L8 : Regulation of gene expression by microRNA Flashcards
What are microRNAs (miRNAs) and why are they important?
Short unstructured RNAs
~22 nt ss RNAs that guide RISC complex to set of specific target mRNAs, leading to transcriptional repression and mRNA decay
Provide general layer of control for gene expression
- Thousands of annotated miRNAs in humans
How were miRNAs first discovered?
In C. elegans model organism
Found to be conserved from C. elegans to humans
Recognised as a class of regulators
What organisms possess miRNAs?
In multicellular organisms
Found in metazoa and plants as a result of parallel evolution (not yeast)
How are miRNAs conserved?
Little sequence conservation outside vertebrates
- Except for few miRNAs key to development
- Not conserved as miRNA must adapt to mRNA sequence, which changes faster than protein sequence
Provide a summary of canonical miRNA biogenesis and function in mammals
- miRNA encoded by genomic sequences
- Undergo two steps of process in nucleus then cytoplasm
- Target Ago complex to selected mRNAs, leading to translational repression and degradation
Compare origins, machinery, and mechanisms of miRNAs and siRNAs
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
Where are miRNA genes found in the genome?
Diverse range of genomic locations
- Exons, 3’ UTR, introns
Can be in isolation or clustered
Explain canonical miRNA biogenesis
- Transcribed by RNA Pol 2
- Primary miRNA undergoes two steps of processing in nucleus then cytoplasm (based on recognition of miRNA precursor structure)
- Pri-miRNA processed by Dorsha (component of microprocessor complex) in nucleus to pre-miRNA
- EXP5 exports to cytoplasm
- DICER converts into mature miRNA and conjugates to RISC
How is nuclear processing performed by drosha?
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
How is cleavage of pri-miRNA performed by microprocessor?
Microprocessor complex recognises length of stem as well as sequences near base of stem
Cleaves pri-miRNAs on the two opposite strands
Describe the structure of the microprocessor complex? Functions of components?
Stoichiometry of 2 DGCR8 for each Drosha
Drosha is larger catalytic protein
- Contains dsRNA binding domain and pri-miRNA specific features
- Catalyses cleavage of two strands
DGRC8 is smaller and shorter
- Contains 2 dsRNA binding domains
- Binds along stem of hairpin (docking)
How has solving structures of microprocessor complexes helped in understanding its function?
Multiple structural states including apo, partially docked, catalytic have been solved
Helped reconstruction of recognition and cleaving process
How is length of pri-miRNA stem measured?
dsRNA binding domains from DGCR8 and Drosha combine to form ‘molecular ruler’ and measure stem length
How are pre-miRNAs exported into the cytoplasm?
- In nucleus, binding of RAN GTP opens (activates) exportin5 to receive pre-miRNA
- Exportin5 recognises stem of hairpin and 3’ end (not sequence specific)
- In cytoplasm, hydrolysis of GTP releases both RANGDP and pre-miRNA
How are pre-miRNAs processed in the cytoplasm?
Dicer complex processes pre-miRNA into ss mature miRNAs by cleavage
Measures length of ds region from 5’ to 3’ ends and cleaves accordingly
How do Dicer complex proteins and domains contribute to docking and cleaving of pre-miRNA?
Dicer protein
- Performs cleavage
- Mediated by 2 RNase III -like domains
- PAZ. domain recognises 3’ end of pre-miRNA for accurate processing
TRBP
- Helps recruit Dicer
- dsRBD1 and 2 interact with RNA and aid docking
What are the key steps of loading mature miRNA onto RISC complex?
- Unwinding of RNA helix
- Strand discrimination
How successful is loading of mature miRNAs onto RISC?
Guide miRNA loaded with >95% discrimination from passenger miRNA
Imperfect fidelity
Describe loading of AGO and discrimination of guide and passenger miRNA strands? Formation of RISC?
- RNA hairpin is cleaved by Dicer complex (~22 nt)
- Cleaved RNA duplex shifts towards helicase domain and dsRBD protein (TRBP or PACT depending on organism)
- AGO made competent to loading by Hsp70/90 protein chaperonine and loads Dicer
- Opens AGO and allows loading of duplex
- AGO checks base pairing between the strands and passenger strand is ejected or cleaved
- Ago now loaded with guide strand forms RNA-induced silencing complex (RISC)
How is the guide strand discriminated from passenger?
- Low stability of base pairing at 5’ end allows strand selection
- Identity of base is specifically recognised and stabilised by Ago protein
What is the model for removal of the passenger miRNA strand?
- Loading is ATP dependent
- Unwinding ATP independent but likely driven by rleease of tension accumulated during loading
- 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
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