What are miRNA?
single stranded, non-coding RNAs (20 - 25 nucleotides)
What is the function of miRNA?
RNA silencing: suppress gene expression in a sequence-specific manner at a post-transcriptional level
How can miRNA silence RNA?
- RNA interference (RNAi)
- posttranscriptional gene silencing (PTGS)
What are RISCs?
RNA-induced silencing complexes
how does miRNA bind to target mRNA?
either in full complementary or partial complementary
What is the role of miRNAs in early drosophila embryos?
suppress apoptosis
How are miRNAs organized in mammals?
- intergenic miRNA genes: defined transcriptional units which encode for one or more miRNAs –> miRNA clusters are transcribed together as polycistronic transcripts
- intragenic miRNA genes: intron encoded or mirton genes
Describe the general biogenesis of miRNA
- intergenic and intronic miRNA genes are transcribed to pri-miRNA which is a large primary transcript containing one or more hairpins structures
- pre-miRNA with stem loop structure and nucleotide 3’ overhang is formed
- formation of miRNA duplex which has incomplete stem complementary
- mature miRNA: single strand miRNA
how can mature miRNA bind to mRNA?
by base pairing
Describe the drosha-dependent miRNA biogenesis pathway
takes place in the nucleus
1. transcription of intergenic and intronic miRNAs into long pri-miRNA with 5’ cap and polyA
2. processing of pri-miRNAs into pre-miRNA by microprocessor consisting of Drosha RNase III endonuclease and 2 dsRNA binding proteins
3. export of the pre-miRNA into the cytoplasm by Exportin-5/Ran-GTP complex
Which protein processes pre-miRNA into miRNA duplexes and how does it work?
Dicer
–> is a RNase III endonuclease in the cytoplasm that cleaves long dsRNA or pre-miRNAs into dsRNA duplexes
What are the molecular hallmarks of Drosha and Dicer cleavage products
- two nucleotide 3’ overhang
- 5’ phosphate ends and 3’ hydroxyl ends are characteristic RNase III cleavage products
How does the formation of the RNA unduced silencing complexes (RISC) work?
- Loading of the miRNA duplex into the complex. The complex contains 1 Argonaute proteins, Dicer and dsRNA binding protein
- selection of the mature miRNA strand by removal of the passenger strabd which leads to the formation of an activated RISC.
- The active RISC contains a single stranded miRNA (guide strand) which directs the complex to the target mRNA for posttrancriptional gene silencing
Name 4 characteristic Domains of the Argonaute proteins
- N region: unwinding guide/passenger duplex
- PAZ domain: binds overhang of miRNA
- MID: binds 5’ end of miRNA
- PIWI: important for cleavage reaction
How does miRNA mediated gene silencing work?
- miRISC binds to 3’ UTR of target mRNA
- AGO recruits scaffold protein GW182
- GW182 interacts with polyA binding protein and de-adenylase complexes
–> 4 ways of gene silencing
- repressio of cap- dependent translation
- de-adenylation by de-adenylase complexes
- removal of 5’ cap by decapping prtoeins
- mRNA degradation by 5’3’ exonucleases
What are the principles of miRNA target recognition and mode of action? (kr!)
- imperfect base pairing with target mRNA
- inhibition of protein translation and/or mRNA degradation
- target sites: 3’ UTR
- inhibits expression of muultiple mRNAs
What are the principles of siRNA target recognition and mode of action? (kr!)
- perfect base pairing with target RNA
- mRNA cleavage by AGO2 (only one cut)
- cleavage position: between nucleotide 10 and 11 starting from the 5’ of the guide strand
- degradation by other ribonucleases
- target sites: ORF, 5’ UTR, 3’ UTR
- inhibits expression of one specific mRNA
Compare miRNA and siRNA in their principles of function/ mode of action
both mechanism involve a RISC complex (argonaute protein with a small ssRNA)
miRNA: post- trancriptional gene silencing –> fast
animals
siRNA: RNA interference –> slow
plants
Why do mammals retain the silencer Ago2
- AGO2 is essential for mouse embryonic development
- in oocytes endogenous siRNA are important for widespreaf regulation of gene expression and proper meiotic maturation
Describe the miRNA-mRNA interaction in mammals
- binding to 3’ UTR
- presence of a bulge in the central region (prevents Ago2 mediated cleavage)
- base pairing at the supplemental region –> stability and directing miRNA family member with the same seed to different target sites
Name types of miRNA binding modes
- a single miRNA (seed region) might bind to more that 200 target mRNAs
- several miRNAs can bind to a single mRNA
- a single miRNA can bind to multiple binding sites on a single targte mRNA
How can computational predicted miRNA target be confirmed?
- gain of miRNA function via transfection of synthetic dicer substrate dsRNA (increased activity of endogenous miRNA)
- loss of miRNA function via transfection of synthetic antisense oligonucleotides
What is necessary to determine the function of miRNA?
identification of the target mRNAs (due to different types of binding modes)
How is miRNA expression in human cancer?
abnormal –> either
1. overexpression (GOF) of oncogenic miRNA –> down regulation of expression of tumor suppressor genes
2. underexpression (LOF) of tumor suppressor miRNA ..> upregulated expression of oncogenes
