Lecture 17

Question 1

Coding RNA:

Answer 1

• RNAs that encode some type of information
• A conduite between the RNA and the protein
• messenger RNAs

Question 2

Functional RNA:

Answer 2

• Ribosomal RNA
• transfer RNAs
• Decoding the information within the mRNA
• short nuclear RNAs
• part of processing RNA, splicing introns out of transcripts in eukaryotes

Question 3

Regulatory (small) RNAs:

Answer 3

• siRNAs (discovered through silencing RNAs)
• miRNAs (discovered developmental mutants)
• piRNAs
• circRNAs

Question 4

Regulatory (long) RNAs:

Answer 4

• IncRNAs

- Involved with X inactivation in placental mammals

Question 5

Gene silencing:

Answer 5

• Trans-acting antisense RNA in E.coli silence their targets by forming dsRNA duplexes
• 100 nt in size, complimentary sequences to mRNA
• Selectively inactivate a gene/nucleotide sequence within a genome

Question 6

Antisense inibition:

Answer 6

Paring between antisense and sense RNA, inhibiting gene expression as the duplex cannot be translated

Question 7

Kemphues group and gene silencing experiment:

Answer 7

• Anti-sense inhibition for silencing Par-1
• Antisense mechanism cannot account for sense inhibition
• Phenotype observed in half of the worms injected
• Injection is easy, but the silencing effect is not easy to replicate

Question 8

What does partition-1 (par-1) gene do?

Answer 8

• It promotes early cell division in the nematode Caenorhabditis embryo

Question 9

Can antisense/sense suppression be induced by aberrant RNA?

Answer 9

• Gene silencing is mediated/induced by dsRNA

- 100x more effective than ssRNA

Question 10

What does the unc22 gene code for?

Answer 10

• Uncordinated-22
• Codes for a muscle microfilament
• Mutations in this gene result in twitching worms

Question 11

Is gene silencing sequence specific?

Answer 11

Yes

Question 12

What gene is unc24?

Answer 12

• Paralysis

Question 13

Fem-1 gene mutant:

Answer 13

• No sperm, so feminises the worm

Question 14

hlh-1 gene mutant:

Answer 14

• Lumpy-dumpy phenotype

Question 15

dsRNA causes

Answer 15

• Loss of target mRNA, either because
• dsRNA is interfering with transcription
• dsRNA is causing degradation of the transcript, post-transcriptionally

Question 16

Does gene silencing occur post-transcriptionally?

Answer 16

• Yes!

- Injecting dsRNAs homologous to promoters and introns does not cause silencing

Question 17

RNA interference:

Answer 17

• RNAi

- Eukaryotic cells perceive dsRNA as a sequence specific signal to inhibit expression of the corresponding mRNA

Question 18

Co-suppresion/post-transcriptional gnee silencing (PTGS)

Answer 18

• Manipulate petal colour by introducing a transgene into plants
  WT flowers and transgenic flowers are produce
• mRNA of transgene/endogenous gene are degraded

Question 19

Virus-induced gene silencing (VIGS)

Answer 19

Induce virus resistance by introducing viral transgene into plants

• Expression of untranslatable viral transgene in plants, these plants are immune to virus as viral RNA/transgene mRNA is degraded
• Non-transgenic plants exhibit disease symptoms as viral RNA accumulates

Question 20

Virus induced gene silencing:

Answer 20

• Natural defence against viruses exists in some plants
• Virus produce dsRNA either via secondary structures in the RNA or as a duplication event
• dsRNA is recognised as being foreign, triggering RNAi

Question 21

RNAi is associated with small RNAs:

Answer 21

• Antisense RNAs
• Short interfering RNAs (siRNAs)
• Target the genes/genetic elements from which they are derived
• Work in trans and complementary to target
• Induces mRNA degrading, post-transcription and sequence specific

Question 22

Discovery of endogenous small RNAs occured via:

Answer 22

• Study of the lin mutant with altered patterns of cell lineage in vulva development

Question 23

Lin-4 mutant:

Answer 23

• Bag of worms phenotype

- Vulva doesn’t form correctly, so eggs cannot come out of the worm

Question 24

Heterochronic mutants affect developmental timing (slowing it down or speeding it up):

Answer 24

• Four stages of larval development
• In lin-4 and lin-14(gf) there is a delay in developmental progression
• Lin-14 and lin-28 loss of function shows developmental process earlier than normal

Question 25

Lin-14 mutation:

Answer 25

- Required for the cell type of the first larval stage - The protein is only required for the first larval stage - But lin-14 mutants do not produce any lin-14 transcript so larval stage 1 is skipped

Question 26

Lin-4 mutation:

Answer 26

- Stuck with L1 cell types in all four larval stages - Limits lin-14 activity to the l1 larval stage - Effects protein abundance

Question 27

Lin-14(gf) mutants with a loss of the 3' UTR region show

Answer 27

- 7 partially complementary sequences | - that the 3' UTR region is essential for the degradation of the lin-4 protein in the 2nd, 3rd and 4th larval stages

Question 28

let-7

Answer 28

- regualtes lin-41 - found in all metozoans - works in larval development, regulating lin-41

Question 29

Primary miRNA is generated by:

Answer 29

- RNA pol II and has a 5' CAP and poly-A tail

Question 30

Primary miRNAs can form:

Answer 30

- an imperfect hairpin stem-loop strucutre throuh the intramolecular interactions

Question 31

Drosher, a type III endonuclease:

Answer 31

- Found in the nucleus - Recognises the hairpin loop, and binds to primary micro RNA transcript - precursor miRNA is exported from the nucleus - Produces a single duplex

Question 32

Dicer, another type III endonuclease:

Answer 32

- In the cytoplasm - Type III endonuclease - Dices the precursor RNA, removes the loop from the stem - Produces multiple duplexes - 5' phosphate and a 2nt 3' overhang - Needs to be processed still further

Question 33

siRNA biogenesis

Answer 33

- long perfectly pair dsRNA - it is processed in the cytoplasm by dicer - produces multiple duplexes - 5' phosphate and a 2nt 3' overhang

Question 34

RNA-induced silencing complex (RISC)

Answer 34

- A complex that modifies dsRNA further - Argonaut degrades the target transcript leading to gene silencing - The end that enters RISC is determined by the sampling of thermodynamic stability of the 5' end - The weakest end of the duplex enters