Lecture 15 - miRNAs and siRNAs

Question 1

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

Answer 1

Enhancer RNA

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

Functions to regulate transcription

Question 2

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

Answer 2

Their regulatory regions (like their promoters) are very conserved

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

Question 3

What is the function of micro RNAs?

Answer 3

Regulate gene expression typically by blocking translation of selective mRNAs

Question 4

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

Answer 4

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

Question 5

Where was RNA interference by miRNAs first observed? Explain.

Answer 5

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

Question 6

In what form is miRNA first transcribed?

Answer 6

Primary miRNA (pri-miRNA)

Question 7

What % of our genome codes for miRNA?

Answer 7

1%

Question 8

How many miRNAs are coded for in the human genome?

Answer 8

200-255

Question 9

What enzymes transcribes miRNAs?

Answer 9

RNA Pol II

Question 10

Where are miRNAs coded for?

Answer 10

Intergenic DNA regions:

1. Coding introns
2. Non-coding introns
3. Non-coding exons

Question 11

Describe the pathway of miRNA synthesis. 5 steps

Answer 11

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)

Question 12

How long are pri-mRNAs?

Answer 12

60-100 nucleotides

Question 13

What does DGCR8 stand for?

Answer 13

DiGeorge syndrome Critical Region gene 8

Question 14

How long are pre-mRNAs?

Answer 14

~70 nucleotides

Question 15

Describe the structure of mature miRNA and its 2 parts

Answer 15

Double stranded RNA:

• Guide strand
• Passenger strand

Question 16

How do miRNAs silence translation? 2 steps

Answer 16

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

Question 17

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

Answer 17

YUP

Pleiotropy

Question 18

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

Answer 18

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

Question 19

Where are translationally repressed by miRNAs mRNAs stored? 2 names

How?

Answer 19

Processing bodies = P-bodies

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

Question 20

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

Answer 20

Depending on cellular conditions and stimuli, stored mRNA can:

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

Question 21

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

Answer 21

Decapping

Question 22

How do plant miRNAs degrade mRNA? 3 steps

Answer 22

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

Question 23

What are siRNAs?

Answer 23

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

Question 24

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

Answer 24

They usually have associated developmental issues

Question 25

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

Answer 25

Hairpin RNA

Question 26

Are siRNAs usually endogenous or exogenous? Provide details.

Answer 26

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

Question 27

What are siRNAs derived from?

Answer 27

dsRNA

Question 28

Where do miRNA and siRNA pathways converge?

Answer 28

At the RISK complex assembly

Question 29

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

Answer 29

5' end phosphorylation

Question 30

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

Answer 30

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

Question 31

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

Answer 31

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

Question 32

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

Answer 32

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

Question 33

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

Answer 33

Suppress genes involved in certain diseases like cancer and viral infections

Question 34

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

Answer 34

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

Question 35

What are 5 ways of delivering siRNAs to cells?

Answer 35

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

Question 36

How do antibody–protamine fusion proteins bind siRNAs?

Answer 36

Non-covalently

Question 37

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

Answer 37

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

Question 38

What is miRNA translation repression directly analogous to? Explain.

Answer 38

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)

Question 39

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

Answer 39

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

Question 40

Provide an example of an miRNA acting as an oncogene?

Answer 40

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

Question 41

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

Answer 41

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