Ferrari: Lecture VIII Flashcards
Gene Regulation by microRNA
Central Dogma
DNA → RNA → proteins
What is the majority of the genome composed of?
ncDNA
What are the elements ncDNA is composed of?
repetitive sequences (DNA and transposons)
DNA that codes for ncRNA (long ncRNA and small ncRNA)
RNA interference leads to…
RNA silencing pathways
What are the best known small interfering RNAs?
small interfering RNAs (siRNA)
miRNA/microRNA
piwi-interacting RNA
small interferring RNA (siRNA)
endogenous or exogenous origin with respect to the cells they act on
miRNA
endogenous; produced in any cell and somatic cells
piwi-interacting cRNA
endogenous, specifically produced only in germline cells (probably to protect the genome of germline from transposable elements)
How was RNA interference discovered?
by chance in C. Elegans
Regulation of gene expression comes from…
the interaction within the genome at the level of DNA or chromatin modifications
How are most fundamental processes regulated?
with the help of proteins and miRNA through development, differentiation, and cancer (cancers express RNAs differentially)
What is RNA interference (RNAi)?
it is a process of gene silencing that regulates gene expression
What is the function of small interferring RNA (siRNA)?
mediate RNAi to direct gene silencing
it has a sequence which is complementary to the mRNA
What complex does the siRNA form?
RNA-induced silencing complex (RISC)
What does RISC cause?
the mRNA to unwind and leads to the complementary functional guide strand
the RISC also cleaves the mRNA and causes it degrade and protein not be synthesized…this leads to reduced gene expression
Describe RNAi
https://www.youtube.com/watch?v=cK-OGB1_ELE
How do miRNA differ from siRNA?
they differ in biogenesis but not function
List miRNA features:
endogenous small RNA guides that repress the expression of target genes
not specific for one gene as they only need a few bases to match (1 miRNA can regulate different mRNAs)
21-24 nt regulatory RNAs with secondary hairpin structure
regulate signalling pathways, apoptosis, metabolism, developmetn, cancer
can regulate >60% of all protein-coding genes
What is the role of miRNAs in biological processes?
regulate abberant transcript in the cell, so that they are not expressed
What are the evidences that show miRNA is involved in biological processes?
genes with tissue-specific expression have longer 3’ UTR that contain more miRNA binding sites
miRNA expression increases during embryonic development
the diversity of miRNA increases with increasing organism complexity
KO mice in which one of the proteins of RISC is KO show a severely impaired development of the embryo
miRNAs’ function is essential for…
sustaining life
If you want to study Argonaute function, how do you overcome lethality?
a conditional KO can be done to delete the expression of Argo2 in a particular phase, so that the adult phase can be studied after the embryo has been developed
What is lin-4?
miRNA
What does lin-4 regulate?
lin-14, which is mRNA
in-4 miRNA regulates…
lin-14 RNA
What is the 1st product of transcription?
pri-miRNA: ss molecule that has a 3’ and 5’ end, extended region of intrastrand homology and a small loop at the end
What processes the pri-miRNA?
Drosha, which is coupled with the protein DGCR8 in mammalians
What is the Drosha-DGCR8 complex responsible for?
cleavage and generation of 3’ overhang, which happens in the nucleus after transcription
What is the result after pri-miRNA is processed by the Drosha complex?
pre-miRNA is formed
What is pre-miRNA a precursor for?
mature miRNA
What exports the pre-miRNA from the nucleus to the cytoplasm?
RAN-GTP/exportin 5 active complex of the nuclear pore
Which enzyme is responsible for processing the pre-miRNA in the cytoplasm?
DICER
What modifications are done from pre-miRNA to mature miRNA?
loop is lost and 3’ overhang on both sides is formed
What does the 3’ overhang act as a marker for?
RISC complex
What are the 2 different strands called once the miRNA is mature?
Duplex miRNA/miRNA
What happens to the Duplex miRNA/miRNA strands?
one is retained in RISC complex and the other is released and degraded
How does the RISC complex decide which strand to keep and which one to degrade?
answer is not clear, but it seems to be based on the sequence, 3’ overhang nature, and thermodynamics
What are the 3 contexts in which miRNA genes are present in the genome?
intergenic position
intragenic position
clusters
intergenic position
there is a specific promoter and a specific regulation: this is a single transcription unit
intragenic positition
miRNAs are in introns
transcription occurs under the control of the promoter of the gene in which they are contained
clusters
not in humans (in Drosophilia)
processed as polycystronic RNAs, which is unusual for eukaryotes
What is responsible for the transcription of miRNAs?
RNA polymerase II
What is the evidence that RNA polymerase II is responsible for transcription?
intragenic location
transcript size of primary transcript can be 1 Kb long
tissue specific expression happens for RNA polymerase II but not RNA polymerase III
What are the players in mRNA biogenesis?
Drosha
DGCR8
Pasha
exportin5
Ran-GTP complex
Drosha & DGCR8 (humans)
nuclear enzyme, which trims long transcripts with the help of nuclear enzyme, DGCR8
DGCR8 (humans)
helps Drosha
Pasha (Drosophilia)
homolog for human DGCR8
exportin5
Drosha-DGCR8 complex with exportin5 recognize the ends and stem structure of the pre-miRNA
this protein then binds this product and works with Ran-GTP complex
What is a feature of the pri-miRNA structure?
loop
Review the picture:
cleavage sites of different enzymes has been mapped (Dicer cuts off the loop and the Drosha cleavage site is visible)
*remember: during processing, inferior stem and loop are lost while the superior stem is retained
Describe Drosha
ds RNA binding domain and 2 domains with RNAse activity, a N-terminal domain and a nuclear localization signal, which is necessary since Drosha has nuclear localization)
What is the length of pre-miRNA?
70 nucleotides
What processes pre-miRNA?
Drosha and it has a 5’ phosphate and a 3’ 2 nt overhang
Describe Dicer
protein with a helicase domain, 2 RNAse-!!! domains, a dsRNA binding domain, and a PAZ domain
What organisms have DICER proteins?
many organisms, including plants and Drosophila, which is the only organism to have 2 different Dicers
What is the function of the 2 Dicers in drosophila?
Dicer1 processes miRNA
Dicer2 processes siRNA
Name the proteins in RISC
helicase
exonuclease
endonuclease
homology searching proteins
When is RISC transformed into an active form?
when the siRNA or miRNA duplex is unwound and 1 strand is lost
What are argonaute proteins a part of?
RISC
What happens when the mRNA is cut?
it is rapidly degraded by endogenous enzymes
Where does RISC assembly occur?
mature 22 nt miRNA
What are the 2 mechanisms of action in regards to the silencing of miRNA?
mRNA is cut at the level of nucleotides complementary to the position of nucleotide 10 and 11 in the miRNA (ONLY occurs when we have quite a perfect match…this means that almost all 22 nt of the miRNA need to find a match)…usually happens in plants
when perfect match is not present, inhibition of translation is the mechanism of action…usually happens in animals
What are the possible mechanisms of action miRISC-mediated repression?
translation blockage
degradation
How do miRNAs control translation initiation?
inhibiting initiation factor 4E/cap and poly(A) tail function
What is the role of the proteins in the EF4 complex (4E, 4A, 4B, and 4G)?
they recognize the cap, bind and then interact with PABP, and leading to the circularization of mRNA, which makes translation efficient
List the 4 ways miRNA might inhibit translation:
binding of cap - miRNA and RISC bind to the 3’ UTR of a mRNA
elongation factors -RISC sequestrates some factors that block translation
blocking of circularization - miRISC mediated repression (cap is not circularized with PABP so translation is attenuated)
induction of ribosome dropoff - RISC complex inhibits the assembly between the small subunit and large subunit
