ML -15 RNAi 1

•the steps in the PTGS pathways that involve small RNAs 

miRNA Pathway PTGS

1. miRNA transcribed by RNA pol 2

2. miRNA transcripts form imperfect stem-loop structures

3. Drosha, DGCR8/Pasha (dsRNA binding protein) join to dsmiRNA

4. pre-miRNA transported to cytoplasm via exportin-5 (transport protein)

5. DICER binds to pre-miRNA, with RNase 3 activity cleaves stem-loop RNAs resulting in  around 21 nucleotide miRNA:miRNA duplex

6. miRNA duplex will bind to arganaute protein but only 1 miRNA stays forming mature miRNA bound to argonaute

siRNA pathway PTGS

Triggers

-viral ssRNA replicating = dsRNA

-Inverted repeat transcription = dsRNA

-Transgene/transposon transcription and rna undergoing rdRP (RNA dependent RNA polymerase) = dsRNA

siRNA pathway (PTGS)

1. dsRNA gets processed by Dicer resulting in short dsRNAs (21-25) with two base pair overhangs at 3' end 

2. argonaute-containg risc interacts initially with ds-siRNA, argonaute turns ds-siRNA into ss-siRNA

3. Loaded risc (risc with mi/siRNA) binds to target mRNA through base paring with mi/siRNA

4.extensive matches between risc and siRNA results in rapid mRNA degradation = silenced

5. partial match between RISC and siRNA results in transfer of mRNA into P-bodies and eventual degradation = translation reduced

•the origins of miRNAs and siRNAs

Cleaing RISC vs non-cleaving RISC dependant on complementarity of mi/siRNA in RISC with target mRNA and Argonaute subtype in RISC (some have slicer acitivity, RNase H-like acitivity)

 •the potential fates of target mRNAs once they are bound by a functional mior siRISC complex

 •how PTGS pathways that involve small RNAs regulate gene expression

-Binding of loaded RISCs to 3' UTR may lead to: Removal of polyA tail, followed by removal of 5'Cap and transfer of mRNA to P-Bodies

Binding of Loaded RISCs= Deadenylation, Initiation block, Proteolysis, Elongation block.

P-bodies: processing bodies, cytoplasmic sites of storage and degradation.

rdRP: RNA dependant RNA Polymerase.

Dicer: Multidomain enzyme, with dsRNA binding domain and RNase 3 activity, cleaves long dsRNA into short dsRNAs.

Argonaute: key protein in RISC, contains dsRNA binding domain and RNase H-like acitivity domain. 

RISC: RNA-induced silencing complex.

Proteolysis: degradation of nascent peptide.

Deadenylation: removal of polyA tail results in mRNA being transferred to P-bodies

Initiation bock: removal of 5' cap --> prevention of 60s ribosomal subunit from binding results in mRNA being transferred to P-Bodies.

Slowing elongation = ribosomes drop off