This document discusses RNA interference (RNAi) mediated gene silencing in plants. It describes how small interfering RNAs (siRNAs) and microRNAs (miRNAs) are central to RNAi and can direct the degradation of messenger RNA (mRNA) to decrease gene expression. The document outlines the key stages of the RNAi mechanism and roles of proteins involved like Dicer and RISC complexes. It provides examples of how RNAi has been used to increase crop traits like shelf life, virus resistance, and fruit development.

1. RNA Interference Mediated Gene Silencing
in Plant
MBB-601
Presented by
Sachin Arunrao Tajne
PhD Scholar
IGKV Raipur

2.  RNA silencing is a novel gene regulatory mechanism that
limits the transcript level by either suppressing transcription
(transcriptional gene silencing) or by activating a sequence-
specific RNA degradation process (post-transcriptional gene
silencing).
 RNA interference (RNAi) is a biological process in
which dsRNA molecules inhibit gene expression or
translation, by neutralizing targeted mRNA molecules.
 RNAi was known by other names, including co-
suppression, post-transcriptional gene silencing (PTGS),

3.  Two types of small ribonucleic acid (RNA) molecules i.e micro
RNA (miRNA) and small interfering RNA (siRNA) are central
to RNA interference.
 RNAs are the direct products of genes, and these small RNAs
can direct enzyme complexes to degrade messenger
RNA (mRNA) molecules and thus decrease their activity by
preventing translation, via post-transcriptional gene silencing.


4.  Jorgensen and Napoli (1986): Discovery of RNA interference
(RNAi).
 Fire, Mello, Xu, Montgomery, Kostas and Driver (1998):
Double stranded RNA demonstrated to be potent mechanism for
silencing genes.
 Baulcombe and Hamilton (1999): Discovery of small
interfering RNA (siRNA), very small stretches of double-
stranded RNA interfere with genes.
 Elbashir and Harborth (2004): Small interfering RNA (siRNA)
shown to be useful tool for switching off certain genes.

5. Source: BBSRC

6. Mechanism of RNAi can be divided into four stages
 Double-stranded RNA cleavage
 Silencing complex formation
 Silencing complex activation
 mRNA degradation.

7. source :Zofia et al
Fig 1: SiRNA Gene silencing Pathway

8.  Small interfering RNA (siRNA), sometimes known as short
interfering RNA or silencing RNA, is a class of double-
stranded RNA, non-coding RNA molecules, 20-25 base pairs in
length.
 It interferes with the expression of specific genes with
complementary nucleotide sequences by degrading mRNA
after transcription, preventing translation.
 siRNA are generally considered to have come from longer
strands of exogenous growing or originating from outside an
organism
 Each siRNA strand has a 5' (five-prime) phosphate group and a
3' hydroxyl (OH) group.

9. The RISC-loading complex (RLC) is
the essential structure required to load
dsRNA fragments into RISC in order to
target mRNA.
The RISC complex consists of dicer,
the trans activating response RNA-
binding protein (TRBP) and Argonaute 2.
Dicer is an RNase
III endonuclease which generates the
dsRNA fragments to be loaded that
direct RNAi.
TRBP is a protein with three double-
stranded RNA-binding domains.
Argonaute 2 is an RNase and is the
catalytic centre of RISC.
 Dicer associates with TRBP and
Argonaute 2 to facilitate the transfer of
the dsRNA fragments generated by
Dicer to Argonaute 2.

10. RNase III family members are among the few nucleases that show
specificity for dsRNAs and cleave them with 3 overhangs 2 to 3
nucleotides and 5-phosphate and 3-hydroxyl termini.
Dicer has four distinct domains:
 an amino terminal helicase domain,
 dual RNase III motifs
 a dsRNA binding domain
 a PAZ domain (a 110-amino-acid domain present in
proteins like Piwi, Argo, and Zwille/Pinhead),
 which it shares with thRDE1/QDE2/Argonaute family of
proteins that has been genetically linked to RNAi by independent
studies.

12.  A miRNA is a ssRNA of ~22 nucleotides in length.
 Generated by the RNase-III-type enzymes Drosha and Dicer
from an endogenous transcript that contains a local hairpin
structure.
 pri-miRNAs contain cap and poly(A) tail and are transcribed
by RNA Polymerase II.
 first discovered in 1993 by Victor Ambros in C. elegans:

13.  RNase III enzyme Drosha, which cleaves the stem ~22 nt
away from the terminal loop to generate an ~65-nt pre-
miRNA hairpin intermediate.
 Drosha leaves a characteristics 2-nt 3` overhang.
 The pre-miRNA is transported to the cytoplasm by Exportin-
5,where it interacts with a second RNase III enzyme called
Dicer.
 Dicer binds the 2-nt 3` overhang found at the base of the pre-
miRNA hairpin and cleaves ~22nt away from the base,
removing loop & leaving another 2-nt 3` overhang.
 The resultant duplex intermediate interacts with RISC
components,

14. SOURCE: SLIDE SHARE

15.  Fruits and vegetables are more prone to spoilage than cereals
due to their nature and composition, and this spoilage results
in inedible waste.
 The shelf life in tomato has been increased by silencing of
genes associated with either ethylene production or ripening.
 Xiong et al. (2005) used RNAi technology to increase shelf
life in tomato., They introduced a unit of dsRNA and blocked
the expression of ACC oxidase gene in tomato.
 The ethylene production rate in ripened fruits and leaves of
transgenic plants was found to be significantly inhibited,
ensuring a prolonged shelf life of tomato.

16. Fig 3: Biosynthesis pathway
of Ethylene

17.  RNAi, targeting the coat protein (CP) gene of viruses is found
to be quite effective in inducing resistance to the plant against
viruses.
 Pradeep et al. (2012) reported that the introduction of inverted
repeats of the CP gene of Tobacco Streak Virus (TSV) may
be an effective and reliable strategy for developing
economically important crops with resistance to TSV.
 Zhou et al. (2012) have created an RNAi construct containing
CP gene and disease-specific protein gene sequences from
Rice Stripe Virus.

18.  Phytohormone plays a key role in regulation of transition
between flowering, fertilization and fruiting.
 De Jong et al. (2009) indicated that SlARF7 acts as a
modifier of both auxin and gibberellin responses during
tomato fruit set and development.
 Reduction of SlARF7 transcript levels by an RNAi approach
may release the repression of the auxin and GA signalling
pathways that are imposed by SlARF7 independently of
pollination and fertilization.

19.  Saurabh S, Vidyarthi AS, Prasad D (March 2014).
"RNA interference: concept to reality in crop
improvement". Planta. 239 (3): 543–64.
 Zamore PD, Tuschl T, Sharp PA, Bartel DP (March 2000).
"RNAi: double-stranded RNA directs the ATP-dependent
cleavage of mRNA at 21 to 23 nucleotide
intervals". Cell. 101(1): 25–33.
 Kamath RS, Ahringer J (August 2003). "Genome-wide RNAi
screening in Caenorhabditis elegans". Methods. 30 (4):
313–21.