Download William Yin

William Yin
Steve Droho
Thursday Lab
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RNAi
RNA interference (RNAi) is a highly potent and specific process where the presence of
certain fragments of double-stranded RNA interferes with the expression of a particular
gene which shares a homologous sequence with the dsRNA. The RNA interference
machinery cuts up double-stranded RNA molecule with an enzyme known as Dicer
which separates it into two strands (cutting dsRNA into 22-25nt siRNAs). It then
proceeds to destroy other single-stranded RNA molecules that are complementary to one
of those segments. The siRNAs that form from dsRNA target RNA-degrading enzymes
(RNAse) through RISC to destroy transcripts complementary to the siRNAs. RISC or
RNA-induced silencing complex is an siRNA protein complex which cleaves incoming
dsRNA and binds the complementary RNA to a protein which seeks out the
complementary strand. When it finds the complementary strand, it activates RNAse
activity and cleaves mRNAs with complementary sequences.
RNAi has been recently applied as a technique to knockout genes in model organisms for
analyzing the functions of any particular gene. Repressing a gene from being expressed
by feeding dsRNA to the animal allows for testing of the protein and its role in the life of
the animal. Because RNAi may not totally abolish expression of a gene, the process of
using RNAi to decrease the function of a gene is often instead referred to as a knockdown.
dsRNAs that trigger RNAi may be usable as drugs. RNAi interferes with the translation
process of gene expression and does not appear to interact with the actual DNA. Thus
RNAi therapy does not involve alterations of the DNA, such as the physical removal of a
gene.
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RESULTS
I. Development of the Pharynx
Figure 1. Developmental stages of C. elegans embryo and early larva with expression of PD4793
myo-GFP patterns. GFP expression shaded in blue. A: Cleavage; B: Comma stage; C: 2-fold stage; D:
3-fold stage; E: L1 stage.
Table 1. Table describing when and where pharyngeal GFP reporter is expressed in C. elegans.
Developmental stage
GFP expression and location
Cleavage stage embryo
No expression
Comma stage
Yes, around area of the comma indentation.
Two-fold stage
Yes, typically localized at only one fold
Three-fold stage
Yes, localized in a section of several folds
Hatched larva (L1)
Yes, localized in the pharynx region of the larva
II. sma-1 RNAi.
Figure 2. Images of embryos comparing control (panel A) and sma-1 RNAi treated (panel B)
nematodes. GFP expression in green fluorescence. The sma-1 GFP expression also had the dumbbell
shape as in the control, but the thin regions of the expression appear to be more truncated (shorter) in the
sma-1 knockdown. The knockdown larva also appears somewhat shorter.
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III.
Differences between PD4793 and AZ217 were slight. The myo-GFP expression of
AZ217 was overall slightly weaker (less bright) than that of PD4793. The sma-1 RNAi
treated on AZ217 animals revealed a clearer knockdown phenotype (more truncated
myo2:GFP expression) than that on the PD4793, presumably because the weaker single
copy expression of AZ217 myo-GFP made the knockdown more obvious.
V. pha RNAi
Figure 3. Comparing control (panel A) and pha RNAi (panel B and C) treated nematodes.
The effect of pha RNAi treatment was very significant. Faint GFP fluorescence was
detected in the posterior region of the larva. The front half of the pharynx structure in
most of the early larva nematode was also no longer expressing GFP fluorescence when
compared to the control (only one part of the dumbbell is expressed and it is expressed in
a clump without the characteristic thick and thin shape). Some larvae had no expression
in the anterior end. Because pha encodes for a transcription factor, knocking down pha
function may interfere with the timing and localization of the transcription into mRNA of
one or more genes related to the morphogenesis of the pharynx in the nematode embryo,
expressing pharynx tissue (identified by myo-2:GFP fusion gene expression) in regions
not found in the control.
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DISCUSSION
VI.
sma-1 encodes a protein called β H spectrin (Wormbase database), one of 3 spectrin
proteins in invertebrates. Spectrin is a cytoskeletal protein that lines the intracellular side
of the plasma membrane of many cell types in pentagonal or hexagonal arrangements.
sma-1 β H -spectrin mutants have a defect in embryonic elongation, but not
postembryonic growth, and organ size measurements reveal a more severe reduction in
pharynx length than in seam cell length (Wang et al., 2002).
VII.
From our results we saw that the loss of sma-1 function led to a shorter pharynx and body
length than the control. The cytoskeletal protein that sma-1 encodes for probably forms a
scaffold and plays an important role in maintaining the integrity and cytoskeletal
structure of the plasma membrane. Because there are more than one spectrin proteins,
the loss of β H spectrin does not mean the complete absence of the scaffold, but rather an
incomplete formation that somehow interferes with the length of the organs and body of
the animal.
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