33 - RNA interference Flashcards

1
Q

What is RNA interference?

A

Antisense RNA that inhibits translation of specific transcripts

Also called post transcriptional gene silencing (PTGS)

RNAi is the process of mRNA degradation that is induced by double-stranded RNA (dsRNA) in a sequence-specific manner

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2
Q

True or false? Double stranded RNAi is more effective than single stranded RNAi?

A

True

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3
Q

What is the molecular basis of co-suppression?

A

RNAi

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4
Q

What type of transcripts does dsRNAi silence?

A

Sequences similar to the dsRNA

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5
Q

What are the basic steps of RNAi?

A
  • Initiation step: Dicer binds dsRNA and cleaves into nucleotide fragments about 22 nt long (these fragments are called siRNAs, small interfering RNAs)
  • RISC (RNA induced silencing complex) binds one siRNA
  • RISC activated (siRNA unwinding, ATP dependent)
  • Effector step: activated RISC binds a target mRNA and degrades it
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6
Q

What is dicer?

A

A member of the RNase III family of double strand specific endonucleases, generates precisely sized fragments with 2-nuc overhands phosphorylated at 5’ (siRNAs).

Is the enzyme for the initiation step of RNAi

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7
Q

What is RISC?

A

RNA induced silencing complex includes Argonaute (another endoribonuclease). RISC recognizes dicer-generated siRNAs and the unwound fragment directs the particle to cognate mRNA by Watson-Crick base pairing.

Gene expression is silenced by mRNA degradation and/or translation inhibition (effector step of RNAi)

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8
Q

What is amplification of RNAi?

A

Some siRNA unwind without being associated with RISC. The antisense strand can bind to its complementary region of the corresponding mRNA, serving as a primer for a RdRP, which will generate new dsRNA of the target gene.

RdRP (RNA dependent RNA polymerase) generates dsRNA as substrate for dicer, and more siRNA is made

siRNA can go from cell to cell, and so silencing can propagate to other tissues.

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9
Q

What do miRNAs (micro interfering RNAs) do?

A

Encode small RNAs that regulate gene expression through post-transcriptional repression

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10
Q

How are miRNA transcripts processed?

A

Sequentially by two RNase III enzymes, Microprocessor and Dicer to yield mature miRNA duplexes, ranging from 18vo to 24bp in length

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11
Q

How do miRNAs prevent proliferation of transposons? What is the consequence of this?

A

Silencing transposon mRNAs, this controls the expansion of mobile genetic elements in the genome. It is possible because miRNA ‘genes’ have dsRNA with similarity to transposon mRNA

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12
Q

What are the steps of translation repression/mRNA degradation by miRNAs?

A
  1. Transcription of pri-miRNAs
  2. Drosha process yields pre-miRNA that is exported out of the nucleus
  3. pre-miRNA loaded onto RISC, dicer cleaves miRNA duplex
  4. miRNA helicase unwinds to yield mature miRNA
  5. Targets selected, if there is near perfect complementation with cytoplasmic mRNA, the mRNA is degraded.
  6. Is there is partial complementarity, translation is repressed on RISC and then mRNA is degraded.
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13
Q

What does miRNA do in normal tissues?

A

Proper miRNA transcription, processing and binding to complementary sequences of mRNA results in the repression of target-gene expression through a block in protein translation or altered mRNA stability. The overall result is normal rates of cellular growth, proliferation, differentiation and cell death.

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14
Q

What happens when there is a reduction or deletion of miRNA?

A

Tumour formation

Leads to inappropriate expression of miRNA target, oncoprotein

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15
Q

What happens when there is overexpression of miRNA in a cell?

A

Tumour formation. Increased miRNA would eliminate the expression of miRNA-target tumour suppressor gene and lead to cancer progression.

This might occur because of amplification, active promoter, increased efficiency in miRNA processing or increased stability of the miRNA.

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16
Q

How did RNAi evolve?

A

Arose at the origin of eukaryotic cell as a defensive mechanism for viruses and to control the deleterious activity of endogenous DNA parasites (eg. transposons)

17
Q

Why was the purple colour gene in petunias silenced when extra transcript was added?

A

The large amounts of transcript of the artificial gene silenced the purple colour gene.