33 - RNA interference Flashcards
What is RNA interference?
Antisense RNA that inhibits translation of specific transcripts
Also called post transcriptional gene silencing (PTGS)
RNAi is the process of mRNA degradation that is induced by double-stranded RNA (dsRNA) in a sequence-specific manner
True or false? Double stranded RNAi is more effective than single stranded RNAi?
True
What is the molecular basis of co-suppression?
RNAi
What type of transcripts does dsRNAi silence?
Sequences similar to the dsRNA
What are the basic steps of RNAi?
- Initiation step: Dicer binds dsRNA and cleaves into nucleotide fragments about 22 nt long (these fragments are called siRNAs, small interfering RNAs)
- RISC (RNA induced silencing complex) binds one siRNA
- RISC activated (siRNA unwinding, ATP dependent)
- Effector step: activated RISC binds a target mRNA and degrades it
What is dicer?
A member of the RNase III family of double strand specific endonucleases, generates precisely sized fragments with 2-nuc overhands phosphorylated at 5’ (siRNAs).
Is the enzyme for the initiation step of RNAi
What is RISC?
RNA induced silencing complex includes Argonaute (another endoribonuclease). RISC recognizes dicer-generated siRNAs and the unwound fragment directs the particle to cognate mRNA by Watson-Crick base pairing.
Gene expression is silenced by mRNA degradation and/or translation inhibition (effector step of RNAi)
What is amplification of RNAi?
Some siRNA unwind without being associated with RISC. The antisense strand can bind to its complementary region of the corresponding mRNA, serving as a primer for a RdRP, which will generate new dsRNA of the target gene.
RdRP (RNA dependent RNA polymerase) generates dsRNA as substrate for dicer, and more siRNA is made
siRNA can go from cell to cell, and so silencing can propagate to other tissues.
What do miRNAs (micro interfering RNAs) do?
Encode small RNAs that regulate gene expression through post-transcriptional repression
How are miRNA transcripts processed?
Sequentially by two RNase III enzymes, Microprocessor and Dicer to yield mature miRNA duplexes, ranging from 18vo to 24bp in length
How do miRNAs prevent proliferation of transposons? What is the consequence of this?
Silencing transposon mRNAs, this controls the expansion of mobile genetic elements in the genome. It is possible because miRNA ‘genes’ have dsRNA with similarity to transposon mRNA
What are the steps of translation repression/mRNA degradation by miRNAs?
- Transcription of pri-miRNAs
- Drosha process yields pre-miRNA that is exported out of the nucleus
- pre-miRNA loaded onto RISC, dicer cleaves miRNA duplex
- miRNA helicase unwinds to yield mature miRNA
- Targets selected, if there is near perfect complementation with cytoplasmic mRNA, the mRNA is degraded.
- Is there is partial complementarity, translation is repressed on RISC and then mRNA is degraded.
What does miRNA do in normal tissues?
Proper miRNA transcription, processing and binding to complementary sequences of mRNA results in the repression of target-gene expression through a block in protein translation or altered mRNA stability. The overall result is normal rates of cellular growth, proliferation, differentiation and cell death.
What happens when there is a reduction or deletion of miRNA?
Tumour formation
Leads to inappropriate expression of miRNA target, oncoprotein
What happens when there is overexpression of miRNA in a cell?
Tumour formation. Increased miRNA would eliminate the expression of miRNA-target tumour suppressor gene and lead to cancer progression.
This might occur because of amplification, active promoter, increased efficiency in miRNA processing or increased stability of the miRNA.
