RNA silencing Jennifer Grier 1 November 2012. Overview Timing Mechanisms Long non-coding RNA – Xist – Air – HOTAIR Short non-coding RNA – siRNA – miRNA.

RNA silencing

Jennifer Grier1 November 2012

Overview

• Timing• Mechanisms• Long non-coding RNA– Xist– Air– HOTAIR

• Short non-coding RNA– siRNA– miRNA– piRNA

Timing/Location of RNA silencing

Nucleus• CDGS: chromatin-dependent gene silencing– TGS: Transcriptional gene silencing– CTGS: Co-transcriptional gene silencing

Cytoplasm• PTGS: post-transcriptional gene silencing

Mechanisms of RNA silencing

• Heterochromatin Formation – TGS, CTGS

• Enhancer - repression of silencing

• Transcript degradation– PTGS

• Translational arrest – PTGS



Adapted from: Kevin V. Morris. Oligonucleotides. 2009 December;19(4):299-305.



• Enhancer - repression of silencing

XX

XAdapted from: Kevin V. Morris. Oligonucleotides. 2009 December;19(4):299-305.


• Transcript degradation– PTGS

• Translational arrest – PTGS

Moazed, D. Nature. 2009 Jan 22;457(7228):413-20.

(arrest)

http://proxy.library.upenn.edu:2082/pubmed/19158787

Long non-coding RNA’s

Abbreviated as lncRNA,or lincRNA (long intergenic non-coding RNA)

• Defined as:– >200 bp in length (can be up to 100 kb)– Not processed – Non-protein coding

• Very prevalent in genome

Mercer et al., NRG 2009

Found in many places in the genome

lncRNAGene transcript

How do they work?• chromatin regulator recruitment


How do they work?• chromatin regulator recruitment• RNA binding protein recruitment

RNA binding protein inhibits HATs


How do they work?• chromatin regulator recruitment• RNA binding protein recruitment• TF recruitment/nuclear import


Enhancer RNA

Some lncRNA in HOX cluster interact with Trithorx resulting in H3K4me3

How do they work?• chromatin regulator recruitment• RNA binding protein recruitment• TF recruitment (triple helix)• interference with binding or activity of the general transcriptional machinery


Koziol and Rinn COGD, 2010

Mechanisms of Chromatin Regulation

Important to note:

Cis or Trans



• Tethers: sequence specificity




• Acts as Scaffold





• Regulates activity






• Mediates long range interactions





• Mediates long range interactions

Means of carrying epigenetic information from mother to daughter cell

X inactivation

Pontier, DB and Gribnau, J. Hum Genet. 2011 August; 130(2): 223–236.

FISH = fluorescent in situ hybridization

polycomb

Magenta: Jpx RNA, green XISTTian et al. Cell 2010

X inactivation: additional players

X-inactivation center4 ncRNAs• Xi : Xist and RepA– RepA binds PRC2

• Xa: Tsix• cis-Xist repressor

• Both: Jpx • cis- and trans-Xist

activator

Xist only expressed in heterochromatinRepA stem loop binds PRC2 – leading to H3K27me3 in cis on XiAllows activation by JPX

Caley et al., The Scientific World Journal 2010

X inactivation: additional playersTsix recruits Dnmt3a– Methylates Xist promoterActivation by JPX is blocked

Ideraabdullah, Mut. Res., 2008

AIR recruits G9a (HMT)

Results in H3K9me at imprinted gene promoters in cis

Igf2r/Air - lncRNA mediated imprinting

HOTAIR

Gupta et al. Nature 2101

• Expressed from HOXC locus

• Represses in trans

Developmentally regulated (Hox genes)

Tsai et al., Science 2010

HOTAIR

• Binds: – PRC2– EZH2 (HMT)– CoREST

(HDAC)– LSD1

(H3K4me demethlase)

Moazed Nature 2009

(RNAi)

Small non-coding RNAs

RNAi = RNA-interference

Double stranded (ds) RNA induces homology-dependent degradation of cognate RNA and depletion of protein over time

Andrew Z. Fire Craig C. Mello1/2 of the prize 1/2 of the prizeUSA USAStanford University University of MassachusettsSchool of Medicine Medical SchoolStanford, CA, USA Worcester, MA, USA

The Nobel Prize in Physiology or Medicine 2006"for their discovery of RNA interference - gene silencing by double-stranded RNA"

Functions:

viral silencing in plants

suppression of transposable elements

silencing of repetitive sequences

heterochromatin formation

transgene silencing

Small interfering RNAs: siRNAs

Source for siRNA:

Endogenous

Exogenous

How are small ncRNA generated?

• siRNA– natural cis antisense siRNAs

– repeat associated siRNA

• Result in dsRNA products

• PolII/V transcribed or bi-directional transcription

Moazed Nature 2009

Moazed Nature 2009


• From aberrant transcripts• By RdRp: RNA-dependent RNA polymerase


• miRNA– from miRNA genes (non-coding)– found within lncRNAs and coding genes– PolII transcribed– Forms a hairpin

Tamari and ZamorePrespectives: machines for RNAiGenes &Dev.19:517-529 (2005)

Processing of small ncRNAs

Processing of miRNAs

DCL Arabidopsisinitially isolated as developmental mutant!

Dicer Ribonuclease III homolog; helicaseDrosophila, C.elegans, mouse, fungi

Role for a bidentate ribonuclease in the initiation step of RNA interferenceEmily Bernstein, Amy A. Caudy, Scott M. Hammond & Gregory J. Hannon

NATURE | VOL 409 | 18 JANUARY 2001


Binds precursor ds or miRNA through PAZ domainCleaves precursor through ribonuclease III domainSpacing between PAZ and RIII domains determine size and cut location resulting in staggered cuts

Moazed Nature 2009


Functions of small ncRNA

• Argonaute family of proteins– bind miRNA or siRNA

or piRNA• At least two classes– AGO-like– PIWI-like

Important Components


• AGO (Argonaute)• PIWI domain binds 5’ end small RNA (RNAse H-like fold)• PAZ domain binds 3’ end small RNA guide strand• Mid domain binds CAP

• Slicer activity (some AGOs)required for siRNA, most plant miRNAmakes a cut in target RNA leading to degradation

• Catalytically inactive AGOs lead to inhibition of translation (stalling)

• Multi turnover enzyme

RNA-directed RNA polymerase RdRP:

AmplificationTransport (systemic RNAi)Heterochromatin formation

Not required for Drosophila or mammalian RNAi


Moazed Nature 2009

RITS complex

RNA induced transcriptional silencingAgo1 (Argonaute, binds siRNAs)Chp1 (chromodomain, binds H3K9me)Tas3 (binds Ago1 and Chp1, spreading)siRNAs (small inhibitory RNA)

Like RISC: effector complex, bind small RNA


Transcriptional Gene Silencing

Djupedal and Ekwall, Cell Research, 2009





initiation

amplification

de novo DNA methylationSimon and Meyers COPB 2011

RNA induced DNA Methylation

-- Role of siRNAs in ESTABLISHMENT of transcriptional gene

-- silencing first discovered in plants

RNA methods of TGS

tasiRNAs


, V

PolIV and V in TGS

PolIV and V in TGSPolIV

PolV

Wierzbicki et al. Cell, 2008


PolIV and V in TGS


PolIV and V in TGS

Haag and Pikaard, Nat. Rev. MCB, 2011

RNA methods of TGS (in plants)

Maintenance

spreading


TGS speading A. thaliana


Khraiwesh et al., Cell 2010

High levels of miRNA:

Cause miRNA:mRNA duplex formation

Trigger DNA methylation

(for example in response to hormone treatment)

Also described in mammalsKim et al, PNAS 2008

In moss: role of miRNA in DNA methylation


Role of siRNAs in silencing (plants)

triggers DNA methylation

recruitment of H3K9me

role in maintenance of DNA methylation

spreading

Lejeune and Allshire, COCB 2011

RNA methods of TGS (S. pombe)

Role of siRNAs in silencing (S. pombe)

initiation of all heterochromatintogether with Clr4 (HMT)!

maintentance of centromeric heterochromatin

tethered via RITS, Clr4-dependent

RITS brings in nascent transcript

RNA methods of TGS (S. pombe)

Potential for Inheritance?

1.siRNAs inherited, trigger H3K9me

2.Positive feedback between siRNAs and H3K9me: amplification and stabilization

YES!

piRNAFound in animalsRole in germline

Silencing of of repetitive DNAtransposons, subtelomeric regionspericentromeric regions

PROTECTING THE GERMLINE

PIWI: role in piRNA generation

Moazed Nature 2009


piRNA

2ndary piRNA

Dicer independent

PIWI-AGO

example of AGO activity

PIWI associates with HP1

D.m. piRNAs from follicle cells into oocytes

also linked to triggering DNA methylation

Accumulate at time of erasure and re-establishment of DNA methylation


Slotkin et al., Cell 2009

Similar observations for female gametophyte


In mammals: TE silencing via DNA methylation

pericentromeric RNA involved

Occurs early after fertilization

Satenard et al. Nat. Cell. Biol. 2010Probst et al. Dev Cell 2010


AGO-linked activities


PolIV

PolII

TGS

PTGS (post transcriptional gene silencing)message cleavage inhibition of translation

(small and large ncRNAs)

PTGSsiRNA

PTGS

• RISC complex RNA-induced silencing complex

• si RNA– AGO– DCR– dsRNA binding protein (TRBP)

• miRNA– AGO– GW182

FEBS letters 2005

PTGS

Small ncRNA movement

some miRNAs can move (short distance)

siRNAs can move: systemic responses

Systemic Silencing

Summarytypes of long noncoding RNAs

definition, where arise

roles of lnc RNAs in chromatin regulation

types of small noncoding RNAsdefinition, biogenesis

roles in chromatin regulation initiation and maintenance

Potential for epigenetic inheritance

Role in germline

Systemic silencing: why important?

RNA silencing Jennifer Grier 1 November 2012. Overview Timing Mechanisms Long non-coding RNA – Xist – Air – HOTAIR Short non-coding RNA – siRNA – miRNA.

Documents

scaffold slide

activity slide

arrest slide

genome slide

trans slide

h3k4me3 slide

sequence specificity

enhancer rna