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Topic 12 (Regulatory RNA) Flashcards

1
Q

What is RNAi?

A

Interference RNA that silences mRNA expression through short RNA molecules generated from longer dsRNA

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2
Q

What are the two types of RNAi?

A
  • siRNA (small interfering RNA)
  • miRNA (microRNA)
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3
Q

What is siRNA?

A

Small non-coding dsRNA of 21-23nt produces from longer dsRNA precursors

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4
Q

What is miRNA?

A

Short non-coding ssRNA of 19-25nt produced from hairpin RNA precursors

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5
Q

What is Dicer?

A

An RNase III like enzyme that recognizes and digests longer dsRNA to form siRNA or stem-loops to form miRNA

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6
Q

What is the microprocessor complex?

A

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

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7
Q

What is the substrate of RNase III like enzymes? What feature unites all products of the reaction it catalyzes?

A

dsRNA; resulting dsRNA product has a 2nt overhang at the 3’ end

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8
Q

True/False? Dicer and Drosha recognition of pri-miRNA is based on sequence

A

False. Based on pri-miRNA structure

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9
Q

What is RISC?

A

A multi-protein complex that includes a guide RNA derived from siRNA or miRNA

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10
Q

What is an argonaut protein?

A

The catalytic subunit of RISC that carries out mRNA cleavage

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11
Q

How long is pri-miRNA? Pre-miRNA? Mature miRNA?

A

~200nt; ~70nt; ~22nt

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12
Q

Describe the steps of RNAi silencing

A
  1. Pri-miRNA is processed by the microprocessor complex in the nucleus to form pre-miRNA
  2. Pre-miRNA is processed by Dicer in the cytoplasm to form mature miRNA
  3. Incorporation of regulatory RNA into RISC
  4. Denaturation of dsRNA into a guide RNA and passenger RNA
  5. Guide RNA brings RISC to target mRNA to promote silencing
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13
Q

What happens to the passenger strand?

A

Degraded

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14
Q

What are the three ways RISC + guide RNA promotes gene silencing?

A
  • target mRNA degradation
  • translational repression
  • transcriptionally silence genes by directing chromatin modification
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15
Q

How many reactions are needed to create mature miRNA? What are they catalyzed by?

A
  1. First is catalyzed by microprocessor complex (Drosha/Pasha), second by Dicer
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16
Q

Where may miRNAs be found in the RNA transcript?

A

Within the coding region (between start and stop codons) and non-coding regions (UTRs or introns) of protein-coding or non-coding RNA

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17
Q

What are seed residues?

A

Sequence between bases 2 and 9 of a miRNA and are important for determining the base pairing between miRNA and its target RNA (involved in the recognition of target mRNA)

18
Q

What practices may be used to detect the presence of miRNA and siRNA?

A

Northern blotting and qPCR

19
Q

What part of the pri-miRNA does the microprocessor complex recognize? Where is it cleaved by Drosha?

A

Single stranded basal segments; ssRNA-dsRNA junction (bulge on both sides) (+1 site)

20
Q

What determines the cleaving specificity of Drosha?

A

The ssRNA-dsRNA junction on pri-miRNA

21
Q

What is the structure of a pri-miRNA from the terminus to the loop?

A

Basal segments, lower stem, upper stem (contains cleavage site), terminal loop

22
Q

Does Drosha make a double or single-stranded cut?

23
Q

How many fragments result from Drosha digestion? What are they?

A

3; F1 and F3 are ssRNA and are degraded, F2 is the pre-miRNA

24
Q

What kind of cut does Drosha make? Why?

A

Sticky end cut (leaves 2nt 3’ overhang) for Dicer recognition

25
What are the three modules of Dicer?
- 2 RNase III domains (blade) - PAZ domain (handle) - dsRNA-binding domain (handle)
26
What is the PAZ domain?
Anchors the 3' overhang of the dsRNA substrate and positions it to the active sites at ~22nt away
27
What is the active form of regulatory RNAi?
Guide RNA
28
How is guide RNA made?
1. short dsRNA (either miRNA or siRNA) is incorporated into RISC 2. dsRNA is denatured into guide and passenger strands 3. passenger strand is removed from the complex 4. RISC + guide strand = mature RISC
29
What is the main difference between miRNA and siRNA other than their structures
siRNA has perfect complementarity with its target mRNA (complete specificity), while miRNA has imperfect complementarity with its target mRNA (incomplete specificity)
30
What are the two domains of the Argonaut protein?
- PAZ domain: recognizes 3' end of guide RNA - RNase domain: cuts and degrades target mRNA where the guide and target are complemented
31
The RNase domain of Argonaut has what kind of activity?
RNase H
32
How does mature RISC inhibit translation?
Although the mechanism is unknown, researchers hypothesize that miRNA may sequester the target mRNA to processing bodies (P-bodies) to suppress translation
33
Describe gene silencing by chromatin modification using RNA
1. repeats in the centromeres are transcribed from both strands by RNA Pol II to form dsRNA 2. dsRNA is processed by RNAi machinery to generate siRNAs 3. siRNAs direct RITS complex to the Pol II-tethered transcripts 4. Swi6 and Clr4 are recruited to modify nucleosomes by methylating H3K9 (histone 3 lysine 9)
34
What are some examples of biological functions of RNAi?
- protects against viral infection in plants - X chromosome inactivation in females (Xist RNA encoded in the Xic recruits chromatin suppressing modifications)
35
How is RNAi used as a research tool?
Used to knock down gene expression in *C. elegans* by feeding them *E. coli* that produce the dsRNA
36
How are small RNAs (sRNAs) used in prokaryotes?
Regulate gene expression and plasmid replication by directly base pairing with target mRNAs to regulate gene expression
37
What is Hfq?
An RNA chaperone that facilitates binding of sRNA to its target mRNA in prokaryotes
38
What are riboswitches?
Regulate gene expression at transcriptional or translational levels by controlling metabolic operons and attenuation in biosynthetic operons through their response to changes in the concentration of small molecules
39
What are the components of riboswitches?
- aptamer (binds to metabolite and undergoes conformational change in expression platform) - expression platform (conformational change regulates gene transcription/translation)
40
Describe transcriptional regulation by a riboswitch
Metabolite (SAM) binds the aptamer, which turns transcription off by causing a conformational change in the expression platform (forms a transcriptional terminator)
41
Describe translational regulation by a riboswitch
Metabolite (SAM) binds the aptamer, which turns translation off by causing a conformational change in the expression platform (formation of sequestering helix does not allow ribosome to bind)

CMMB 411 (12 decks)

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