L9 : miRNA Regulatory Networks Flashcards
(18 cards)
How can miRNAs act together to regulate a gene expression program?
On average mRNA has 4 binding sites for miRNAs
- Different miRNAs often found to regulate same mRNA
- Same miRNA often found to target different genes
What is the effect of miRNA regulation on mRNA with single or multiple targets?
Single target
- Normally ~2x decrease in genetic expression
Multiple target sequences:
- Leads to stronger regulatory effect (>10x)
How can miRNAs produce a network of regulatory events and reasons?
Target 1 gene with several miRNA
1 miRNA targeting multiple genes
- Confers robustness
- Can multiply regulatory effect
- miRNA can target several genes involved in specific cellular pathway
Explain how Let7 miRNA family can regulate cellular pathway
Let7 downregulates genes involved in cell proliferation and apoptosis, promoting cell differentiation instead
Targets network of diverse genes leading to global pathway regulation
Let7 family membranes are clustered across different genomic lofi, often with related miRNAs
Eg. Let7 a1/2/3 are repeated at distinct sites with highly conserved sequences
How is Let7 miRNA regulated and what is the role of Lin28?
Lin28 protein downregulates Let7 miRNA in negative feedback loop
In stem cells, Lin28 and Let7 are anticorrelated
What are the 3 binding events required for Let7 regulation?
- Lin28 - adaptor protein
- TUT4 - modifying enzyme
- Dis132 - nuclease
Which domains help Lin28 recognise pre-Let7 miRNA?
Recognises terminal loop using 2 RNA binding domains
ZnF domain (ZKD)
- Recognises GGAG seq in Let7 apical loop
Cold shock domain (CSD) recognises rest of loop
What experiment investigated how Lin28 recognises pre-Let7 miRNA?
EMSA
Native gel so structure and interactions are maintained
Lin28 binds and shifts Let7 but no miR16 (not a target of Lin28)
When loop of Let7 attached to stem of miR16 to create hybrid
Informs on where Lin28 binds
What is the role of TUT4 and how is it recruited?
- Lin28 recruits non-templated polyU polymerase TUT4
- Promotes 3’ poly uridylation
What experiment investigated the role of TUT4?
Western blotting and pull down assay (antibody)
- Presence of Lin28 stiulates poly uridylation of pre-miRNAs containing pre-Let7 apical loop
What experiment showed that presence of Lin28 stabilises Let7-TUT4 interaction?
Single molecule techniques
1. Measuring Cy5-labelled (fluorescent) pre-Let binding to surface tethered TUT4
2. In absence and presence of Lin28
- TUT4 binds Let7 in presence and absence
- In absence, binding is transient and complex quickly dissociates
What is the role of Dis312 nuclease and how is it recruited?
- Recognises specifically the polU tail of poly-pre-let7
- These RNAs are then specifically degraded
How does the structure of Dis312 allow for polyU recognition?
Broad, funnel-like entrance to nuclease channel allows insertion of structured RNAs
These are unfolded by interactions with CSD and S1 domains
How does addition of KSRP affect Dicer activity?
Pre-miRNA assay in vitro
- Addition of KRSP to DICER complex increases processivity
- Immunodepletion of KSRP decreases processivity
What experiment revealed the key domain for KSRP-pre-Let7 recognition?
BioLayer Interferomety (BLI)
1. Mutations for individual domains are tested for RNA binding affinity in biophysical assays
2. Knock down of KH3 results in complete loss of binding affinity
What mutation of the KSRP KH3 structure alters specificity?
K368R
Mutation of Lys to Arg changes specificity of domain in one position of target RNA sequence
Specificity switched from purine to pyrimidine
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