Transgene Silencing

1

Lecture- 37

• Gene silencing is a general term describing epigenetic process of gene regulation. The term gene silencing is generally used to describe the “switching off” of a gene by a mechanism other than mutation. That is , a gene which would be expressed under normal circumstances is switched off by machinery in the cell.

2

• Genes are regulated at either the transcriptional or post-transcriptional level.

3

Phenomena of gene silencing was

first observed in Petunia

An attempt was made to overexpress chalone synthase (anthrocyanin pigment

gene) in Petunia to darken flower color by introducing another copy of chalone

synthase gene (transgene)

The result was the loss of pigment.

4

Co-suppression:

Suppression of the expression of

both endogenous gene and

transgene.

5

Two mechanisms can explain this transgene-

mediated gene silencing

•Transcriptional gene silencing(TGS)

•Post Transcriptional Gene Silencing

(PTGS)

mRNA is made, but then degraded

6

• Transcriptional gene silencing is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc. ).

7

• Post-transcriptional gene silencing is the result of mRNA of a particular gene being destroyed or blocked. The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein). A common mechanism of post-transcriptional gene silencing is RNA interference (RNAi).

8

Other names of post-transcriptional gene

silencing (PTGS) :

– RNA silencing

•– RNA interference (RNAi)

•– In certain fungi: quelling

9

Before RNAi was well characterized, it was called by

other names, including post transcriptional gene

silencing (PTGS) and transgene silencing. Only after

these phenomena were characterized at the molecular

level it was observed that they were the same

phenomena.

10

History of post-transcriptional gene silencing

Definition: the ability of exogenous double-stranded

RNA (dsRNA) to suppress the expression of the gene

which corresponds to the dsRNA sequence.

•1990 Jorgensen

Introduction of transgenes homologous to

endogenous genes often resulted in plants with both

genes suppressed.

Called Co-suppression

Resulted in degradation of the endogenous and the

transgene mRNA11

•1995 Guo and Kemphues

Injection of either antisense or sense RNAs in the

germline of C. elegans was equally effective at

silencing homologous target genes

•1998 Mello and Fire

Extension of above experiments, combination of

sense and antisense RNA (dsRNA) was 10 times

more effective than single strand RNA

Contd….

12

Experiment demonstrating RNAi (conducted by Mello and Fire in 1998)

Phenotypic effect after injection of single-stranded or double-stranded unc-22 RNA into the gonad of C. elegans. The unc-22 gene encodes a myofilament protein. Decrease in unc-22 activity is known to produce severe twitching movements. Injected double-stranded RNA, but not single-stranded RNA, induced the twitching phenotype in the progeny. 13

The Nobel Prize in Physiology or Medicine 2006 was awarded jointly to Andrew Z. Fire and Craig C.

Mello "for their discovery of RNA interference - gene silencing by double-stranded RNA"

Andrew Z. Fire Craig C. Mello 14

What is RNA interference /PTGS?

• dsRNA needs to be directed against an exon, not an

intron in order to be effective

• homology of the dsRNA and the target gene/mRNA is

required

• targeted mRNA is lost (degraded) after RNAi

• the effect is non-stoichiometric; small amounts of

dsRNA can wipe out an excess of mRNA (pointing to

an enzymatic mechanism)

15

• Double-stranded RNA triggers cleavage of homologous

mRNA

• The life cycle and replication of many RNA virusesinvolves a double-stranded RNA stage, so it is likelythat part of the RNA interference machinery evolved as a defense against these viruses.

• PTGS-defective plants are more sensitive to infection by RNA viruses

• In RNAi defective nematodes, transposons are much more active

16

Double-stranded RNAs are produced by:

– transcription of inverted repeats

– viral replication

– transcription of RNA by RNA-dependent RNA-

polymerases (RdRP)

17

RNAi can be induced

18

RNA interference process and the biochemical machinery involved

ds RNA is cut into short double-stranded fragments called small interfering RNAs (siRNAs) by Dicer, a ribonuclease III-like nuclease .

siRNAs are then separated into single strands and integrated into an active RNA-induced silencing complex (RISC)

The antisense strand is loaded into RISC and links the complex to the mRNA strand by base-pairing.

The RISC complex cuts the mRNA strand, and the mRNA is subsequently degraded.

• Exogenous, e.g. a virus with an RNA genome • Endogenous, e.g. Stem-loop structure of pre-microRNAs

19