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Regulation of Gene Expression > Ferrari: Lecture VIII > Flashcards

Ferrari: Lecture VIII Flashcards

Gene Regulation by microRNA

1
Q

Central Dogma

A

DNA → RNA → proteins

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2
Q

What is the majority of the genome composed of?

A

ncDNA

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3
Q

What are the elements ncDNA is composed of?

A

repetitive sequences (DNA and transposons)
DNA that codes for ncRNA (long ncRNA and small ncRNA)

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4
Q

RNA interference leads to…

A

RNA silencing pathways

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5
Q

What are the best known small interfering RNAs?

A

small interfering RNAs (siRNA)
miRNA/microRNA
piwi-interacting RNA

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6
Q

small interferring RNA (siRNA)

A

endogenous or exogenous origin with respect to the cells they act on

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7
Q

miRNA

A

endogenous; produced in any cell and somatic cells

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8
Q

piwi-interacting cRNA

A

endogenous, specifically produced only in germline cells (probably to protect the genome of germline from transposable elements)

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9
Q

How was RNA interference discovered?

A

by chance in C. Elegans

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10
Q

Regulation of gene expression comes from…

A

the interaction within the genome at the level of DNA or chromatin modifications

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11
Q

How are most fundamental processes regulated?

A

with the help of proteins and miRNA through development, differentiation, and cancer (cancers express RNAs differentially)

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12
Q

What is RNA interference (RNAi)?

A

it is a process of gene silencing that regulates gene expression

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13
Q

What is the function of small interferring RNA (siRNA)?

A

mediate RNAi to direct gene silencing

it has a sequence which is complementary to the mRNA

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14
Q

What complex does the siRNA form?

A

RNA-induced silencing complex (RISC)

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15
Q

What does RISC cause?

A

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

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16
Q

Describe RNAi

A

https://www.youtube.com/watch?v=cK-OGB1_ELE

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17
Q

How do miRNA differ from siRNA?

A

they differ in biogenesis but not function

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18
Q

List miRNA features:

A

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

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19
Q

What is the role of miRNAs in biological processes?

A

regulate abberant transcript in the cell, so that they are not expressed

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20
Q

What are the evidences that show miRNA is involved in biological processes?

A

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

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21
Q

miRNAs’ function is essential for…

A

sustaining life

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22
Q

If you want to study Argonaute function, how do you overcome lethality?

A

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

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23
Q

What is lin-4?

A

miRNA

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24
Q

What does lin-4 regulate?

A

lin-14, which is mRNA

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25
in-4 miRNA regulates...
lin-14 RNA
26
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
27
What processes the pri-miRNA?
Drosha, which is coupled with the protein DGCR8 in mammalians
28
What is the Drosha-DGCR8 complex responsible for?
cleavage and generation of 3' overhang, which happens in the nucleus after transcription
29
What is the result after pri-miRNA is processed by the Drosha complex?
pre-miRNA is formed
30
What is pre-miRNA a precursor for?
mature miRNA
31
What exports the pre-miRNA from the nucleus to the cytoplasm?
RAN-GTP/exportin 5 active complex of the nuclear pore
32
Which enzyme is responsible for processing the pre-miRNA in the cytoplasm?
DICER
33
What modifications are done from pre-miRNA to mature miRNA?
loop is lost and 3' overhang on both sides is formed
34
What does the 3' overhang act as a marker for?
RISC complex
35
What are the 2 different strands called once the miRNA is mature?
Duplex miRNA/miRNA
36
What happens to the Duplex miRNA/miRNA strands?
one is retained in RISC complex and the other is released and degraded
37
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
38
What are the 3 contexts in which miRNA genes are present in the genome?
intergenic position intragenic position clusters
39
intergenic position
there is a specific promoter and a specific regulation: this is a single transcription unit
40
intragenic positition
miRNAs are in introns transcription occurs under the control of the promoter of the gene in which they are contained
41
clusters
not in humans (in Drosophilia) processed as polycystronic RNAs, which is unusual for eukaryotes
42
What is responsible for the transcription of miRNAs?
RNA polymerase II
43
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
44
What are the players in mRNA biogenesis?
Drosha DGCR8 Pasha exportin5 Ran-GTP complex
45
Drosha & DGCR8 (humans)
nuclear enzyme, which trims long transcripts with the help of nuclear enzyme, DGCR8
46
DGCR8 (humans)
helps Drosha
47
Pasha (Drosophilia)
homolog for human DGCR8
48
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
49
What is a feature of the pri-miRNA structure?
loop
50
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
51
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)
52
What is the length of pre-miRNA?
70 nucleotides
53
What processes pre-miRNA?
Drosha and it has a 5' phosphate and a 3' 2 nt overhang
54
Describe Dicer
protein with a helicase domain, 2 RNAse-!!! domains, a dsRNA binding domain, and a PAZ domain
55
What organisms have DICER proteins?
many organisms, including plants and Drosophila, which is the only organism to have 2 different Dicers
56
What is the function of the 2 Dicers in drosophila?
Dicer1 processes miRNA Dicer2 processes siRNA
57
Name the proteins in RISC
helicase exonuclease endonuclease homology searching proteins
58
When is RISC transformed into an active form?
when the siRNA or miRNA duplex is unwound and 1 strand is lost
59
What are argonaute proteins a part of?
RISC
60
What happens when the mRNA is cut?
it is rapidly degraded by endogenous enzymes
61
Where does RISC assembly occur?
mature 22 nt miRNA
62
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
63
What are the possible mechanisms of action miRISC-mediated repression?
translation blockage degradation
64
How do miRNAs control translation initiation?
inhibiting initiation factor 4E/cap and poly(A) tail function
65
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
66
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

Regulation of Gene Expression (28 decks)

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