Dicer cleaves long exogenous dsRNA into ds siRNA. Dicer is a dsRNA-spec RNAse III. siRNA duplexes are d 21nt strands. Each has a 5' phosphate and 3' OH group. 2 nt overhangs at 3' strands. Guide strand directs silencing. Passenger strand is ultimately destroyed.
RISC mediates target regulation by siRNA. RISc has Ago protein, a small RNA guide and auxiliary proteins. Mammals and C elegans have one Dicer to make miRNAs adn siRNAs. Dosophila has 2 Divers. DCR1 makes miRNAs and DCR2 makes si RNAs. Reduces competition between precursor miRNA and viral ds RNA.
Arabidopsis thaliana has 2 DCL proteins and 10 Agos. In plants, inverted repeate transgenes or coexpressed sense and antisense transcripts produce 2 sizes of siRNA: 21 and 2 4nt.
In plants, cis acting siRNAs (casiRNAs) originate from transposons, repetitive elements and tande, repeats. Bulk of endosiRNAs. casiRNAs promote heterochromatin formation. They direct DNa methylation and histone modification at loci from which they originate.
Trans acting sirNAs are generated by convergence of miRNA and siRNA pathways in plants.
Heterochromatin is inherited from one cycle to next. Silencing genes next to heterochromatin is position effect variegation PEV.
Silencing transposons make a large portion of heterochromatin sequences in eukaryotes.
Heterochromatin has histone modifications and DNA. Histone modifications specific to heterochromatin are more conserved and found in organisms that lack DNa methylation eg fisionyeast and C elegans and most animals and plants where DNA methylation is widespread.
Histone modifications include H3K9, H3K27 methylation and demethylation of H3K4.
dsRNA can trigger PTGS through sequence specific recognition of endogenous transcripts. dsRNA can be generated endogenously eg S. pombe.
It was discovered that in fission east, dramatic loss of reporter gene silencing near centromeres in absence of RNAi was accompanied by accumulation of transcripts derived from heterochromatic repeats. This suggest heterochromatic silencing was mediated by RNAi. Heterochromatic in S pombe exists at centromeres, telomeres and silent mating type locus.
Proper heterochromatin assembly at centromeres and telomeres is required for chromosome stbility. Heterochromatin in mating type locus is required for cell fate determination. Heterochromatin at centromeres recruits cohesin.
Heterochromatic regions eg S pombe centromeric repeats are normally enriched in chromodomain containing Swi6 and H3K9me2. Sites of active transcription are associated with H3K4 me2.
RITS is associated with heterochromatin. RITS bps with siRNAs with heterochromatic nascent transcripts. RITS recruits other protein complexes to heterochromatin.
RNA-directed RNAP complex (RDRC) consists of Rdp1, RNA helicase Hrr1 and Cid12. It generates dsRNA on single stranded RNA templates.
Ago1 endonuclease activity is used when one strand of siRNA duplex is sliced to ss siRNA. This occurs in ARC complex, which chaperones siRNA during complexes generated by Dicer to RITS complex.
In RITS complex, slicer activity of Ago1 on heterochromatin transcripts generates 3' ends to act as substrates for RdRP.
RITS catalytic activity recruits and spreads histone modification complex CLCR complex. CLRC contains sole S pombe lysine 9 histone methyltransferase Clr4 and H3K4 demethylase Lid 2.
RITS association with heterochromatin amplifies siRNA and modifies nucleosomal histones. This reinforces heterochromatin in seq spec manner. Clr4 can bind H3K9me2 through its chromodomain.
In some systems RNAi is ampified by positive feedback mechanism. dsRNA is synthesis by RDRP. In A thaliana, dsRNA promotes methylation of homologous DNA regions. DNA methylation and histone H3K9 methlyation requires an Ago family member.
Dicer, Ago and RDRP are conserved in S pombe. Deletion of genes encoding Dcr1, Ago1 and Rdp1 causes loss of H3K9 methylation, Swi6 localisation and silencing in centromeric DNA repeats.