L8 : Regulation of gene expression by microRNA

Question 1

What are microRNAs (miRNAs) and function?

Answer 1

Short unstructured RNAs

~22 nt ss RNAs that guide RISC complex to set of specific target mRNAs

Leading to transcriptional repression and mRNA decay

Question 2

Why are miRNAs important?

Answer 2

Provide general layer of control for gene expression
- Thousands of annotated miRNAs in humans

Question 3

How were miRNAs first discovered?

Answer 3

In C. elegans model organism, discovered as class of ncRNA in 2001
Found to be conserved from C. elegans to humans
Recognised as a class of regulators

Question 4

When and what was the first identified oncogenic miRNA?

Answer 4

Oncomir miR-122 discovered 2006

Question 5

What is the important of miRNA in medical contexts?

Answer 5

miRNAs can act as:
- Biomarkers in diagnostic tests
- Therapeutic targets

Question 6

Give examples of the medical context for miRNAs?

Answer 6

Early stage lung cancer test based on miRNA signatures in 2014

Range of therapeutic compounds targeting miRNAs currently in development

Question 7

What organisms possess miRNAs?

Answer 7

In multicellular organisms
Found in metazoa and plants as a result of parallel evolution (not yeast)

Question 8

How are miRNAs conserved?

Answer 8

Little sequence conservation outside vertebrates
- Except for few miRNAs key to development

Question 9

Why is miRNA often not conserved?

Answer 9

Must adapt to mRNA sequence, which changes faster than protein sequence

miRNA can evolve quickly to match organism complexity

Question 10

Provide a summary of canonical miRNA biogenesis and function in mammals

Answer 10

1. miRNA encoded by genomic sequences
2. Undergo two steps of process in nucleus then cytoplasm
3. Target Ago complex to selected mRNAs, leading to translational repression and degradation

Question 11

Compare origins, machinery, and mechanisms of miRNAs and siRNAs

Answer 11

Different origins:
- siRNA has exogenous origin
- miRNA encoded by genomic seq

Overlapping machinery:
- Pathways share proteins required for biogenesis and function

Different mechanisms of action:
- Modulate RNA targets using different mechanisms

Question 12

Where are miRNA genes found in the genome?

Answer 12

Diverse range of genomic locations
- Exons, 3’ UTR, introns

Can be in isolation or clustered

Question 13

What might be the purpose of miRNA gene clustering?

Answer 13

Allows regulation

Question 14

Explain canonical miRNA biogenesis

Answer 14

1. Transcribed by RNA Pol 2
2. Primary miRNA undergoes two steps of processing in nucleus then cytoplasm (based on recognition of miRNA precursor structure)
3. Pri-miRNA processed by Dorsha (component of microprocessor complex) in nucleus to pre-miRNA
4. EXP5 exports to cytoplasm
5. DICER converts into mature miRNA and conjugates to RISC

Question 15

How is nuclear processing performed by drosha?

Answer 15

Large pri-miRNA transcript is cleaed to shorter hairpin precursors
Size of pre-miRNA hairpin defined by cleavage point from both loop and basal junction

Question 16

How is cleavage of pri-miRNA performed by microprocessor?

Answer 16

Microprocessor complex recognises length of stem as well as sequences near base of stem
Cleaves pri-miRNAs on the two opposite strands

Question 17

Describe the structure of the microprocessor complex? Functions of components?

Answer 17

2 core protein components
Stoichiometry of 2 DGCR8 for each Drosha

• Drosha is larger catalytic protein
• DGRC8 is smaller and shorter

Question 18

What are the functions of components within microprocessor complex?

Answer 18

Drosha:
- Contains dsRNA binding domain and pri-miRNA specific features
- Catalyses cleavage of two strands

DGCR8:
- Contains 2 dsRNA binding domains
- Binds along stem of hairpin (docking)

Question 19

How has solving structures of microprocessor complexes helped in understanding its function?

Answer 19

Multiple structural states including apo, partially docked, catalytic have been solved
Helped reconstruction of recognition and cleaving process

Question 20

How is length of pri-miRNA stem measured?

Answer 20

dsRNA binding domains from DGCR8 and Drosha combine to form ‘molecular ruler’ and measure stem length

Question 21

How are pre-miRNAs exported into the cytoplasm?

Answer 21

1. In nucleus, binding of RAN GTP opens (activates) exportin5 to receive pre-miRNA
2. Exportin5 recognises stem of hairpin and 3’ end (not sequence specific)
3. In cytoplasm, hydrolysis of GTP releases both RANGDP and pre-miRNA

Question 22

How are pre-miRNAs processed in the cytoplasm?

Answer 22

Dicer complex processes pre-miRNA into ss mature miRNAs by cleavage
Catalytic core component measures length of ds region from 5’ to 3’ ends and cleaves both strands accordingly

Question 23

What sequence feature improves Dicer cleavage processes?

Answer 23

GYM sequence motif
Recent analysis identified as important for precise efficient cleavage

Question 24

What are the two major protein components of Dicer?

Answer 24

Dicer
- Performs cleavage
TRBP
- Assists Dicer recruitment

Question 25

How do domains of Dicer-TRBP contribute to docking and cleaving of pre-miRNA?

Answer 25

Dicer protein - PAZ domain recognises 3' end of pre-miRNA for accurate processing - Cleavage mediated by 2 RNase III -like domains TRBP - dsRBD1 and 2 interact with RNA and aid docking

Question 26

What are the key steps of loading mature miRNA onto RISC complex?

Answer 26

1. Unwinding of RNA helix 2. Strand discrimination

Question 27

How successful is loading of mature miRNAs onto RISC?

Answer 27

Guide miRNA loaded with >95% discrimination from passenger miRNA Imperfect fidelity

Question 28

Describe loading of AGO and discrimination of guide and passenger miRNA strands? Formation of RISC?

Answer 28

1. RNA hairpin is cleaved by Dicer complex (~22 nt) 2. Cleaved RNA duplex shifts towards helicase domain and dsRBD protein (TRBP or PACT depending on organism) 3. AGO made competent to loading by Hsp70/90 protein chaperonine and loads Dicer 4. Opens AGO and allows loading of duplex 5. AGO checks base pairing between the strands and passenger strand is ejected or cleaved 6. Ago now loaded with guide strand forms RNA-induced silencing complex (RISC)

Question 29

How is the guide strand discriminated from passenger?

Answer 29

- Low stability of base pairing at 5' end allows strand selection - Identity of base is specifically recognised and stabilised by Ago protein

Question 30

What is the model for removal of the passenger miRNA strand?

Answer 30

1. Loading is ATP dependent 2. Unwinding ATP independent but likely driven by rleease of tension accumulated during loading 3. Ago removal depends on structure of RNA duplex - Chaperonine bound Ago shifts duplex against NTD that can act as wedge to separate strands - If strand highly complementary, passenger strand is cleaved by Ago

Question 31

What role do AGO proteins play in miRNA function?

Answer 31

Argonaute proteins Present mature miRNAs for target recognition - Stabilise miRNA - Facilitate pairing with mRNA

Question 32

How does AGO recognise the miRNA structure?

Answer 32

AGO binds the A-form RNA helix Interacts mainly wit phosphate backbone, not bases

Question 33

What is the 'seed' sequence in miRNAs and why is it important?

Answer 33

7 nt sequence at 5' end of miRNA Primary determinant for target recognition

Question 34

What is the nature and function of the AGO seed chamber?

Answer 34

Seed chamber is restricted Holds seed region in structured conformation for rapid, precise target scanning and binding

Question 35

How does AGO allow recognition beyond seed region?

Answer 35

AGO undergoes a conformational change to open a supplementary chamber for pairing at miRNA nts 13–16

Question 36

What is the nature of the RNA duplex in the supplementary chamber?

Answer 36

More mobile Allows flexible base pairing

Question 37

What effect does additional bp have on miRNA-target binding?

Answer 37

Few fold increase in affinity and repression - Dependent on strength of base pairing and length of bridge

Question 38

What are the 2 ways that miRNAs mediate gene expression?

Answer 38

1. Translational repression 2. mRNA decay

Question 39

How was increased miRNA repression experimentally observed?

Answer 39

Through reporter assays (e.g. luciferase) - Increased repression correlated with stronger miRNA-mRNA pairing

Question 40

Can translational repression and mRNA decay be uncoupled?

Answer 40

Yes, experimentally they can be uncoupled BUT In most physiological conditions they are closely linked

Question 41

Which protein is recruited by AGO to mediate miRNA-induced repression?

Answer 41

GW182 Has both AGO-binding and silencing domain

Question 42

How does GW182 mediate translational repression?

Answer 42

Binds PABPC via its PAM2 domain Competing with eIF4G - disrupting mRNA circularisation needed for translation initiation

Question 43

What complex does GW182 recruit to promote mRNA decay?

Answer 43

CCR4-NOT complex Causes deadenylation, decapping, and mRNA degradation