rna-interferenca (rnai) v temeljnih raziskavah in biomedicini

Genetski koncepti 2011-12 1

RNA-interferenca (RNAi)

v temeljnih raziskavah

in biomedicini

1 Genetski koncepti 2011-12,

doc. dr. Tanja Kunej

1. Koncept “Forward” in “Reverse” genetike

2. Mejniki pri odkrivanju mehanizma RNAi

3. Mehanizem fenomena RNAi

4. Primeri uporabe RNAi v genomiki, molekularni biologiji in

biomedicini

5. Nekodirajoče RNA

siRNA

miRNA

piRNA

snoRNA

Vsebina

2


PTGS: potranskripcijsko utišanje genov; Post-Transcriptional Gene Silencing

RNAi: RNA-interferenca

dsRNA: double stranded; dvoveriţna RNA, ssRNA: single stranded; enoveriţna RNA

siRNA: mala interferenčna RNA; small interfering RNA;

shRNA: kratka lasnična RNA; short hairpin RNA

miRNA: mikro RNA; microRNA

Dicer: citoplazemski proteinski kompleks, ki cepi dsRNA

RISC: z RNA inducirani kompleks za utišanje genov; RNA-Induced Silencing Complex

Drosha: proteinski kompleks, ki cepi primarne miRNA (primary miRNA; pri-miRNAs), da

nastanejo prekurzorske miRNA (precursor miRNAs; pre-miRNAs)

RdRP: od RNA odvisna RNA polimeraza; RNA-dependent RNA polymerase

Novi izrazi:

3

Argonavt: katalitična komponenta kompleksa RISC

piRNA : Piwi-interacting RNA

PIWI: P-element induced wimpy testes

• Hiwi: homolog pri človeku

• Miwi, Mili: homolog pri miši

Novi izrazi:

4


Odkritje leta, breakthrough of the year for the most significant development in scientific research

Science

1996 understanding HIV

1997 Dolly, the first mammal to be cloned from adult cells

1998 the accelerating universe, dark matter

1999 capturing the promise of youth with stem cells

2000 full genome sequencing

2001 nanocircuits

2002 small RNAs

2003 dark energy

2004 Spirit rover landed on Mars

2005 evolution in action

2006 proof of the Poincaré conjecture

2007 human genetic variation

2008 cellular reprogramming

2009 Ardipithecus ramidus

2010 The first quantum machine

5

Molekula leta

1989 PCR and DNA polymerase

1990 the manufacture of synthetic diamonds

1991 buckminsterfullerene

1992 nitric oxide

1993 p53

1994 DNA repair enzyme

2002 All-trans-Retinoic Acid

2003 Adenovirus GP&NP of the Ebola Vaccine

2004 Imidazoleacetic acid-ribotide

2005 4E10

2006 hsa-mir-155 and hsa-let-7a-2

2007 PKD2L1

2008 anti-SAG 421-433 catalytic IgA

2009 Sleeping Beauty Transposase SB 100X

2010 FOXM1

Awarded by the International

Society for Molecular and Cell

Biology and Biotechnology

Protocols and Researchers

Yanaihara in sod. Unique microRNA molecular profiles in lung cancer diagnosis and

prognosis. Cancer Cell. 2006;9:189-98.

• miRNA expression profiles are diagnostic and prognostic markers of lung cancer

• High hsa-mir-155 and low hsa-let-7a-2 expression correlated with poor survival in

lung cancer.

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biomedicini


Vsebina

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“Forward genetics”

Genetska analiza

od fenotipa do genotipa:

-analiza kandidatnih genov.

“Reverse genetics”

Genetska analiza

od genotipa do fenotipa:

-tehnologija “knock-out”,

-tehnologija “antisense”,

-tehnologija z ribocimi,

-RNAi. McManus in Sharp, 2002

Koncept “Forward” in “Reverse” genetike

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biomedicini


Vsebina

9

Namen: pridobiti petunije s cvetovi temnejše barve.

Metode: vnos gena za ključni encim pigmentacije.

Rezultat: Rastline z dodatno kopijo gena za pigmentacijo imajo bele in belo-vijolične cvetove in ne temno vijolične.

Zniţano izraţanje endogena in transgena.

Pojav so poimenovali:

ko-supresija izraţanja genov;

"co-suppression of gene expression“.

Napoli in sod. Plant Cell 1990;2: 279-289.

Jorgensen in sod. Trends Biotechnol. 1990;8: 340-344.

Odkritje RNAi

Griffiths in sod., str. 445 10

http://en.wikipedia.org/wiki/Image:PetuniaHybridaPiebald.jpg


Inaktivacija mex-3B ustavi embrionalni

razvoj: a: negativna kontrola,

b: embrio starša brez injicirane dsRNA

(normalen vzorec endogene mex-3 RNA),

c: embrio starša z injicirano mex-3B

antisense RNA,

d: embrio starša z injicirano mex-3B dsRNA

(ni detekcije mex-3 RNA).

Odkritje RNAi

Guo in Kemphues, 1995

Vnos protismerne “antisense” molekule RNA za inaktivacijo gena.

Enak rezultat z vnosom smerne “sense” molekule RNA!

Fire in Mello, 1998:

Vnos dvoveriţne RNA (dsRNA) v C. elegans, kar povzroči

sekvenčno-specifično utišanje gena.

Pojav poimenujejo RNA interferenca (RNAi). Caenorhabditis elegans

Fire A. in sod. Potent and specific genetic interference by double-stranded RNA in

Caenorhabditis elegans. Nature 1998; 391: 806-811. 5209 citatatov (11/2011).

11

Mejniki v odkrivanju mehanizma RNAi

-1990: Ko-supresija vijolične barve cvetov pri petunijah

Napoli in sod. Plant Cell 1990; 2:279-289.

-1995: “Antisense” in “sense” mRNA zavirata izraţanje (ekspresijo)

Guo in Kemphues. Cell 1995; 81:611-620.

-1998: Vnos dvoveriţne RNA v celico sproţi utišanje genov (RNAi)

Fire A in Mello CC. Nature 1998; 391:806-811.

-2000: Vnešena dsRNA se razcepi na 21-23 bp dolge molekule (siRNA)

Zamore in sod. Cell 2000; 101:25-33.

-2001: Vnos siRNA omogoči RNAi v sesalskih celičnih kulturah

(vnos dsRNA je sproţal interferonski odgovor).

Elbashir in sod. Nature 2001; 411:494-498.

-2001: Encim Dicer cepi dsRNA v siRNA

Bernstein in sod. Nature 2001; 409:363-366. 12


-2001: Odkrit nov razred majhnih RNA s potencialno regulatorno vlogo, ki

jih poimenujejo mikro RNA (microRNA; miRNA)

Bartel,Tuschl, Ambros Lab

-2006: Nobelova nagrada za fiziologijo in medicino

Fire A in Mello CC.

-2006: Odkritje fenomena RNAa (small RNA-induced gene activation)

Li LC s sod. Small dsRNAs induce transcriptional activation in

human cells. PNAS 2006; 103:17337-42.

http://www.rnaiweb.com/RNAi/RNAi_Timeline/ 13

Mejniki v odkrivanju mehanizma RNAi

Obstoj mehanizma RNAi so lahko potrdili pri večini

evkariontov in ugotovili vpletenost v različne funkcije:

-obramba celice pred virusi,

-preprečevanje razširjanja transpozonov,

-potranskripcijsko uravnavanje (post-transcriptional regulation),

fino uravnavanje količine mRNA,

-embrionalni razvoj.

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biomedicini


Vsebina

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Mehanizem RNAi se sproţi ob vstopu dolge dvojnovijačne RNA

(dsRNA) v obliki:

-endogene dvojnovijačne RNA (A),

-virusa (B),

-mikro RNA (C),

-siRNA, shRNA. Ţe majhne količine siRNA

sproţijo mehanizem RNAi. 16


Mehanizmi za tvorjenje dsRNA – dvoveriţne RNA

A) Replikacija viralne enoveriţne RNA,

B) prepisovanje (transkripcija) obrnjenih ponovitev (inverted repeats),

C.1) delovanje RdRP (od RNA odvisna polimeraza RNA) pri rastlinah in

C. elegans,

C.2) prepisovanje s promotorjev na nasprotnih straneh (trans)gena ali

transpozona (nastaneta dve komplementarni verigi RNA).

Matheu in

Bender, 2004 17

Processes

pre-miRNA to

mature miRNA

Initiates formation of

RNA-induced silencing

complex (RISC) –

responsible for gene

silencing.

He in Hannon 2004

Shared biosynthetic

pathways of siRNAs

and miRNAs

processes primary

transcripts (pri-miRNA) to

60 bp long pre-miRNA

Drosha

Dicer

18


a) siRNA

b) DICER razcepi

dsRNA v siRNA.

“Antisense” veriga siRNA

se vgradi v

RISC (RNA-Induced

Silencing Complex)

in omogoči

prepoznavanje

tarčne mRNA.

McManus in Sharp. Nature 2002;

3:737-747.

siRNA in RNAi

19

RNAi je oblika potranskripcijskega utišanja genov

RNase H

RISC

Kurreck J. Antisense technologies.

Eur J Biochem. 2003, 270:1628- 44.

RNase H

Ribozymes and DNA enzymes

possess catalytic activity and cleave

their target RNA. Ribozymes are

RNA molecules that act as enzymes

in the absence of proteins.

DNA

mRNA

Transcription

Regulation Translation

blocked

Ribozyme/

DNA enzyme

AO = Antisense

Oligonucleotide

Translation

miRNA

Translation

Attenuation

siRNA-induced

gene silencing

Degradation

of target

mRNA

siRNA

RISC

RISC

mRNA

cleavage

RNAi

AO (single-stranded DNA or RNA)

block translation of mRNA or induce

its degradation by RNase H.

PTGS= Post-Transcriptional

Gene Silencing

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biomedicini


Vsebina

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-analiza funkcije genov,

-analiza metabolnih poti,

-gensko kartiranje,

-zdravljenje bolezni.

Uporaba tehnologije RNAi

22


Tehnologija RNAi je koristno orodje v funkcijski genomiki

•tehnologija RNAi:

-enostavnejša, cenejša

-isto ţival lahko v poskusu

uporabimo večkrat

-utišanje genov; “knock down”

-5-10% izraţanja gena ostane

-poskus traja 1 teden

Sirna Therapeutics preimenovana

•tehnologija antisense

•tehnologija z ribocimi

•izničenje genov; “knock out”

-izraţanja genov ni

-poskus traja 6 mesecev ali več

Ribozyme Pharmaceuticals 23

Primer uporabe tehnologije RNAi pri genskem kartiranju

Identifikacija gena za vitamin K epoksid reduktazo (VKOR),

ki je tarča za antikoagulant varfarin (zdravljenje infarktov in

tromboz).

Rost, S. in sod.

Mutations in VKORC1 cause

warfarin resistance and

multiple coagulation factor

deficiency type 2. Nature 2004; 427: 537-41

Li, T. in sod.

Identification of the gene for

vitamin K epoxide reductase.

Nature 2004; 427: 541-44

Klasične metode kloniranja za

lokalizacijo gena.

siRNA proti kandidatnim

genom in testiranje aktivnosti

za inhibicijo VKOR. 24


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM 25

VK

OR

activity (

%)

siRNA pools

26


Podjetje Področje raziskav za zdravjenje z RNAi

Acutty Pharmaceuticals makularna degeneracija, diabetična retinopatija

AGY Therapeutics RNAi v nevronih

Alnylam Pharmaceuticals makularna degeneracija, Parkinsonova bolezen, cistična

fibroza, gripa

Atugen AG rak, očesne bolezni, koţne bolezni

Benitec Australia hepatitis C, HIV, rak, diabetes, debelost

Cytrx Corporation diabetes/debelost

Devigen diabetes/debelost

Genesis R&D alergije

Genta Incorporated rak

Intern. Therapeutics HIV, hepatitis B

Intradigm Corporation rak, SARS, artritis

Nucleonics, Inc. hepatitis B, hepatitis C

Sirna Therapeutics hepatitis C, astma, diabetes, rak, gluhost,

Huntingtonova bolezen

Raziskave terapevtske genske supresije

Uprichard, 2005

27

RNAi – velik potencial za zdravljenje bolezni

-Specifičnost

-alelno-specifično utišanje genov (selektivno zaviranje

izraţanja mutantnega alela),

-onkologija in zdravljenje nevromiopatij, ki so posledica

nukleotidne zamenjave.

-Učinkovitost

Utišanje gena je moţno z niţjimi koncentracijami efektorskih

molekul kot pri “antisense” tehnologiji (RNAi je naravni

biološki proces in predstavlja bolj naravno strategijo

manipulacije genov).

-Širok spekter delovanja

RNAi lahko sproţimo z vnosom več siRNA za utišanje

enega gena ali več genov hkrati.

Uprichard, 2009. The therapeutic potential of RNA interference

28


Brummelkamp s sod. Stable suppression of tumorigenicity by virus-mediated RNA

interference. Cancer Cell 2002; 2:243-247.

Selektivno utišanje alela K-rasV12 povezanega z razvojem raka jeter

(nukleotidna zamenjava G>T).

Uporaba retroviralnih vektorjev za prenos siRNA, ki utiša alel K-rasV12,

na alel K-rasWT pa nima vpliva.

ni inhibicije

inhibicija

Za alel-specifično utišanje z RNAi pri zdravljenju raka

kodon 12:

wild-type (wt) = GGT (glicin)

K-ras = GTT (valin)

29

Prenos efektorskih molekul do tarčnega gena

-RNAi efektorji:

-siRNA vnešena kot zdravilo,

-z vektorji posredovano intracelularno izraţanje shRNA, ki se

procesira v aktivno siRNA v gostiteljski celici (dolgotrajnejši efekt

utišanja genov).

-Lokalni vnos:

-vnos v oči, intranazalno, v centralni ţivčni sistem.

-Sistemski vnos:

-stabilizacija molekul siRNA (kemijske spremembe molekul,

pakiranje v liposome),

-prenos do tarčnega tkiva in vstop v celico (na efektorsko

molekulo vgrajeni/konjugirani ligandi ali protitelesa),

-uporaba viralnih vektorjev: lentivirusi, adenovirusi, retrovirusi.

30


Pridobivanje siRNA

Razgradnja in vitro z

dicerjem ali RNAzo III

dicer

transkripcija in vitro/

kemična sinteza plazmid,

retrovirus,

adenovirus,

lentivirus

transkripcija

in vivo

siRNA

RISC

Vektorji, ki vsebujejo

zapis za shRNA

(short hairpin RNA).

Kurreck, 2003

Griffiths in sod., str. 533. 31

Clark in Whitelaw, 2003

Vnos shRNA v embrije miši z lentivirusom

1. V ţivali so vnesli gen za

GFP (Green Fluorescent

Protein) (transgene ţivali).

2. Lentivirus z zapisom za

GFP shRNA so vnesli v

oocite transgenih ţivali s

funkcionalnim transgenom

GFP.

3. Očitno zmanjšanje

fluorescence GFP v

embrijih in transgenih

ţivalih.

32


Omejitve pri uporabi RNAi

-Zgrešitev tarče; učinek “off-target”

Problem, da bi prišlo do utišanja ne-tarčnega gena (zaradi delne

komplementarnosti). Ta efekt je odvisen od koncentracije in se mu

izognemo z niţanjem koncentracije siRNA.

-Nespecifičen učinek

-Interferonski odziv, ki je lahko citotoksičen.

-Izogibanje nevarnim zaporedjem pri izbiri zaporedja za siRNA;

“9 bp danger motif” (Hornung in sod., 2005).

-Zasičenost mehanizma RNAi

-siRNA vstopajo v naravne celične poti miRNA in lahko motijo

njihovo regulatorno vlogo. Za miRNA so ugotovili vpletenost v

razvoj nevroloških bolezni in raka.

-Potrebno bo določiti največjo še varno količino siRNA, ki jo lahko

vnesemo v določen celični tip. 33

Učinkovitost terapije z RNAi

-Rezistenca

-Za RNAi je značilna visoka (z nukleotidnim zaporedjem

pogojena) specifičnost, ki pa je lahko tudi njena pomanjkljivost;

RNA in DNA virusi lahko zelo hitro razvijejo mutacije za rezistenco

na siRNA; “escape mutations”.

-Nekateri virusi kodirajo proteine, ki inhibirajo mehanizem RNAi. Bennasser in sod. Evidence that HIV-1 encodes an siRNA and

suppressor of RNA silencing. Immunity 2005; 22:607-619.

-Pri nekterih vrstah raka je okvarjen dicer in spremenjeno

izraţanje miRNA, terapija z RNAi je zato neučinkovita.

-Nepopolno utišanje genov

RNAi utiša in ne izniči izraţanja genov. Za zdravljenje nekaterih

bolezni je to dovolj, pri drugih pa RNAi samo zmanjša

napredovanje bolezni (potrebna kombinacija z drugimi oblikami

zdravljenja).

34


Haasnoot in sod. Inhibition of virus replication by RNA interference. J. Biomed. Sci.

2003; 10:607-616.

RNAi v raziskavah preprečevanja infekcij s HIV

Tarča siRNA je lahko :

-viralna RNA ali

-geni gostitelja* (za celične receptorje; preprečen vstop virusa v celico)

Tarčni gen RNAi efektor Tip celic x zmanjšana

replikacija virusa

35

Xu in Xia. RNAi therapy: dominant disease gene gets silenced.

Gene Ther. 2005;12:1159-60.

Terapija z RNAi Potencial za zdravljenje bolezni,

kot je Amiotropna Lateralna

Skleroza (ALS) (1:50.000);

degeneracija motoričnih

nevronov.

ALS je posledica mutacij v genu

SOD1 (SuperOxide Dismutase).

36


RNAi pri zdravljenju nevromiopatij, ki so posledica

nukleotidne zamenjave

Ding in sod., 2003; Ralph in sod., 2005; Xia in sod., 2005.

siRNAG256C

siRNAWT

sod1G256C

sod1wt

Načini terapije za ALS:

1. Selektivno utišanje mutiranega alela z sod1 G256C siRNA.

2. Na mišjem modelu za ALS so z lentiviralnimi vektorji vnesli shRNA

za sod1 G93A ter podaljšali ţivljenjsko dobo ţivali.

3. ALS lahko povzroči

> 100 različnih mutacij

v genu SOD1, zato so

izvedli

“Replacement RNAi

strategy”

(inhibicija obeh alelov

in vnos funkcionalnega

gena SOD1 ).

37

RNAi pri zdravljenju nevromiopatij, ki so posledica

povečanja števila trinukleotidnih ponovitev

Normalno: 10-35 ponovitev CAG

Mutacija: >40 ponovitev CAG

-Sindrom fragilnega X (CGG)n, (1:2.000)

-Spinobulbarna mišična atrofija; SBMA (CAG)n, (<1:50.000)

-Miotonična distrofija (CTG)n, (1:8.000)

-Huntingtonova bolezen (CAG)n, (1:10.000)

-Spinocerebralna ataksija tipa 3; SCA3 (CAG)n

Alelno specifičnost doseţemo s

siRNA proti SNP, ki je/ni

funkcionalno povezan z

bolezenskim alelom.

Griffiths in sod. Miller in sod. 2003. Allele-specific

silencing of dominant disease genes 38


VEGF= vascular endothelial growth factor;

ţilni endotelni rastni dejavnik

Za VEGF-specifična siRNA - pomen za zdravljenje raka, saj je

vaskularizacija prisotna pri nastanku tumorjev.

vaskularizacija

rast tumorja

sinteza VEGF

VEGF

tumor

RNAi pri zdravljenju raka

39

RNAi pri zdravljenju očesnih bolezni

VEGF je vzrok za vaskularizacijo pri očesnih boleznih

(diabetična retinopatija, makularna degeneracija).

Vnos vegf-specifičnih siRNA zmanjša obseg vaskularizacije pri

miškah.

Kontrola Vegf siRNA

µm2 Kontrola Vegf siRNA

Reich in sod. Mol. Vision 2003; 9: 210-216. 40






biomedicini


Vsebina

41

• Razredi nekodirajočih RNA

• siRNA/miRNA – podobnosti in razlike

• miRNA

• Trije razredi genetske variabilnosti:

• miRNA geni

• miRNA tarče

• mehanizem, ki sodeluje pri utišanju genov

(silencing machinery)

• piRNA

• snoRNA

Vsebina

42


RNA

Coding RNA

mRNA Involved in translation

rRNA tRNA

Non-coding RNA

UCR

siRNA

piRNA

miRNA

rasiRNA

snRNA snoRNA

tasiRNA scnRNA

ribosomal

RNA

transfer

RNA

messenger

RNA

Druţina RNA

Prirejeno po:

-Buckingham. The major world of microRNAs. 2003.

-Ferdin et al. 2010

siRNA small interfering RNA

snRNA small nuclear RNA

miRNA microRNA

snoRNA small nucleolar RNA

tasiRNA trans-acting siRNA

scnRNA small-scan RNA

rasiRNA repeat-associated siRNA

piRNA Piwi-interacting RNA

UCR ultraconserved regions

+ rapidly growing list of new ncRNA classes

43

Nekodirajoče RNA (nkRNA)

Small non-coding

RNAs;

sncRNA

Long non-coding

RNAs;

lncRNA

21-23 bp

miRNA

siRNA

500 bp

Telomerase RNA

17 kb

Xist

108 kb

Air RNAs

24-31 bp

piRNA

>200 bp

UCRs

rasiRNA

• Nomenclature and classification is still in progress.

• Number of ncRNA increases with developmental complexity;

mainly protein-coding mRNAs > mainly non-coding RNAs

70-200 bp

snoRNAs

(X-inactive

specific transcript)

44


Porast števila člankov, ki opisujejo nkRNA

*through

8/2009

Taft in sod., 2010 45

Matera in sod., 2007

Mattick in Makunin, 2006

Non-coding RNA comprise a

hidden layer of internal signals

that control various levels of gene

expression in physiology and

development, including chromatin

architecture/epigenetic memory,

transcription, RNA splicing, editing,

translation and turnover.

46


http://research.imb.uq.edu.au/rnadb/

http://www.mirbase.org/

http://www-snorna.biotoul.fr/

http://pirnabank.ibab.ac.in/

http://rfam.sanger.ac.uk.

Podatkovne zbirke nkRNA

47



• miRNA


• miRNA geni

• miRNA tarče



• piRNA

• snoRNA

Vsebina

48


Processes

pre-miRNA to

mature miRNA

Initiates formation of

RNA-induced silencing

complex (RISC) –

responsible for gene

silencing.

He in Hannon 2004

Shared biosynthetic

pathways of siRNAs

and miRNAs

processes primary

transcripts (pri-miRNA) to

60 bp long pre-miRNA

Drosha

Dicer

49

Types of small RNA-associated Argonaute proteins, and the

origin and mechanism of action of small RNAs

Kim in sod., 2009. Biogenesis of small RNAs in animals.

The name Argonaute comes from phenotypes

observed for AGO1 mutants in Arabidopsis

thaliana. The plants resembled the tentacles of

the pelagic octopus, Argonauta argo.

Bohmert in sod.,1998 50


• Andrew Fire and Craig Mello

• among the most important

discoveries in molecular biology in

the past few decades

• Nobel prize in physiology/medicine,

2006.

• Victor Ambros

• equally important as RNAi

• deserves recognition by the Nobel

committee

Fire in sod.

Potent and specific genetic interference by

double-stranded RNA in Caenorhabditis

elegans.

Nature 1998; 391: 806-811.

Lee in sod.

The C. elegans heterochronic gene lin-4

encodes small RNAs with antisense

complementarity to lin-14.

Cell 1993; 75, 843–854.

• odkritje RNAi • odkritje miRNA

51

52


primarna

miRNA

>1000 bp

prekurzorska

miRNA

~60 bp

zrela

miRNA

21-23 bp

miRNA-protein

complex = miRISC

MiRNAs - translational silencing

Novina CD, Sharp PA. The RNAi revolution. Nature 2004;430:161-4.

1. miRNA gen

2. miRNA tarče

3. Mehanizem za utišanje genov

53

Georges M. Polymorphic miRNA-mediated gene regulation: contribution to phenotypic variation

and disease. Curr Opin Genet Dev. 2007;17:166-76

miRNA tarče miRNA geni Mehanizem za utišanje

DSPs altering miRNA

recognition sites in the target

Altering existing target sites

Creating illegitimate target sites

DSPs altering the 3'UTR of the

target (e.g. polymorphic

polyadenylation)

DSPs altering the sequence of

the miRNA

Stabilizing or destabilizing the

interaction with the target

DSPs altering the concentration

of the miRNA

CNVs encompassing the pri-

miRNA

DSPs altering the transcription

rate of the pri-miRNA

DSPs altering the amino acid

sequence of silencing

components

DSPs altering the concentration

of silencing components

CNVs encompassing silencing

components

1. 2. 3.

CNV = Copy Number Variation; različica v številu kopij, DSP: DNA sequence polymorphisms

54

Kategorije genetske variabilnosti, ki vplivajo na

z miRNA posredovano uravnavanje genov


Iwai N, Naraba H. Polymorphisms in human pre-miRNAs. BBRC; 2005 331:1439–44.

Saunders MA. Human polymorphism at microRNAs and microRNA target sites. PNAS 2007;104:3300–5

1. miRNA geni

stem loop

miRNA

miRNA*

stem*

seed

Polymorphisms in both pre-miRNA and mature miRNA modify

various biological processes by influencing the processing

and/or target selection of miRNAs

55

Polimorfizmi v pre-miRNA in zreli miRNA

vplivajo na procesiranje in/ali izbiro tarč

za miRNA in s tem na številne biološke

procese.

Iwai N, Naraba H. Polymorphisms in human pre-miRNAs. BBRC; 2005 331:1439–44.

pre-miRNA

zrela miRNA

hsa-mir-154

(rs41286570)

miRNA geni

56


Garzon R. et al. Targeting microRNAs in cancer: rationale, strategies and challenges. Nature Reviews Drug

Discovery 2010;9:775-789.

Some miRNAs (miR-29b)

coordinately regulate multiple

targets in different pathways

miR-15a induces

apoptosis by targeting

B-cell lymphoma

protein-2 (BCL-2)

miR-29b suppresses cell

proliferation by blocking

CDK6

Ocenjeno je, da ima miRNA v povprečju 200 tarč. Krek in sod., 2005

57

Lewis in sod.

An ENU-induced mutation of miR-96

associated with progressive hearing

loss in mice.

Nature Genetics 2009;41:614–618.

Mencia in sod.

Mutations in the seed region of human

miR-96 are responsible for

nonsyndromic progressive hearing

loss.

Nature Genetics 2009;41:609-613.

SNP-ji znotraj seed miRNA regij – povezava z gluhostjo

Mouse

A>T substitution in mmu-miR-96

Human

GC>AA substitution in hsa-miR-96

58


1. growth

associated

quantitative trait

locus (QTL)

2. body mass

associated host

gene Gpc3.

3. conserved

miRNA gene -

Mir717

4. mature miRNA

seed SNP

identified in the

lean mouse strain

129/Sv

Kunej T, Jevsinek Skok D, Horvat S, Dovc P, Jiang Z. The glypican 3-hosted murine Mir717 gene:

sequence conservation, seed region polymorphisms and putative targets. Int J Biol Sci. 2010; 6:769-772.

Seed mmu-mir-717 SNP – nalaganje maščobe

59

2. miRNA tarče

3'-UTR gena

POU5F1 (OCT4)

SEED Out-seed 60

Analiza s programom TargetScan: ohranjeno in delno ohranjeno mesto za vezavo

miRNA na 3„-UTR gena POU5F1.


2. miRNA tarče

Target site for miR-141/200a in the FTO gene is

conserved in six species.

• Patrocles database

• polymorphic

miRNA target site

• TargetScan

• conserved miRNA target site

3'-UTR of the NANOG gene 3'-UTR of the FTO gene

61

Interakcija med miR-127-3p in tarčo BCL6

je bila ţe eksperimentalno dokazana.

miRecords

1) Eksperimentalno potrjene (validirane) miRNA tarče

• Trenutna verzija vsebuje: 2286 interakcij med 548 miRNA in 1579 tarčnimi geni pri

devetih ţivalskih vrstah.

2) Predvidene miRNA tarče

• Integracija 11 bioinformacijskih orodij

http://mirecords.umn.edu/miRecords/

Samo PITA in RNA22 sta

napovedala interakcijo

med miR-127-3p in BCL6.

2. miRNA tarče

The prediction of the likelihood of formation

of a productive miRNA–mRNA duplex:

-sequence complementarities,

-thermodynamic stability calculations,

-evolutionary conservation.

62


Exceptional muscularity of the belgian texel sheep

A point mutation (G>A) that creates an illegitimate miRNA target site

for mir-1 and mir-206 in the 3' UTR of MSTN; myostatin gene.

muscular hypertrophy

of Texel sheep

Clop in sod. A mutation creating a potential illegitimate microRNA target site in the myostatin

gene affects muscularity in sheep. Nature Genetics 2006;38:813-818.

2. miRNA tarče

highly expressed in

skeletal muscle controls skeletal muscle mass;

restrains muscle growth

63

3. Mehanizem za utišanje

genov (miRNA silencing

machinery)

nucleus

cytoplasm

=slicer

argonauts

=DiGeorge syndrome

critical region gene 8

=TAR (HIV-1) RNA

binding protein 2

64




• miRNA


• miRNA geni

• miRNA tarče



• piRNA

• snoRNA

Vsebina

65

Watanabe et al.,

Genes and

Development,

2006

Grivna et al.,

Genes and

Development,

2006 Girard et al.,

Nature, 2006

Aravin et al.,

Nature, 2006

piRNA; Piwi-interacting RNA - odkritje

66


Types of small RNA-associated Argonaute proteins, and the

origin and mechanism of action of small RNAs

Kim in sod., 2009. Biogenesis of small RNAs in animals.

Piwi:

• P-element induced wimpy testis, which refers to the strong

spermatogenesis and sterility defect observed in piwi

mutant males.

• discovered in Drosophila (Lin in Spradling, 1997).

67

Loss of murine Piwi homologues leads to spermatogenic arrest

Deng and Lin. miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis.

Kuramochi-Miyagawa et al. Mili, a mammalian member of piwi family gene, is essential for spermatogenesis.

sg: spermatogonia

sc: spermatocyte

r: round spermatid

e: elongated spermatids Spermatogenesis in miwi -/-

mice is blocked at the early

round spermatid stage.

68


piRNA; vloga pri razvoju bolezni

-no direct role for piRNAs in human disease,

-hiwi (human piwi homolog), has been implicated in several diseases:

• The hiwi gene locus is linked to the onset of testicular germ cell tumors

• Hiwi is overexpressed in testicular seminomas

Qiao et al., Oncogene, 2002

• Loss of the hiwi gene locus has been associated with ambiguous genitalia and

testicular atrophy.

• Expression of Hiwi is positively correlated with cell proliferation in the setting of

pancreatic and gastric cancers.

69

Lakshmi S, Agrawal S. piRNABank: a web resource on classified and clustered Piwi-interacting

RNAs. Nucleic Acids Research 2008; 36: D173–D177. 70




• miRNA


• miRNA geni

• miRNA tarče



• piRNA

• snoRNA

Vsebina

71

snoRNAs; small nucleolar RNAs, male nukleolarne RNA

Delujejo kot ribonukleoproteinski (RNP) kompleksi, ki vodijo encimatsko modifikacijo

tarčnih RNA z RNA:RNA interakcijami.

H/ACA box snoRNA

100–200 bp

vodi psevdouridilacijo tarčne RNAs

C/D box snoRNA

70–120 bp

vodi metilacijo tarčne RNAs

Matera in sod. 2007; Makarova in Kramerov; 2007

Pseudo-

uridylated

nucleotide

UGAUGA CUGA ACA ANANNA

pseudouridylated

nucleotide

snoRNA tarče: tRNA, rRNA, snRNA 72


Znotraj delecije 15q11-13:

• SNORD64

• SNORD107

• SNORD108

• Dve kopiji SNORD109

• 29 kopij SNORD116 (HBII-85)

• 48 kopij SNORD115 (HBII-52)

snoRNAs - povezava s sindromom Prader-Willi

C/D BOX, 116;

SNORD116

Sahoo in sod. Prader-Willi phenotype caused by paternal deficiency for the HBII-85 C/D box small

nucleolar RNA cluster. Nat Genet; 2008;40:719–21. 73

http://www.nature.com/focus/rnai/animations/index.html 74

http://www.nature.com/focus/rnai/animations/index.html

http://www.nature.com/focus/rnai/animations/index.html


75

76


77

78


The Argonaute family of proteins, specifically Argonaute 2 (also known as Ago2,

eIF2C2 or Slicer) are essential components of RISC, which are involved in mRNA

cleavage. 79

http://www.abcam.com/Ago2-eIF2C2-antibody-ab5072.html

rna-interferenca (rnai) v temeljnih raziskavah in biomedicini

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