Lecture 20; RNA interference:

Question 1

What type of mechanism are RNAi or RNA interference?

Answer 1

A post-transcriptional mechanism to regulate gene expression

Question 2

What type of RNA does RNAi involve?

Answer 2

Involves double-stranded RNA

Question 3

What 2 things does RNAi involve to regulate gene expression post-trancriptionally?

Answer 3

Can involve degradation of mRNA shortening its lifespan in the cell or prevention of translation

Question 4

Instead of gene deletion, what is a common method used in labs to inactivate a gene?

Answer 4

RNAi

Question 5

When does RNAi occur?

Answer 5

RNAi occurs when a cell encounters various types of double-stranded RNA (dsRNA) from different sources:
1) Viruses (e.g. some RNA viruses have dsRNA genomes)
2) Transposons
3) Transgenes that express dsRNA

Question 6

What are the 2 paths where RNAi may occur?

Answer 6

1) RNAi resulting from processing of small interfering RNA (siRNA)
2) RNAi resulting from the processing of micro RNA (miRNA)

Question 7

What is the difference between small interfering RNA (siRNA) and micro RNA (miRNA)?

Answer 7

Difference is the source:
1) siRNA = exogenous source of RNA
2) miRNA = encoded in the cell itself

Question 8

How was RNAi identified as a mode of post-transcriptional gene regulation?

Answer 8

In experiments with petunia flowers:

Compared to the wild type flower which is purple throughout, when the transgenic plants were made by inserting genes that produced iRNAs, they saw some parts of the flower had an interference in the gene that produced the purple colour leading to some regions without the purple pigmentation

Question 9

What type of RNA is found to be involved in RNAi through 2 controls and 2 experiments with nematodes injected with complementary ssRNA or dsRNA to regulate the mex-3 gene involved in embryonic development?

Answer 9

Controls:
(a) Embryo without hybridization probe = no detection of mRNA transcript
(b) Embryo with hybridization probe = detect RNA

Experiments with injected RNA:
c) Embryo injected with ssRNA complementary to mex-3 = doesn’t knock down the translation from the mRNA → no degradation
d) embryo injected with dsRNA (antisense and sense strand) corresponding to mex-3 = saw that all the RNA was degraded

Question 10

How does RNAi occur by siRNAs in 3 steps?

Answer 10

1) dsRNA from an exogenous source is cut into siRNAs by a nuclease called “Dicer”
2) After being cut by Dicer double stranded siRNA is separated into single strands: Passenger strand and guide strand
3) The guide strand assembles with proteins in a complex known as RNA-induced silencing complex (RISC), which cuts mRNA complementary to the guide strand using a component nuclease called “Argonaute” or “Slicer”

Question 11

What is the nuclease that cuts exogenous dsRNA into siRNA?

Answer 11

a nuclease called “Dicer”

Question 12

When siRNAs are made, what are the 2 single strands created and what are the fates of the 2 strands?

Answer 12

1) Passenger –> discarded
2) Guide –> assembles with RNA-induced silencing complex (RISC), which cuts mRNA complementary to the guide strand using a component nuclease called “Argonaute” or “Slicer”

Question 13

What nuclease component of the complex with RISC and the guide strand cuts mRNA complementary to the guide strand?

Answer 13

cuts mRNA complementary to the guide strand using a component nuclease called “Argonaute” or “Slicer”

Question 14

What occurs if complementarity is imperfect and what occurs if it is perfect to the guide strand created from siRNA?

Answer 14

1) If complementarity is imperfect, repression of translation results, followed sometimes by mRNA degradation (but not always)
2) Perfect base pairing with the transcript results in cleavage of the mRNA and rapid degradation of those mRNA transcripts

Question 15

What is an example of an RNAi pathway evolved to regulate viral gene expression?

Answer 15

• Incorporation of viral derived siRNA into the cell’s RISC machinery results in specific targeting of viral RNA for degradation
• Some plant viruses (e.g., tombusvirus that infects carnations) have evolved to counter-act this with a protein, p19, the binds siRNA preventing it from associating into RISCs

Question 16

What protein in plant viruses can bind to RISC to prevent it from degrading the viral RNA?

Answer 16

Some plant viruses (e.g., tombusvirus that infects carnations) have evolved to counter-act this with a protein, p19, the binds siRNA preventing it from associating into RISCs

Question 17

How does miRNAs lead to gene silencing in 4 steps?

Answer 17

1) Micro RNAs (miRNAs) are 18-25 nt RNAs produced naturally in plant and animal cells by cleavage from 75-nt stem-loop precursor RNA
2) In last step of miRNA synthesis, Dicer RNase cleaves double-stranded stem part of the precursor to yield miRNA in double-stranded form
3) Strands of miRNA are separated and a guide strand complexes with proteins in RISC control gene expression by base-pairing to their mRNAs
4) Single-stranded miRNA (as part of RISC) by base-pairing with the transcript can direct the degradation of complimentary mRNA (like siRNA) or just block its translation

Question 18

What are and how are miRNAs made?

Answer 18

Micro RNAs (miRNAs) are 18-25 nt RNAs produced naturally in plant and animal cells by cleavage from 75-nt stem-loop precursor RNA

Question 19

What enzyme cleaves the double-stranded stem part fo the precursor transcript to yield miRNA?

Answer 19

Dicer RNase

Question 20

How do both siRNAs and miRNAs bind to a complementary strand to recognize and cleave it?

Answer 20

Both miRNAs (microRNAs) and siRNAs (small interfering RNAs) function by base-pairing with target mRNA sequences to regulate gene expression

Question 21

What is the “Drosha” pathway of miRNA maturation in humans (and primary pathway in animals) in 5 steps?

Answer 21

1) “Primary RNA” (pri-RNA) is transcribed by RNA polymerase II and forms a stem loop (transcript may have more than one miRNA stem loop)
2) Cleavage at the stem results from processing by a complex of proteins called “microprocessor” that includes the ribonuclease called “Drosha”
3) Export from the nucleus
4) Cleavage of the loop by nuclease called “Dicer”
5) Separation of strands and assembly of RISC including one miRNA guide strand to direct translation inhibition/mRNA degradation, with the passenger strand getting removed

Question 22

What cleaves at the stem of the miRNA stem loop in the Drosha pathway?

Answer 22

Cleavage at the stem results from processing by a complex of proteins called “microprocessor” that includes the ribonuclease called “Drosha”

Question 23

What is Drosha?

Answer 23

Drosha is an RNase III that recognizes dsRNA and cleaves at the stem of the stem loop

Question 24

What are the 2 enzymes involved in the “Drosha” pathway of miRNA maturation in humans and animals?

Answer 24

1) Cleavage at the stem results from processing by a complex of proteins called “microprocessor” that includes the ribonuclease called “Drosha”
2) Cleavage of the loop by nuclease called “Dicer”

Question 25

What is the Mirtron pathway of miRNA formation in 2 steps?

Answer 25

1) In some introns of protein coding genes after splicing, the exons are joined together, but the introns remain as a lariat structure which can be further processed if they include a miRNA (may have more than one miRNA on introns). 2) After the lariats are unbranched, they form a hairpin which can be further processed by Dicer to cleave them into miRNAs which then can join the proteins that form the RISC complex to silence genes.

Question 26

What is the common pathway to gene silencing by miRNAs in animals?

Answer 26

- In animals, miRNAs tend to base-pair imperfectly to the 3’-UTRs of their target mRNAs - This leads to inhibition of protein product accumulation from the miRNA targeted mRNA

Question 27

What is the common pathway to gene silencing by miRNAs in plants?

Answer 27

In plants, miRNAs tend to base-pair perfectly or near-perfectly with target mRNAs and cause mRNA cleavage

Question 28

How can RNAi be used to study gene function?

Answer 28

Knocking down a gene if you deliver RNA into a cell through a viral vector, this virus could directly introduce dsRNA which might be then processed into siRNA to target specific genes for silencing.

Question 29

What is a common therapeutic use of RNAi?

Answer 29

treatment for HIV