RNAI - TSG

TSG: transcriptional Gene silencing

Chromatin modifcation Pathway : Transcriptionally silencing translation via methylation and formation of heterochromatin.

RdDM: RNA-Directed DNA Methylation, gene silencing specific to only regions with RNA-DNA complementarity

Methylation targets: CG, CNG, CNN

RdDM steps:

1.dsRNA via RDR2

2. cut up into shorter siRNA via Dicer-like protein.

3. chromatin remodelling proteins open  chromatin allowing siRNA to bind to region of chromatin with base complementarity 

4. Leads to recruitment of DNA-Cytosine-Methyltransferases, adding methyl groups to the cytosines

5. addition of these methyl groups results in inactivation of gene.

Maintenance of methylation:

CG Maintenance of methylation: Chromatin remodelling complex(DDM1) and Histone deacetylase --> chromatin stays methylated.

CNG maintenance of methylation: DNA methyltransferases, Histone methyltransferase, Argonaute protein

CNN maintenance of methylation: Cannot be maintained in methylated state and requires continuous presence of inducing RNA.

Reactivation: glycosylases/lyases, needed for loss of methylation.

RdDM Targets: transposons and repeats in heterochromatin and some euchromatin, genes containing transposon or transposon-like elements in their promoters and introns, genes containing tandem repeats in regulatory regions. 

RNAi-Mediated Heterochromatin assembly: methylation of histones.

RNAi-Mediated Heterochromatin targets: transposable elements, LTRs, regions of genome containing transposonderived sequences, centromeric repeats.

RNAi-Mediated Heterochromatin pathway:

1. dsRNA produced by RdRP

2. Dicer generates shorter siRNA

3. RITS binds siRNA and directs it to complementary region on chromosome.

4. Binding recruits histone methyltransferase resulting in methylation of H3.

Maintenance in TGS: Heterochromatin binding protein associates with HMT and RITS, aids in maintaining chromatin silenced state, aids in spreading of silenced state.

HMT: Histone methyltransferase

RITS: RNA-Induced intiation of transcriptional Gene silencing

siRNAs derived from region of chromosome being silenced

Biological functions of miRNAs: development, differentiation, proliferation, apoptosis, oncogenesis.

miRNA control development by: clearing previous gene reulatory trancripts to make room for new expression programs.

miRNA control example: Plant development of leaf shape/patterning

siRNA control example: Protection of the genome from mutagenic agents such as transposable elements, siRNA detroyed or repressed transcripts, Chromatin modification suppresion of transcription and movement.

Chromatin modifactions: stabilisation of genome by preventing rearrangement of centromeric regions, ensuring normal chromosome segregation.

RNAi used in genome-wide approaches for analysing gene expression, help determine function of genes.

RNAi in Cotton: modification of oil content, fatty acid desaturase gene targeted.

RNAi in coffee: reduction in caffeine content, caffeine synthase gene targeted