Topic 12

Question 1

Small interfering RNAs (siRNAs)

Answer 1

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

Question 2

MicroRNAs (miRNAs)

Answer 2

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 3

Dicer

Answer 3

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

Question 4

Microprocessor complex

Answer 4

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

Question 5

RNase III enzymes are specific for…

Answer 5

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

Question 6

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

Answer 6

The pri-miRNA structure rather than its specific sequence

Question 7

RNA-induced silencing complex (RISC)

Answer 7

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

Question 8

Argonaute protein

Answer 8

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

Question 9

Describe the steps of RNAi silencing (5 steps)

Answer 9

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 10

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

Answer 10

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

Question 11

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

Answer 11

Does, does not

Question 12

Mature miRNA is generated from which reactions?

Answer 12

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 13

Do introns or exons encode miRNAs?

Answer 13

Both

Question 14

Seed residues

Answer 14

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

Question 15

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

Answer 15

Northern blotting and qPCR

Question 16

Describe the pri-miRNA structure (4 regions)

Answer 16

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 17

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

Answer 17

True

Question 18

Describe how the microprocessor complex processes pri-miRNA

Answer 18

• 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 19

What are the 3 modules of Dicer?

Answer 19

• 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 20

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

Answer 20

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 RISK

Question 21

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

Answer 21

3’UTR

Question 22

Describe the structure of the Argonaute (2 domains)

Answer 22

1. A PAZ domain: specifically recognizes the 3’ end of the guide RNA
2. An RNase domain (RNase H activity)

Question 23

The RNase active site of the argonaute is positioned…

Answer 23

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

Question 24

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 24

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

Question 25

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

Answer 25

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 26

Describe RNAi used for X-chromosome inactivation

Answer 26

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 27

Describe RNAi as a research tool

Answer 27

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

Question 28

What are small RNAs in prokaryotes? Describe their structure

Answer 28

- 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 29

What do riboswitches regulate?

Answer 29

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 30

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

Answer 30

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 31

Describe regulation of transcription termination by riboswitch using SAM

Answer 31

SAM: S-adenosyl mehionine 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 32

Describe regulation of translation initiation by riboswitch using SAM

Answer 32

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 33

sRNA is said to be a...

Answer 33

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