Lecture 15 - miRNAs and siRNAs Flashcards Preview

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Flashcards in Lecture 15 - miRNAs and siRNAs Deck (41):
1

What is eRNA? Where is it coded? How does it work?

Enhancer RNA

Coded for near the promoter by enhancer sequences and transcribed by RNA Pol II

Functions to regulate transcription

2

What is the degree of evolutionary conservation of miRNAs? How come?

Their regulatory regions (like their promoters) are very conserved

They regulate genes that are highly conserved and need to complement them

3

What is the function of micro RNAs?

Regulate gene expression typically by blocking translation of selective mRNAs

4

How do miRNAs work in plants vs in animals? What is this difference due to?

Plants: mRNA degradation

Animals: translation repression

Difference due to the degree of homology between the miRNA and the mRNA: in plants it's perfect and in animals it's partial

5

Where was RNA interference by miRNAs first observed? Explain.

In pigmentation experiments with petunias because pigmentation is silenced by miRNAs by inhibition of chalcone synthase

6

In what form is miRNA first transcribed?

Primary miRNA (pri-miRNA)

7

What % of our genome codes for miRNA?

1%

8

How many miRNAs are coded for in the human genome?

200-255

9

What enzymes transcribes miRNAs?

RNA Pol II

10

Where are miRNAs coded for?

Intergenic DNA regions:
1. Coding introns
2. Non-coding introns
3. Non-coding exons

11

Describe the pathway of miRNA synthesis. 5 steps

1. RNA Pol II transcribes primary miRNA which folds into stem–loop structure
2. Drosha, an RNase III interacts with its cofactor DGCR8 crops pri-mRNA to pre-mRNA with a 5' phosphate group and 2 nucleotide overhang at the 3' end
3. Pre-miRNAs are transported to the cytoplasm by binding to exportin-5–RanGTP
4. Dicer, another RNase III, processes pre-miRNA into smaller miRNAs that assemble into a 22 nucleotide duplex which is bound by the protein complex Argonaute
5. miRNAs assemble into the RNA-induced silencing complex (RISC) for translation silencing (guide strand only)

12

How long are pri-mRNAs?

60-100 nucleotides

13

What does DGCR8 stand for?

DiGeorge syndrome Critical Region gene 8

14

How long are pre-mRNAs?

~70 nucleotides

15

Describe the structure of mature miRNA and its 2 parts

Double stranded RNA:
- Guide strand
- Passenger strand

16

How do miRNAs silence translation? 2 steps

1. RISC complexes that are loaded with miRNA (miRISC) bind to target complementary mRNA by forming a bulge sequence
2. Proteins that are part of the RISC complex chaperone the mRNA to P-bodies

17

Can 1 miRNA bind to different mRNAs? What is this called?

YUP

Pleiotropy

18

Describe the binding of miRNA to mRNA in humans. What does this lead to?

In humans, the guide strand will form an imperfect complement to the mRNA, meaning it does not hybridize completely (usually does not extend beyond a short 7-nucleotide “seed” region near the 5’ end of the miRNA)

This leads to translation suppression and does not lead to mRNA degradation like it does in plants

19

Where are translationally repressed by miRNAs mRNAs stored? 2 names

How?

Processing bodies = P-bodies

Proteins that are part of the RISC complex chaperone the mRNA to these

20

What are the 2 fates of the repressed mRNAs stored in P-bodies? What do their fate depend on?

Depending on cellular conditions and stimuli, stored mRNA can:

1. Re-enter the translation pathway
2. Enter the mRNA-decay pathway

21

What does the mRNA degradation pathway in the P-body start with?

Decapping

22

How do plant miRNAs degrade mRNA? 3 steps

1. Slicing
2. Release of RISC with ATP hydrolysis
3. Rapid mRNA degradation

23

What are siRNAs?

Experimental method that mimics the plant miRNA pathway, leading to mRNA degradation

24

Why would researchers use siRNAs instead of knocking out genes or using repressors?

They usually have associated developmental issues

25

What is the stem-loop structure of primary miRNA derived from?

Hairpin RNA

26

Are siRNAs usually endogenous or exogenous? Provide details.

Usually exogenous and can be of viral origin that attacks germ cells

27

What are siRNAs derived from?

dsRNA

28

Where do miRNA and siRNA pathways converge?

At the RISK complex assembly

29

What first happens to exogenously introduced siRNA before it's assembled into a RISK complex?

5' end phosphorylation

30

Other than through mRNA degradation, how else can siRNA suppress gene expression? What does this pathway use instead of the Argonaute-RISC complex?

By inducing transcriptional silencing via heterochromatin formation through epigenetic mechanisms like histone and DNA methylation

Uses Argonaute and RNA-Induced Transcriptional Silencing (RITS) complex instead

31

In what 2 forms can exogenous siRNA be introduced in a cell? What is done to each prior to the RISC complex assemble?

1. dsRNA: Dicer cleavage
2. siRNA: 5' phosphorylation

32

What have miRNA-433 and miRNA-127 been implicated in? What do they regulate?

Gastric carcinoma

They regulate the expression or Grb-2 (adaptor that binds GEF to RTKs), aka they regulate Ras

33

What is a potential use for siRNAs? What 2 big classes of disease are targeted?

Suppress genes involved in certain diseases like cancer and viral infections

34

Why is it hard to insert siRNAs for therapeutic use? How can this be avoided?

Cells will readily phagocytosis siRNAs (or any type of nucleic acid) in the extracellular space

This can be avoided by modifying the siRNAs in a way that will stimulate their uptake by cells and avoid their degradation

35

What are 5 ways of delivering siRNAs to cells?

1. Naked siRNA
2. Direct conjugation of siRNA to a natural ligand (e.g. cholesterol)
3. Aptamer-conjugated siRNA
4. Liposome-formulated siRNA
5. Antibody-protamine-complexed siRNA

36

How do antibody–protamine fusion proteins bind siRNAs?

Non-covalently

37

What is an example of a therapeutic use of siRNAs? How do they observe results?

RNA interference using siRNA introduced in humans via targeted nanoparticles (transferrin) to subunit of ribonucleotide reductase gene in cancer cells (over-expressed)

Results: decreased mRNA and protein expression (not near 100% though) and detection of siRNA-induced mRNA cleavage fragments

38

What is miRNA translation repression directly analogous to? Explain.

Regulation of transcription by transcription factors

They share:
1. Pleiotropy
2. Combinatorial and cooperative activity
3. Accessibility (both easily synthesized from DNA)
4. Regulation (both can be modified)
5. Involved in network motifs (e.g. feedforward loops, other feedback loops)

39

What is an important function of miRNAs in vivo? How?

In the balance in cancer between cell death and cell proliferation, a number of miRNAs function as oncogenes leading to tumor development and a number of them serve as tumor suppressors by stimulating apoptosis

40

Provide an example of an miRNA acting as an oncogene?

c-Myc stimulates miRNA-17-92 and E2F1 expression => miRNAs inhibit E2F1 => apoptosis inhibition => lung cancer and lymphomas

41

Can plant miRNAs regulate human gene expression? What could this suggest?

There is some evidence: a paper showed is there evidence that miRNAs in rice can pass through the GIT and enter the serum and organs and can be detected (proportionally to the amount ingested)

In this case it leads to a rise in cholesterol because these miRNAs inhibit the expression of an LDL receptor adaptor protein causing an decrease in LDL removal from plasma

Could suggest that part of why fruits and vegetables are beneficial may be because they're expressing miRNAs that are positively influencing our health