RNA interference

Hepatitis B

protein x interferes with DNA repair mechanisms and over time can cause cancer

measles

ss - sense enveloped uses hemmaglutinin to bind to cells in respiratory tract epithelial cells then infects macrophages, dendritic cells etc

RNA interference

destoryes mRNA to silence expression, triggered by dsRNA in the cell to defend from viruses

mammal cell response to RNA

anything longer than 30 illicits an immune response

mRNA decay

most do this, is constantly happening

deadenylases

remove mRNA poly A tail

decapping enzymes

decap the 5’ cap

RNAi

RNA takes ds conformation which is substrate for DICER family of proteins to cut into siRNAs

Petunia pigmentation study

First report of RNAi, were trying to make black petunias but the mRNA kept not being expressed because of RNAi

fire and mello

E. Elegans experiment, purified and injected sense and antisense ssRNA and dsRNA which was much more effective

Micro RNA

similar to siRNA, found in c. Elegans, transcribed to pri-miRNA thats cleaved by drosha enzyme into pre-miRNA

pri-miRNA

primary mRNA with long multiple hairpins and imperfect internal sequences, gets cleaved by Drosha

pre-miRNA

precursor miRNA 70nt long, exported from nucleus

MiRNA function

regulate gene expression, guard genes from interruption by jumping genes and protect from viral attacks

SiRNA origins

exogenous uptake by cells, dsRNA

siRNA pairing

highly specific target with only one mRNA target

miRNA target

inhibit translation of multiple mRNA targets

MiRNA and RNAi

both share protein components and are made with similar processes

argonaute proteins

mammalian proteins, all bind miRNA and siRNA

Mammal dicer protein

mainly for miRNA biogenesis

Helicase domain of dicer

highly homologous to RIGI/MDA5/LGP2, with three subdomains held, hel2i, and hel2

interferon stimulated genes

encode proteins capable of inhibiting viral replication, virus spread

mammalian IFN response

commonality between RNAi pathway and the IFN pathways to keep cell in homeostasis with viruses, but presence of some response limit others

RNAi initiation

generation of mature siRNA or miRNA

RNAi execution

silencing of target gene, degradation or inhibition of translation

siRNA biogenesis

dicer cleaves long dsRNA into siRNA 21-23 nt long (ds exogeneous)

drosha protein

processes pri-miRNA into pre-miRNA laving 3’ overhangs on pre miRNA

nuclear RNAse III enzyme

tandem RNAse II domains, finds pre miRNA with hairpin terminal loop and stem structure, hairpin flanking sequences

cytoplasmic RNAse III enzyme

cleaves sdRNA or pre miRNA, cleaves at termini

dicer functional domains

putative Helicase, PAZ domain, tandem RNAse II domains, ds RNA binding domain

Dicer function

dfines the length of small RNA by the distance between PAZ domain and RNase III cleavage sites

argonaute (AGO) proteins

loading of small RNA onto protein is key in forming RISC complex, binds both ends of siRNA and 5’ end in pocket and 3’ anchored to PAZ

RISC complex

AGO exposes nucleotides for target pairing, and promotes conformational change to expose nt 2-8 and 13-16 for target recognition, central 3’ tail regions of the siRNA provide a Helicase geometry for catalyze

c. Elegans

intoruction of dsRNA sufficient for RNAi, in vitro transcription

RNAi in plants

key regulator of infections, have pathogen recognition receptor proteins that recognize pathogens and place the plant in defense mode of patter triggered immunity

cleaved mRNA in plants

act as template of another round of dsRNA formation by rdRP protein starting the second cycle of RNAi and producing secondary siRNAs

Viral suppressors of RNA silencing

virus strategy to block RNAi mechanisms by inhibiting function of various components