L27 miRNA siRNA Flashcards
Recap of gene silencing pathways
- miRNA and siRNA utilize the same cellular machinery
RISCs that slice target transcript
- extensive complementarity to mRNA in ANY region of target transcript (5’ UTR/coding region/3’ UTR)
- near perfect pairing between miRISC/siRISC and target transcript
- repression associated with endonucleolytic cleavage of target mRNA
- slicing of target mRNA occurs at site complementary to residues 10-11 of the guide
- cleaved mRNA subsequently degraded (resulting products unprotected from exonucleases => degraded rapidly)
- many siRISC, most plant miRISC, a few animal miRISC
RISC that block protein synthesis
- Complementary to short repetitive sequence ALMOST ALWAYS in 3’ UTR of target transcript
- imperfect pairing between miRISC and target transcript
- number/efficiency of miRNA binding sites in 3’ UTR determines extent of repression
- repression associated with a block in protein synthesis or mRNA deadenylation and decay (mRNA destabilisation)
- most animal miRISC
miRISC associated with mRNA and recruits PABC-interacting proteins and deadenylase complex
See onenote slide
- shortens polyA tail/decapping/degradation
- deadenylase complex recruit factors that block formation of the 43S preinitiation complex (PIC)
miRNA target recognition in plants
- comparisons between cloned plant miRNAs and genomic sequence reveals almost complete complementarity with target mRNA
- approach: scan genomes for regions with 4 or less mismatches to cloned miRNAs
General observations:
- target one gene/closely related members of a gene family
- miRNA target site typically located in coding sequence of targeted transcript
- mainly transcription factors involved in developmental patterning, cell differentiation and nutrient homeostasis
miRNA target recognition in animals
see onenote slide
- typical miRNA recognition site in 3’ UTR - based on analysis of lin-4 and let-7 targets
- strict complementary over 7nt of seed region
- may be binding at position 1/8
- often central bulge
between nt 9-12 of miRNA sequence, prevents AGO from slicing its target (bulge due to mismatch) - moderate complementarity nt 13-16 (<5%)
miRNA target recognition in animals - approach and general observations
Approach:
- search for 3’ UTR that have equal/greater than 2 seed match sites (if seed match site is conserved, suggests that it performs a function)
- use genomic sequences to identify related genes and align 3’ UTRs
- search within aligned 3’ UTRs for occurrence of conserved seed match sites
General observations:
- one miRNA can have many unrelated targets
- one gene can be targeted by multiple miRNAs with different seeds
- targets are enriched for TF involved in cell differentiation and physiological pathways
hundreds of conserved targets per miRNA family
>50% human protein coding genes maintain conserved miRNA sites in their 3’ UTR
miRNA expression tends to be tissue specific
- miRNA expression is specific and diverse
- miRNA not detected during early development => miRNAs have roles in differentiation and maintenance of tissue identity
regulatory roles of miRNAs
- Genetic switch:
1. completely suppress gene activity
2. temporal regulation of gene activity
3. spatial regulation of gene activity - insulators of gene expression
- tuning of gene expression - limit gene activity, control levels of gene activity within cells/tissues
miRNA as a switch - spatial
see onenote slide
Spatial switch example
see onenote slide
- blood development in zebrafish
Loss of spatial switch
see onenote slide
Mutate miRNA that is acting as a spatial switch
=> Target transcript can accumulate
Spatial switch is common in plants, a lot rare in animals systems
regulatory roles of miRNA - insulators of gene expression
- limit gene activity
- reinforce transcriptional regulation/activity of promoter
miRNAs as insulators
See onenote slide
- reciprocal expression of miRNA and target transcript
BUT…
Why accumulate miRNA there when the target transcript is not present as its promoter is not active = insulators confers precision and robustness to gene expression, promoter may be slightly leaky , slight activity may have some serious consequences
Insulator example
See onenote slide
Drosophila miR123 and repo
loss of an insulator
see onenote slide
Tuner example
see onenote slide
- miRNA ensures optimal gene activity in tissue
Loss of a tuner
see onenote slide
Take home message
See onenote slide
miRNA sculpt transcripts to give particular expression level and pattern of expression, depend on which miRNA are accumulating, number of binding sites in 3’ UTR and efficiency of binding
Optimisation more common in animal systems
Approach to investigate miRNA function
see onenote slides for examples
How would you distinguish between switch, insulator and tuner?
- look at expression of miRNA/targets and/or miRNA-sensors
- look at miRNA mutants and lines expressing miRNA resistant targets
