Epigenetics:RNAi and Heterochromatin formation

RNAi pathways

• RNAi (RNA interference) -> silencing by dsRNA• dsRNA -> siRNA -> target mRNA ->

PostTranscriptional Gene Silencing (PTGS)• dsRNA -> siRNA -> target chromatin ->

Transcriptional Gene Silencing (TGS)• Dicer: RNase III class ribonuclease -> convert

dsRNA -> siRNA• Argonaute/PIWI family: RNAse H -> cleaving

target mRNA

Three main RNAi complexes

1 RISC (RNA-Induced Silencing Complex): siRNA,Argonaute, -> remove target mRNAs (PTGS)

2 RITS (RNA-Induced Transcriptional Silencing Complex): siRNA, target chromatins (TGS)

3 miRNA-mediated silencing mechanism: endogenous non-coding gene -> miRNA by Dicer and Argonaute -> suppress translation

Sources of dsRNA

Early indications

• PSTV (potato spindle tuber viroid): a 359-nt RNA virus triggers DNA methylation on any transgenic PSTV sequences in the host genome, and this requires the viroid transcription

• In plants, aberrant transcripts trigger DNA methylation of similar sequences

Connection of RNAi with Heterochromatin formation

Mutations in RNAi complex (dcr1, ago1, rdp1) in S. pombe caused loss of heterochromatin and accumulation of repeat-derived transcripts

Sequencing of a small RNA library (~22 nt long) derived many hit from centromeric repeats

dsRNA and siRNA generation

• Bi-directional transcription of repeats• Transcription of inverted repeats• In S. pombe, RDRC (RNA-directed RNA

polymerase Complex) is required for siRNA production: Rdp1, Cid12, Hrr1

Conservation of RNAi and Heterochromatin assembly proteins

siRNA target via RNA-RNA models

Two models for targeting RITSRNA-DNA modelVsRNA-RNA model

Disturbance in transcription causes failure in PTGS

Localizing RITS complex to centromeric regions, which is also siRNA dependent

RNAi-mediated Chromatin and DNA modification in Arabidopsis

RNA-directed RNA polymerases(RdRP, RdR2, RNA POL IV)

siRNA production by DCL3

H3K9 methylation (KYP/SUVH4)

Chromatin remodeling (DDM1)

DNA methylation (CMT3, MET1,DRM1/2)