Lecture 11: Gene Silencing/RNA Interference

Question 1

What is the silencing process after transcription in plants and animals?

Answer 1

1. Plants: Post-transcriptional gene silencing
   (PTGS)
2. RNA interference (RNAi)

Question 2

Explain the process of transgene silencing in Petunia

Answer 2

1. Petunia colour is determined by:
   a. Enzyme - chalcone synthase (CHS);
   increase enzyme = deeper colour
2. Dark flower was transformed with
   additional copy CHS gene
3. HOWEVER - they found providing a
   transgene led to BOTH copies not
   being expressed
4. Transcription in nucleus was unchanged
   
   • THEREFORE mechanism was post-
     transcriptional
     a. CHS mRNA was only missing in
     white flowers - THEREFORE
     mechanism is
     sequence specific (because only
     CHS expression changed)
     b. process called CO-SUPPRESSION;
     because transgenic and
     endogenous mRNA was
     suppressed at same time

Question 3

What is the process of RNAi in C. elegans?

Answer 3

1. antisense RNAs were used to suppress 
   gene expression in worms
2. (was found that injection of sense RNA 
     was just as efficient as antisense)
3. They found that is was              
    double-stranded RNA (dsRNA) that 
    works
   a. found using GFP bound dsRNA in C. 
       elegans

Question 4

What is the process of RNAi?

Answer 4

Has been found to be present in all other organisms.

1. dsRNA in cell (not common)
2. DICER (dsRNA specific RNase)
   a. cuts dsRNA into 21-25nt dsRNA’s
   (very similar fragments) - (various
   DICER genes in plants hence different
   nt sizes - usually only 21 in animals)
3. Sequence specific nuclease - RISC
   a. “RNA Induced Silencing Complex”
   b. protein complex
   c. Takes up dsRNA fragment to be
   active
   d. Single strand of it is taken up and
   has specific sequence
   e. binds to complementary strand -
   causing cleavage of mRNA
   f. Sides not protected by polyA or
   capping get broken down

Question 5

What are the applications for RNAi?

Answer 5

1. Knock out gene expression of any gene
   within a short period of time
2. long dsRNA can be used in plans and
   lower animals including model
   organisms like C. elegans and Drosophila
3. MAMMALIAN CELLS
   a. orignally excluded from RNAi due to
   long RNA pathways often being shut
   down by processes which result in the
   entire transcriptom being degraded
   b. cell dies
   c. thought to be a mechanism of viral
   defence
   d. HOWEVER; RNAi began use in 2001
   when a group used siRNAs to
   target genes in HeLa human cells

Question 6

What are potentials for RNAis in mammals/humans?

Answer 6

1. Much quicker process than gene
   knockout (especially for human/mouse
   experiments)
2. siRNAs as therapeutics against viruses
   (HIV, hepatitis, etc.) and other diseases
   (cancer, arthiritis, etc)
3. promising results in cell cultures
4. NEXT STEP: delivery to whole
   organism - much more difficult than
   delivering siRNAs into cell cultures

Question 7

Why did the RNAi machinery evolve?

Answer 7

1. lin-4 cloned by genetic mapping from C.
   elegans in 1993
2. regulates developmental timing by
   blocking expression of heterochromatin
   gene without reducing levels of mRNAs
3. lin-4 not a protein coding gene -
   ENCODES 21 NUCLEOTIDE RNA
   TRANSCRIPT
4. DICER produces si and miRNAs
   a. lin-4 and let-7 forms a semi- dsRNA for
   DICER to detect (miRNA = microRNA)

Question 8

Is miRNAs and siRNAs a C. elegans specific process?

Answer 8

1. 20-25nt RNA isolated, cloned and
   sequenced from C. elegans, Drosophila,
   HeLa cells and Arabidopsis
2. Many diff. miRNA sequences found in
   all organisms
3. 1917 miRNAs are published from
   human and more than 300 in plants

Question 9

How are miRNAs produced?

Answer 9

1. pri-miRNA
2. transcription
3. pre-miRNA (~70 nt)
4. Export out of nucleus
5. mature miRNA (~22 nt)