65 and intranodal injections 66,67,68 . Notably, a recent report showed that repeated intranodal immunizations with naked, unmodified mRNA encoding tumour-associated neoantigens generated robust T cell responses and increased progression-free survival 68 (discussed further in Box 2).

Physical delivery methods in vivo. To increase the efficiency of mRNA uptake in vivo, physical methods have occasionally been used to penetrate the cell membrane. An early report showed that mRNA complexed with gold particles could be expressed in tissues using a gene gun, a microprojectile method 69 . The gene gun was shown to be an efficient RNA delivery and vaccination method in mouse models 70,71,72,73 , but no efficacy data in large animals or humans are available. In vivo electroporation has also been used to increase uptake of therapeutic RNA 74,75,76 however, in one study, electroporation increased the immunogenicity of only a self-amplifying RNA and not a non-replicating mRNA-based vaccine 74 . Physical methods can be limited by increased cell death and restricted access to target cells or tissues. Recently, the field has instead favoured the use of lipid or polymer-based nanoparticles as potent and versatile delivery vehicles.

Protamine. The cationic peptide protamine has been shown to protect mRNA from degradation by serum RNases 77 however, protamine-complexed mRNA alone demonstrated limited protein expression and efficacy in a cancer vaccine model, possibly owing to an overly tight association between protamine and mRNA 36,78 . This issue was resolved by developing the RNActive vaccine platform, in which protamine-formulated RNA serves only as an immune activator and not as an expression vector 52 .

Cationic lipid and polymer-based delivery. Highly efficient mRNA transfection reagents based on cationic lipids or polymers, such as TransIT-mRNA (Mirus Bio LLC) or Lipofectamine (Invitrogen), are commercially available and work well in many primary cells and cancer cell lines 9,13 , but they often show limited in vivo efficacy or a high level of toxicity (N.P. and D.W., unpublished observations). Great progress has been made in developing similarly designed complexing reagents for safe and effective in vivo use, and these are discussed in detail in several recent reviews 10,11,79,80 . Cationic lipids and polymers, including dendrimers, have become widely used tools for mRNA administration in the past few years. The mRNA field has clearly benefited from the substantial investment in in vivo small interfering RNA (siRNA) administration, where these delivery vehicles have been used for over a decade. Lipid nanoparticles (LNPs) have become one of the most appealing and commonly used mRNA delivery tools. LNPs often consist of four components: an ionizable cationic lipid, which promotes self-assembly into virus-sized (

100 nm) particles and allows endosomal release of mRNA to the cytoplasm lipid-linked polyethylene glycol (PEG), which increases the half-life of formulations cholesterol, a stabilizing agent and naturally occurring phospholipids, which support lipid bilayer structure. Numerous studies have demonstrated efficient in vivo siRNA delivery by LNPs (reviewed in Ref. 81), but it has only recently been shown that LNPs are potent tools for in vivo delivery of self-amplifying RNA 19 and conventional, non-replicating mRNA 21 . Systemically delivered mRNA–LNP complexes mainly target the liver owing to binding of apolipoprotein E and subsequent receptor-mediated uptake by hepatocytes 82 , and intradermal, intramuscular and subcutaneous administration have been shown to produce prolonged protein expression at the site of the injection 21,22 . The mechanisms of mRNA escape into the cytoplasm are incompletely understood, not only for artificial liposomes but also for naturally occurring exosomes 83 . Further research into this area will likely be of great benefit to the field of therapeutic RNA delivery.

The magnitude and duration of in vivo protein production from mRNA–LNP vaccines can be controlled in part by varying the route of administration. Intramuscular and intradermal delivery of mRNA–LNPs has been shown to result in more persistent protein expression than systemic delivery routes: in one experiment, the half-life of mRNA-encoded firefly luciferase was roughly threefold longer after intradermal injection than after intravenous delivery 21 . These kinetics of mRNA–LNP expression may be favourable for inducing immune responses. A recent study demonstrated that sustained antigen availability during vaccination was a driver of high antibody titres and germinal centre (GC) B cell and T follicular helper (TFH) cell responses 84 . This process was potentially a contributing factor to the potency of recently described nucleoside-modified mRNA–LNP vaccines delivered by the intramuscular and intradermal routes 20,22,85 . Indeed, TFH cells have been identified as a critical population of immune cells that vaccines must activate in order to generate potent and long-lived neutralizing antibody responses, particularly against viruses that evade humoral immunity 86 . The dynamics of the GC reaction and the differentiation of TFH cells are incompletely understood, and progress in these areas would undoubtedly be fruitful for future vaccine design (Box 3).

Box 2: Personalized neoepitope cancer vaccines

Sahin and colleagues have pioneered the use of individualized neoepitope mRNA cancer vaccines 121 . They use high-throughput sequencing to identify every unique somatic mutation of an individual patient's tumour sample, termed the mutanome. This enables the rational design of neoepitope cancer vaccines in a patient-specific manner, and has the advantage of targeting non-self antigen specificities that should not be eliminated by central tolerance mechanisms. Proof of concept has been recently provided: Kreiter and colleagues found that a substantial portion of non-synonymous cancer mutations were immunogenic when delivered by mRNA and were mainly recognized by CD4 + T cells 176 . On the basis of these data, they generated a computational method to predict major histocompatibility complex (MHC) class II-restricted neoepitopes that can be used as vaccine immunogens. mRNA vaccines encoding such neoepitopes have controlled tumour growth in B16-F10 melanoma and CT26 colon cancer mouse models. In a recent clinical trial, Sahin and colleagues developed personalized neoepitope-based mRNA vaccines for 13 patients with metastatic melanoma, a cancer known for its high frequency of somatic mutations and thus neoepitopes. They immunized against ten neoepitopes per individual by injecting naked mRNA intranodally. CD4 + T cell responses were detected against the majority of the neoepitopes, and a low frequency of metastatic disease was observed after several months of follow-up 68 . Interestingly, similar results were also obtained in a study of analogous design that used synthetic peptides as immunogens rather than mRNA 177 . Together, these recent trials suggest the potential utility of the personalized vaccine methodology.

Box 3: The germinal centre and T follicular helper cells

The vast majority of potent antimicrobial vaccines elicit long-lived, protective antibody responses against the target pathogen. High-affinity antibodies are produced in specialized microanatomical sites within the B cell follicles of secondary lymphoid organs called germinal centres (GCs). B cell proliferation, somatic hypermutation and selection for high-affinity mutants occur in the GCs, and efficient T cell help is required for these processes 178 . Characterization of the relationship between GC B and T cells has been actively studied in recent years. The follicular homing receptor CXC-chemokine receptor 5 (CXCR5) was identified on GC B and T cells in the 1990s 179,180 , but the concept of a specific lineage of T follicular helper (TFH) cells was not proposed until 2000 (Refs 181, 182). The existence of the TFH lineage was confirmed in 2009 when the transcription factor specific for TFH cells, B cell lymphoma 6 protein (BCL-6), was identified 183,184,185 . TFH cells represent a specialized subset of CD4 + T cells that produce critical signals for B cell survival, proliferation and differentiation in addition to signals for isotype switching of antibodies and for the introduction of diversifying mutations into the immunoglobulin genes. The major cytokines produced by TFH cells are interleukin-4 (IL-4) and IL-21, which play a key role in driving the GC reaction. Other important markers and functional ligands expressed by TFH cells include CD40 ligand (CD40L), Src homology domain 2 (SH2) domain-containing protein 1A (SH2D1A), programmed cell death protein 1 (PD1) and inducible T cell co-stimulator (ICOS) 186 . The characterization of rare, broadly neutralizing antibodies to HIV-1 has revealed that unusually high rates of somatic hypermutation are a hallmark of protective antibody responses against HIV-1 (Ref. 187). As TFH cells play a key role in driving this process in GC reactions, the development of new adjuvants or vaccine platforms that can potently activate this cell type is urgently needed.


MRNA Splicing


Transcription and processing (which includes splicing) of the newly made mRNA occurs in the nucleus of the cell.
Once a mature mRNA transcript is made it is transported to the cytoplasm for translation into protein.

Figure (PageIndex<1>). (CC BY-NC-SA)

Most eukaryotic genes and their pre-mRNA transcripts contain noncoding stretches of nucleotides or regions that are not meant to be made into protein. These noncoding segments are called introneand must be removed before the mature mRNA can be transported to the cytoplasm and translated into protein. The stretches of DNA that do code for amino acids in the protein are called exons. During the process of splicing, introns are removed from the pre-mRNA by the spliceosome and exons are spliced back together. If the introns are not removed, the RNA would be translated into a nonfunctional protein. Splicing occurs in the nucleus before the RNA migrates to the cytoplasm. Once splicing is complete, the mature mRNA (containing uninterrupted coding information), is transported to the cytoplasm where ribosomes translate the mRNA into protein.

A Detailed Look at mRNA Splicing

The pre-mRNA Transcript
The pre-mRNA transcript contains both introns and exons. The introns are removed during the process of splicing. In this example, the pre-mRNA contains two exons and one intron.

Introns contain several important and conserved sequences that guide the splicing process: a 5&rsquo GU sequence (the 5&rsquo splice site), an A branch site located near a pyrimidine-rich region (a region with many cytosine and uracil bases) and a 3&rsquo AG sequence (the 3&rsquo splice site).

The Spliceosome
A large protein complex known as the spliceosome controls mRNA splicing. The spliceosome is composed of particles made up of both RNA and protein. These particles are called small nuclear ribonucleoprotein of snRNPs (pronounced &ldquosnurps&rdquo) for short. The snRNPs recognize the conserved sequences within introns and quickly bind these sequences once the pre-mRNA is made and initiate splicing.

The spliceosome is built in distinct steps. Eerstens, die U1 snRNP binds the 5&rsquo splice site and the U2 snRNP binds the branch site.

A number of other snRNPs (U4, U6 en U5) bind the pre-mRNA transcript forming the mature spliceosome complex. This causes the intron to form a loop and brings the 5&rsquo splice site and 3&rsquo splice site together.

Now that the spliceosome is assembled, splicing can begin. First the 5&rsquo end of the intron is cut. The 5&rsquo GU end of the intron is then connected to the A branch site, which creates a lariat structure.

At this stage the U1 and U4 snRNPs are released and the 3&rsquo splice site is cleaved. Once the intron has been fully cleaved, the two exons are attached to each other. The intron in the form of a lariat is released along with U2, U5 and U6 snRNPs.

The intron will be degraded and the snRNPs are used again to splice other pre-mRNAs. The mature mRNA transcript is now ready to be exported to the cytoplasm for translation.

Alternatiewe Splicing

The example of a gene with a single intron and two exons used above is a very simple model of RNA splicing. Many genes contain multiple exons as well as multiple introns. A process known asalternative splitsing allows for different combinations of exons to be included in the final mature mRNA, making different versions of proteins (called isovorme) that are all encoded by the same gene. Alternative splicing of mRNA allows for many proteins to be made, with different functions, all produced from a single gene. One of the most dramatic examples of alternative splicing is the Dscam gene in Drosophila melanogaster (a fruit fly). This single gene contains 116 exons! Some exons are always included, others may or may not be included. Over 18,000 different proteins from this single gene have been found in Drosophila! Theoretically, this system is capable of producing 38,016 different proteins all from a single gene!

Below is an example of alternative splicing of a pre-mRNA transcript. In this case, there are two different, alternatively spliced mRNAs that can be made from this pre-mRNA. The two mature mRNAs can contain either the yellow or the green exon. This produces two distinct protein isoforms when the mRNAs are translated into protein.

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mRNA Splicing Tutorial by Dr. Katherine Harris is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.