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Transcript of RNA Interference
Emily Camras RNA Interference and its Applications dsRNA(4) DICER siRNA RISC RISC-RNA Complex RNAi Can Occur Naturally or be Used in the Lab(4) Natural RNAi In the Lab Background RNAi is a gene-silencing mechanism, a process that prevents expression of targeted genes (4)
It takes place naturally in eukaryotic cells, as a mechanism of targeting and destroying viral RNA strands (7)
The process, however, can be used as a laboratory technique to target mRNA, to silence desired genes to gain insight into gene fucntion (3) Discovery and Evidence Description Andrew Fire & Craig Mello -Used RNAi to in C. elegans to determine that double stranded RNA, or dsRNA, is responsible for RNAi
-The dsRNA approach was compared to the effects of injecting DNA with only an "antisense" or "sense" strands
-Only worms in which dsRNA was introduced had a changed phenotype than the controls
-Therefore, Fire and Mello found that only dsRNA could successfully degrade mNRA and inhibit certain genes (4) RNAi Occurs Naturally in Plant and Animal Cells Some plants and animals have evolved genes that code for interfering RNA which is complimentary to viral RNA strands. The interfering RNA targets the viral strands, thereby deactivating the viruses. Therefore, RNAi is used as an evolved protective mechanism. (3) 2006 Nobel Prize(4) 1) Endogenous - genes in the cell code for pri-miRNAs (7) 2) pri-miRNAs processed by DROSHA, a ribonuclease enzyme
which cuts them into pre-miRNAs (7) 3) pre-miRNAs leave nucleus, enter cytoplasm(7) 2) DICER cuts foreign dsRNA into small interfering
RNA, known siRNAs(3) 3) siRNAs are processed and leave the nucleus(3) Consists of domains (3) -
Dicer belongs to the helicase family
DUF283: a domain of unknown function of about 100 amino acids
PAZ: recognizes dsRNA ends with a 3', 2 nucleotide overhang
RNase III domain: space between this domain and PAZ determines length of the dsRNA
dsRBD: binding region for double stranded RNA
DICER in Action(2) Results in cleavage of target mRNA! long double-stranded RNA that is injected into the cell. This RNA is complementary to strands of mRNA that will be targeted. enzyme from the Ribonuclease (RNase) III family that cleaves dsRNA into short segments(3) the resulting segments are called ~20 nucleotides in length
siRNA taken apart into one "passenger" and one "guide" strand (3) passenger strand
incorporated into: RNA-Induced
Slicing Complex(7) -guide strand base pairs with mRNA
-a protein Argonaute 2 induces cleavage play from 1:22(6) Used to investigate
gene function(7), dsRNA introduced
by feeding or injection Often used in C. elegans or
Drosophila(7) 1) Exogenous - dsRNAs are introduced into cell(3) RNAi Medical Applications (7)
Macular degeneration - RNAi can be used to inhibit expression of a protein, vascular endothelial growth factor, that promotes growth of blood vessels in the eye. As a result, excess growth can be stopped, inhibiting progression of the disease
HIV- RNAi can be used to target the virus
Huntington's Disease - RNAi can be used to target the dominant gene responsible for the disease
(7) dsRNA is not as easily introduced into mammalian cells as these cells have an interferon response to foreign DNA (7)
treatments show promise ->
RNAi in Plasmodium Background research by molecular biologists at the University of Leads
indicated that a chloroplast-like organelle could be essential for
Malaria population growth. They used RNAi to determine
the degree to which this organelle was necessary for growth (5). Introducing dsRNA into
C. elegans (7) Methods An RNAi was run by adding dsRNA to infected red blood cells via plasmids(5).
P. falciparumdhodh and Aroc, two genes they knew to be essential to this rare organelle's structure, were silenced in this RNAi (5). Effects of RNAi can be quantified and used to determine gene function(7). Results The results for the RNAi are shown in the graph below. Fig 1.
A comparison of protein levels in the experimental population to protein levels in the control population. The graph shows the experimental population's protein concentration as a percentage of the control population at 24, 48, and 72 hours after the dsRNA was administered(5). The organelle was shown to be neccesary for Malaria population growth(5). Bibliography RNAi is both a naturally occurring cellular process as well as a research technique Occurs when
is degraded Silences targeted
expression Valuable research tool
gene function While RNAi results are often analyzed by comparing protein content in the experimental population to the control population, comparing experimental phenotype (such cell as color) to the control phenotype can also provide information(5). Overview Transfer Question In a study, RNAi was used to silence expression of the gene that codes for a protein Ls.Act in nematodes.
The nematodes were soaked in a dsRNA solution for 24 hours, then removed from the solution. Measurements of the percent Ls.Act were taken at given time intervals, at 24 and 48 hours after removal from the solution. 24 hours after removal and 48 hours after removal are labled 48 and 72 respectively because time is meassured from the beginning of the RNAi. The dsRNA used in this study silences the mRNA associated with Ls-act.
The graph below compares the amount of Ls.Act in nematodes based on the time elapsed since their incubation in a dsRNA solution.
Does this data suggest anything about
the time frame for Ls.Act synthesis?
Why or why not? Give an interpretation for this
graph that does not pertain to
the time frame of Ls.act synthesis. Pfarr, K., Heider, U., Hoerauf, A. (2006). RNAi mediated silencing of actin expression in adult Litomosoides sigmodontis is specific, persistent and results in a phenotype. Molecular and Cellular Biology of Helminth Parasites, 36, 661-669. doi: 10.1016/j.ijpara.2006.01.010