Topic 12

Question 1

What is the function of RNAi (RNA Interference in Eukaryotes)?

Answer 1

RNAi silences mRNA expression through short RNA molecules (~19-25 nucleotides) generated from longer double-stranded RNAs (dsRNAs)

Question 2

What are the five components of the RNAi machinary?

Answer 2

• miRNA or siRNA
• Dicer
• Microprocessor complex
• RNA-induced silencing complex (RISC)
• Argonaute protein

Question 3

What are small interfering RNAs (siRNAs)

Answer 3

Small non-coding RNAs of ~21-23 nucleotides produced from longer dsRNA precursors

Question 4

What are micro RNAs (miRNAs)?

Answer 4

Short single-stranded, non-coding RNAs of ~19-25 nucleotides produced from hairpin RNA precursors (stemloop structure generated as a ssRNA that was folded back into a hairpin)

Question 5

What is Dicer?

Answer 5

An RNase-III like enzyme that recognizes and digests longer dsRNA to form siRNAs or the stem-loop structures to form miRNAs

Question 6

What is the Microprocessor complex?

Answer 6

Consists of Drosha (an RNase-III like enzyme similar to Dicer) and Pasha/DGCR8 responsible for initiating the biogenesis of miRNA from pri-miRNA to form pre-miRNA

Question 7

RNase III enzymes are specific for…

Answer 7

dsRNA and the resulting product has 2 nucleotides overhanging on the 3’ end

Question 8

Both Dicer and Drosha recognize and cleave pri-miRNA on the basis of…

Answer 8

The pri-miRNA structure rather than its specific sequence

Question 9

What is the RNA-induced silencing complex (RISC)?

Answer 9

A multi-protein complex that includes a guide RNA, which is the active form of the regulatory RNA

Question 10

What is the Argonaute protein?

Answer 10

The catalytic (i.e. slicer) subunit of RISC that carries out the mRNA cleavage

Question 11

Describe the steps of RNAi silencing (5 steps)

Answer 11

1. pri-miRNAs are processed in nucleus by microprocessor complex (Drosha+Pasha) to form pre-miRNA
2. Pre-miRNAs or dsRNA are processed by Dicer in cytoplasm to form mature miRNAs or siRNAs
3. Incorporation of ds-regulatory RNA into RISC
4. De-naturation of dsRNA into a single-stranded guide RNA and a passenger RNA (degraded complement of guide RNA)
5. The guide RNA base pairs with the target mRNA to promote silencing of gene expression by triggering the argonaut in the RISC to cleave mRNA

Question 12

What are the 3 ways that RNAi can silence gene expression?

Answer 12

1. Through mRNA degradation
2. Translational repression
3. Transcriptionally silence genes by directing chromatin modification

Question 13

siRNA (does/does not) fully complement the target mRNA. miRNA (does/does not) fully complement the target mRNA.

Answer 13

Does, does not

Question 14

Mature miRNA is generated from which reactions?

Answer 14

pri-miRNA by 2 RNA cleavage reactions:
- pri-miRNA (hairpin structure) -> first cleavage by microprocessor complex -> pre-miRNA (stem loop)
- pre-miRNA -> second cleavage by Dicer -> mature miRNA

Question 15

Do introns or exons encode miRNAs?

Answer 15

Both

Question 16

What is the “Seed residues”?

Answer 16

The sequence between bases 2 and 9 of miRNA and are important for determining the base pairing between miRNA and its target RNA

Question 17

What techniques can be used to detect the presence of an miRNA?

Answer 17

Northern blotting and qPCR

Question 18

Describe the pri-miRNA structure (4 regions)

Answer 18

1. Basal segments don’t base pair: important for microprocessor complex (Pasha/Drosha) recognition
2. Lower stem region
3. Mature miRNA region/upper stem that isn’t perfectly complementary, seed residue contained within this region
   - ssRNA-dsRNA junction (where ssRNA meets dsRNA) contained at start of this region, which determines Drosha cleavage specificity
   - 2 helical turns
4. Terminal loop
   - Variable in size
   - Sequence is not important for processing

Question 19

True or false: the miRNA sequence is not important for processing

Answer 19

True

Question 20

Describe how the microprocessor complex processes pri-miRNA

Answer 20

Note: All occurs in the nucleus!

• Drosha makes 2 cleavages at the ssRNA-dsRNA junction
• 3 fragments are generated
• The F2 pre-miRNA has 2 nucleotides overhanging on its 3’ end, which is important for Dicer recognition in the cytoplasm

Question 21

What happens to the Drosha/Pasha processed pre-miRNA?

Answer 21

It gets exported to the cytoplasm, where the second RNA cleavage reaction gets carried out by Dicer

Question 22

True or False: short siRNAs are also formed by Dicer cleavage from long dsRNA

Answer 22

True

Question 23

What are the 3 modules of Dicer?

Answer 23

• 2 RNase III domains
• PAZ (Piwi, Argonaute and Zwille) domain: dsRNA-binding domain; anchors the 3’ end of the dsRNA substrate and positions it to the active site at ~22 nt away

Question 24

What is the guide RNA?

Answer 24

It is the active, single-stranded form of the regulatory RNA

Question 25

What are the 3 steps of formation of guide strand RNA and RISC activation?

Answer 25

1. The short (~21-25 nt) double-stranded RNA generated by Dicer is incorporated into RISC 2. The dsRNA is de-natured into a guide strand and a passenger strand 3. The passenger strand is removed from the complex 4. The resulting RISC is called the mature RISC

Question 26

What does the Guide RNA do in the mature RISC?

Answer 26

It recognizes and base pairs with the target mRNA - Causing gene silencing!

Question 27

siRNA and miRNA usually bind to which region of the mRNA?

Answer 27

3'UTR

Question 28

Describe the structure of the Argonaute (2 domains)

Answer 28

1. A PAZ domain: specifically recognizes the 3' end of the guide RNA 2. An RNase domain (RNase H activity) - degrade target mRNA

Question 29

True or False: Most members of the Argonaute protein family do not have RNA slicing activity

Answer 29

False

Question 30

The RNase active site of the argonaute is positioned...

Answer 30

To cleave in the middle of the paired region between small RNA and mRNA

Question 31

Mature RISC can inhibit translation, but the mechanism remains to be elucidated. What are some potential ways that researchers think this is done? (2)

Answer 31

1. miRNA may sequester the target mRNA to processing bodies (P-bodies or P-granules), where translation is repressed in the cytoplasm 2. miRNA binding can also destabilize the polyA tail of the targeting mRNA

Question 32

Describe how genes are silenced by chromatin modification in S.pombe (4)

Answer 32

1. Each centromere in S.pombe has a central unique sequence flanked by common repeats that are important for heterochromatin formation and transcriptional silencing 2. The repeats are transcribed from both strands by RNA Pol II to form dsRNA, which is then processed by the RNAi machinery to generate siRNAs 3. siRNAs direct RITS complex to the Pol II-tethered transcripts 4. The complex further recruits factors (Swi6 and Clr4) to modify nucleosomes by adding the H3K9 silencing markers

Question 33

Describe RNAi used for X-chromosome inactivation

Answer 33

Female mammals: XX Male mammals: XY - females need to inactivate one of the 2 X chromosomes for dosage compensation to ensure both sexes express comparable amounts of X chromosome gene products. - Xist RNA is encoded in the Xic on the X chromosome - Xist RNA recruits other factors to modify and condense X chromosome, which leads to X chromosome inactivation

Question 34

Describe RNAi as a research tool

Answer 34

RNAi is a powerful tool for manipulating gene expression through knockdowns - C. elegans eat plasmids with shRNA inserts

Question 35

What are small RNAs (sRNA) in prokaryotes? Describe their structure

Answer 35

- Involved in regulating gene expression and plasmid replication - 80-110 nucleotides long - Encoded by small genes - Mostly work by direct base pairing with the target mRNAs to regulate gene expression - Hfq, an RNA chaperone, facilitates binding of sRNA to its target mRNA

Question 36

What do riboswitches regulate?

Answer 36

Regulate gene expression at transcription or translational levels by controlling metabolic operons and attenuation in biosynthetic operons through their response to changes in the concentration of small molecules

Question 37

What are the two components of cis-regulator RNAs (riboswitches)?

Answer 37

1. Aptamer: binds to the small molecule ligand and undergoes conformational changes 2. Expression platform: undergoes secondary structural changes upon aptamer-controlling metabolite binding (after comformational change)

Question 38

Describe regulation of transcription termination by riboswitch using SAM

Answer 38

SAM: S-adenosyl methionine is a metabolite - In bacteria, SAM is bound to the SAM sensing riboswitch, which in turn regulates genes involved in methionine or cysteine biosynthesis - After SAM binds aptamer, regions 1-2 and 3-4 start base pairing instead of 2-3 in the aptamer, and the 3-4 stem loop acts as a transcriptional terminator

Question 39

Describe regulation of translation initiation by riboswitch using SAM

Answer 39

When SAM binds, conformational change in aptamer moves ribosome binding site (RBS) on region 4, making RBS inaccessible to the ribosome and inhibiting translation

Question 40

sRNA is said to be a...

Answer 40

Trans-acting regulatory RNA - It can activate and repress translation