BCH3031 Lecture Notes - Lecture 11: Heterochrony, Forward Genetics, Genetic Screen
25 May 2018
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Lecture 11-12: World of RNA
Diversity of RNA Functions
• RNA interference (RNAi)
o Discovered ~15 years ago
o Revloutionarised how we think about
▪ Gene regulation
▪ Development
▪ Disease
o Significant promise for medical applications
▪ Research
▪ Diagnositcs
▪ Therapeutics
o Non-medical applications
▪ Crop modifications
Colour modification via over expression of key enzymes in colour biosynthesis
pathway
• Colour change segregated with transgene
• Co-suppression: both endogenous and introducing genes silenced
• Added sense RNA and anti-sense RNA
o When separate sense and anti-sense, C.elegans acted as wild type
o When combined: sense + antisense, C.elegans phenocopied mutant
• Conclusion drawn:
o Double stranded RNA
o To promoter region → wildtype
o To intron → wildtype
o Interacting with mature mRNA to drive phenotype
▪ Likely to be in cytoplasm
o When inject double strand RNA – cant detect
▪ Double strand RNA injected, targets maturemRNA → leads to
degradation of RNA
Biogenesis of microRNAs (miRNAs)
• Non-coding RNAs
• Part of our own genome: we make them by transcription, RNA polymerase 2
• Short, 19-24 nucleotide, non-coding RNAs
• Negatively regulate (generally) gene expression post transcriptionally through
sequence specific base pairing with their targets
• Effect is at level of mRNA translation or stability in cytoplasm
• In broad consequences of their action might include
o Temporal or spatial switching
o Fine tuning of expression
o Defining tissue identity
• Identification of miRNAs
o Discovered first miRNA, non-coding 22nt RNA through conventional
forward genetic screens in C. elegans for heterochronic mutants
o Lin-4 (lineage 4) regulates lin-14 translation via an antisense RNA-
RNA interaction (can bind together)
find more resources at oneclass.com
find more resources at oneclass.com
▪ Lin-4 mutant doesn’t differentiate into second cell type (all
blue) → heterochronic phenotype
o Second miRNA: let-7
▪ Genetic screen for mutations that suppress synthetic sterile
phenotype (forward approach)
▪ Non coding 21nt RNA
▪ Regulates lin-41
o Amount of protein encoded by miRNA will decrease as expression of
the appropriate miRNA goes up
• How to identify microRNAs
o Look for conservation of sex between organisms
o Similar expression in different organisms suggests an evolutionary
conserved function
▪ Zebrafish: brain specific miRNAs show distinct in situ
expression patterns
• Nuclear processing of pri-miRNAs (biogenesis)
o Pri-miRNAs are processed by RNAseII enzyme Drosha and DGCR8
→ produces pre-miRNAs
▪ DGCR8 acts as molecular ruler to measure and determine
Drosha cleavage site
▪ Drosh and DGCR8 are part of Drosha Microprocessor
Complex
o Pre-miRNAs are 70-80 nucleotides stem-loop structures
▪ 5’ end has phosphate and 3’ end at 2 nt overhang
▪ What will be mature miRNA is located on one of stem arms of
pre-miRNA
▪ Pre-miRNA as exported to cytoplasm (by exportin 5) for
further processing
o High fidelity
• Alternative nuclear processing for mirtrons
o Mirtrons: pri-miRNAs encoded by introns that don’t contain 11 bp
o Processed by spliceosomes instead of Drosha
o Processed in cytoplasm → released as lariat structure
• Cytoplasmic processing of pre-miRNAs
o Second RNase III enzyme, Dicer – associated with TRBP
▪ Occurs in cytoplasm
▪ Job: to recognise and cut pre-miRNA → generate mature
miRNA as part of short RNA duplex (21bp)
• Duplex RNA is unwound by
helicase activity (HGO)
• Unwinding starts at end with
lowest thermodynamic stability
▪ Strand that has 5’ terminus is future
mRNA; other strand is degraded
o Dicer
▪ Checks distance from 3’ over-hang to
terminal loop of pre-miRNA via its
PAZ and helicase domains
▪ Dicer-1 cuts pre-miRNA at a fixed
distance (non-specific manner) from 3’
find more resources at oneclass.com
find more resources at oneclass.com
