L27 miRNA siRNA

Question 1

Recap of gene silencing pathways

Answer 1

• miRNA and siRNA utilize the same cellular machinery

Question 2

RISCs that slice target transcript

Answer 2

• 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

Question 3

RISC that block protein synthesis

Answer 3

• 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

Question 4

miRISC associated with mRNA and recruits PABC-interacting proteins and deadenylase complex

Answer 4

See onenote slide

• shortens polyA tail/decapping/degradation
• deadenylase complex recruit factors that block formation of the 43S preinitiation complex (PIC)

Question 5

miRNA target recognition in plants

Answer 5

• 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:

1. target one gene/closely related members of a gene family
2. miRNA target site typically located in coding sequence of targeted transcript
3. mainly transcription factors involved in developmental patterning, cell differentiation and nutrient homeostasis

Question 6

miRNA target recognition in animals

Answer 6

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%)

Question 7

miRNA target recognition in animals - approach and general observations

Answer 7

Approach:

1. 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)
2. use genomic sequences to identify related genes and align 3’ UTRs
3. search within aligned 3’ UTRs for occurrence of conserved seed match sites

General observations:

1. one miRNA can have many unrelated targets
2. one gene can be targeted by multiple miRNAs with different seeds
3. 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

Question 8

miRNA expression tends to be tissue specific

Answer 8

• miRNA expression is specific and diverse

- miRNA not detected during early development => miRNAs have roles in differentiation and maintenance of tissue identity

Question 9

regulatory roles of miRNAs

Answer 9

• 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

Question 10

miRNA as a switch - spatial

Answer 10

see onenote slide

Question 11

Spatial switch example

Answer 11

see onenote slide

- blood development in zebrafish

Question 12

Loss of spatial switch

Answer 12

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

Question 13

regulatory roles of miRNA - insulators of gene expression

Answer 13

• limit gene activity

- reinforce transcriptional regulation/activity of promoter

Question 14

miRNAs as insulators

Answer 14

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

Question 15

Insulator example

Answer 15

See onenote slide

Drosophila miR123 and repo

Question 16

loss of an insulator

Answer 16

see onenote slide

Question 17

Tuner example

Answer 17

see onenote slide

- miRNA ensures optimal gene activity in tissue

Question 18

Loss of a tuner

Answer 18

see onenote slide

Question 19

Take home message

Answer 19

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

Question 20

Approach to investigate miRNA function

Answer 20

see onenote slides for examples

How would you distinguish between switch, insulator and tuner?

1. look at expression of miRNA/targets and/or miRNA-sensors
2. look at miRNA mutants and lines expressing miRNA resistant targets