CSB349H1 Lecture Notes - Lecture 11: Locked Nucleic Acid, Rna Virus, Microrna
14 May 2018
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RNA Virus
Lecture 11(b): miRNA, RNAi & ncRNA:
Triggering RNAi:
• RNAi can be triggered by various sources of RNA molecules:
o Endogenous miRNAs
o Exogenous constructs [siRNA]
o RNA viruses [siRNA]
o Transposons [siRNA]
Experimental Transgenes:
• To create a construct to target our gene of interest we need to have:
o Promotor: [Sense Orientation] – [Loop] – [Antisense Orientation] – [Terminator]
• Once injected, it will be transcribed it will generate a stem
loop structure; transcript will be complementary to each
other and then be processed with dicer to generate siRNA
ð This is beneficial as it is inducible; often causes a knock-down
and not a knock-out:
o Therefore, controllable when we want to see the silencing
of a specific mRNA (i.e. developmental pathway)
RNA Virus:
• Some RNA viruses have dsRNA intermediates
o dsRNA intermediates are recognized by cells as ‘foreign’
and will be via RISC which will function to destroy
viral RNA transcripts
Transposons:
• DNA transposons:
o Transcribed into RNA and hybridize into dsRNA
and are capable of ‘jumping’ into actively transcribed
loci in a random orientations
• Retrotransposons:
o Integrate into DNA in reverse orientation; RNA intermediates hybridize into dsRNA
ð dsRNA is recognized by RISC and Dicer functions to process them into siRNA
RISC versus RITS:
• RITS = RNA Induced Transcriptional Silencing Complex – blocks transcription
o Return back to the nucleus and binds to the nascent mRNA and target the DNA itself
leading to DNA histone methylation
• RISC = RNA Induced Silencing Complex – blocks translation
o Found in the cytosol and binds to the mature mRNA leading to its cleavage
Document Summary
Triggering rnai: rnai can be triggered by various sources of rna molecules: Lecture 11(b): mirna, rnai & ncrna: endogenous mirnas, exogenous constructs [sirna, rna viruses [sirna, transposons [sirna] This is beneficial as it is inducible; often causes a knock-down and not a knock-out: therefore, controllable when we want to see the silencing of a specific mrna (i. e. developmental pathway) Rna virus: some rna viruses have dsrna intermediates, dsrna intermediates are recognized by cells as foreign" and will be via risc which will function to destroy viral rna transcripts. Transposons: dna transposons, transcribed into rna and hybridize into dsrna and are capable of jumping" into actively transcribed loci in a random orientations. Retrotransposons: integrate into dna in reverse orientation; rna intermediates hybridize into dsrna. Dsrna is recognized by risc and dicer functions to process them into sirna. Detecting mirna expression: direct, mirna are too small for conventional in-situ hybridization (ish)
