1. Epigenetics: RNAi and Heterochromatin formation

2. 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

3. 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

4. Sources of dsRNA

5. 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

6. 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

7. 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

8. Conservation of RNAi and Heterochromatin assembly proteins

9. siRNA target via RNA-RNA models
Two models for targeting RITS
RNA-DNA model Vs
RNA-RNA model
Disturbance in transcription causes failure in PTGS
Localizing RITS complex to centromeric regions, which is also siRNA dependent

10. 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)