Lecture 12: RNAi 1 UNFINISHED

Question 1

RNA Interference:

RNAi

Answer 1

1* RNAi

2 * double-stranded (ds) RNA-mediated gene regulation

3 * RNA silencing
4* silencing of genes

Question 2

RNA Interference: organisms

Answer 2

• animals
• plants
• protists

Question 3

RNA Interference: targets

= 2

Answer 3

• endogenous genes
• exogenous genes

Question 4

RNA Interference: roles = 3

Answer 4

1 * development

2 * genome stability

3 * adaptive responses to biotic and abiotic stresses

Question 5

Orthodox and new view of Gene Expression diagram

Answer 5

diagram slide 4

GENE A –> transcription –> mature mRNA —> Protein

Question 6

Orthodox and new view of Gene Expression:

‘Factors controlling gene expression:’ = 3

Answer 6

1. • transcription factors (TFs; proteins)
2. • mRNA processing and stability
3. • translational and post-translational control

Question 7

Orthodox and new view of Gene Expression:

‘Fine Tuning of gene expression’

Answer 7

RNAi-mediated pathways controlling gene
expression are distinct

Question 8

RNA Interference - sense and antisense technologies… wild type

diagram =3

Answer 8

WILD TYPE:

1. ENDOGENOUS GENE
2. TRANSCRIPTION
3. PROTEIN

slide 5

Question 9

RNA Interference - sense and antisense technologies…antisense technology

SENSE (OVEREXPRESSION) TECHNOLOGY

+
diagram =

Answer 9

1. ENDOGENOUS GENE —> TRANSCRIPTION —> PROTEIN
2. ENDOGENOUS GENE —> ‘ADD A COPY OF THE GENE’—> TRANSCRIPTION —> PROTEIN

slide 5

Question 10

RNA Interference - sense and antisense technologies…

ANTISENSE TECHNOLOGY

= + DIAGRAM

Answer 10

1.On a chromosome…NORMAL GENE ‘X’

2. GENE ‘ADDITION’ + GENE X ‘ALTERED’ to make ANTISENSE RNA
3. TRANSCRIPTION
4. Normal mRNA + Antisense RNA
5. = RNA double Helix
6. FORMATION OF THE mRNA/RNA Helix Prevents the synthesis of a protein product from Normal GENE X.

slide 5

Question 11

RNA Interference - first indications of RNAi

• about PETUNIA

Answer 11

… Petunia: “co-suppression” : Jorgensen et al Plant Molecular Biology 31: 957-973, 1996 and reviewed in Stam et al. (97) Ann Bot 79:3

Question 12

RNA Interference - first indications of RNAi

• ABOUT NEUROSPORA

Answer 12

DIAGRAMS

WILDE-TYPE PETUNIA V26 - PURPLE COLOUR

FLOWERS OF CHALCONE SYNTHASE SILENCED TRANSFORMATIONS

Neurospora: wild type (left); albino mutant (right)
“quelling”Cogoni & Macino (97) Trends Plant Sci 2:438

Neurospora: wild type (left); albino
mutant (right)
“quelling”Cogoni & Macino (97)
Trends Plant Sci 2:438

Question 13

Double-stranded RNA can be more effective at suppressing the ‘expression of a gene’ than the antisense RNA

WHAT WAS THE EXPERIMENT? = 2

Answer 13

1 * Fire et al used ‘C. elegans’ animals and inject various mex3 RNA constructs.

2 * Analyse the offspring embryos for EXPRESSION OF ENDOGENOUS ‘mex3’ RNA using ‘in situ’ hybridisation with a probe that can hybridise to endogenous mex3 RNA

Question 14

Double-stranded RNA can be more effective at suppressing the ‘expression of a gene’ than the antisense RNA =

TEST: MEX3 PROBE : OUTCOME =4

Answer 14

1. NEGATIVE CONTROL - NO Mex3 Probe = NO STAINING
2. EMBRYO FROM ‘UNINJECTED’ PARENT - YES Mex 3 Probe = ENDOGENOUS ‘mex-3’ RNA ‘DETECTED’
3. EMBRYO FROM A PARENT INJECTED WITH PURIFIED ‘mex-3’ ANTISENSE RNA- YES Mex3 Probe = ENDOGENOUS ‘Mex-3’ RNA DETECTED
4. EMBRYO FROM A PARENT INJECTED WITH ‘ds’ mex 3 mRNA- YES Mex3 probe = ENDOGENOUS mex-3 RNA ‘NOT DETECTED’

Question 15

1998 Fire and Mello and coworkers observed in experiments using ‘C. elegans’:

WHAT WAS DISCOVERED: 2

Answer 15

1. • that ‘NEITHER mRNA NOR ANTISENSE RNA INJECTION’ had an EFFECT on the production of
     several proteins.
2. • that DOUBLE-STRANDED RNA SUCCESSFULLY SILENCED THE TARGETED GENE

Question 16

The discovery of RNA interference -1998 Fire and Mello and coworkers observed in experiments using C. elegans:

THEY DISCOVERED RNA INTERFERENCE OR RNAi = 3

Answer 16

1. They further discovered that an ‘RNA MOLECULES CAN INHIBIT GENE EXPRESSION BY ‘CONTRIBUTING TO THE DEGRADATION’ OF ENDOGENOUS mRNA MOLECULES

2 * They named this ‘RNA interference or RNAi’

3 * Fire and Mello received the 2006 Nobel Prize in Physiology or Medicine for this discovery

Question 17

WHAT IS RNA SILENCING? = 2

Answer 17

1. RNA silencing and RNA interference are BIOLOGICAL PROCESSES in which RNA
   MOLECULES INHIBIT GENE EXPRESSION
2. RNA silencing is a broader category that INCLUDES RNA interference

Question 18

RNAi pathways = 2

Answer 18

1. Post-transcriptional gene silencing (PTGS)

2.Transcriptional gene silencing (TGS)

Question 19

RNAi pathways = Post transcriptional gene silencing (PTGS) = 2

Answer 19

1. ▪ micro (mi) RNAs
2. ▪ small interfering (si) RNAs

Question 20

RNAi pathways: Transcriptional gene silencing (TGS): 3

Answer 20

1. ▪ small interfering (si) RNA silencing
   
   2. • chromatin modification
        3. methylation of DNA and/or histones

Question 21

RNAi pathways OCCURS WHERE? = 2

Answer 21

1. Not all are known to occur in all organisms
2. Not all components or steps identical in all organisms