Download The Unfolded Protein Response in C. Elegans

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Signal transduction wikipedia , lookup

SR protein wikipedia , lookup

Magnesium transporter wikipedia , lookup

Protein moonlighting wikipedia , lookup

List of types of proteins wikipedia , lookup

Gene expression wikipedia , lookup

Epitranscriptome wikipedia , lookup

Transcript
The Unfolded Protein
Response in C. Elegans
Biology 314, Advanced Cell
Biology, Spring 2004
Characterization of a Mutation in the Unfolded
Protein Response Signal Pathway of the
Nematode Worm C. elegans
Jillian Anibaldi, Chris Binner, Jo’Vonna Grayson & Artemio Linares
BIO 314, Advanced Cell Biology
The unfolded protein response (UPR) is a transcriptional and translational
intracellular signaling pathway activated by the accumulation of unfolded proteins in
the lumen of the endoplasmic reticulum (ER). Protein folding in the endoplasmic
reticulum is sensitive to agents that interfere with protein glycosylation or the
formation of disulfide bonds (Tunicamycin or DTT). A strain of mutant C.elegans,
SJ17, has been identified as having a flaw in the UPR. We have further characterized
the mutant strain by: (i) assaying the mutants for expression of hsp-4 gene, a
characteristic of the UPR. We have shown that the mutants are incapable of inducing
hsp-4 when stressed with tunicamysin, (ii) The mutants, treated with DTT and
Tunicamycin, exhibit slow growth and do not progress beyond the L2 larval stage.
The observed deficiency in UPR, could lie in ire-1 since this gene codes for the
endonuclease that signals ER stress by splicing (and activating) the mRNA for Xbp-1.
Xbp-1 is a transcription factor, which activates the hsp-4 and other genes, required
for the UPR. We are currently attempting to determine if the xbp-1 mRNA in the
mutant strain is spliced in response to stress. If xbp-1 is spliced, then ire-1 is not the
gene in which the mutation lies. The mutation, then, might be in the xbp-1 gene or
other distal elements of the pathway.
Introduction
• The Endoplasmic Reticulum (ER) is the compartment in cells where
membrane and secreted proteins are folded and glycosylated.
• When proteins fail to fold properly in the ER they can potentially
aggregate and eventually kill the cell.
• Cells prevent aggregation by synthesizing molecular chaperone
proteins which assist proper folding.
• When the ER is stressed by agents which interfere with
glycosylation or folding, the cell responds with the synthesis of
additional chaperones. This response is called the UPR (See next
panel for description).
• Our task was to identify the genetic defect in a mutant strain of C.
elegans (SJ17) which apparently is unable to mount an unfolded
protein response.
The Unfolded Protein Response (UPR)
XBP-1
IRE-1
• The UPR occurs when proteins are misfolded in the endoplasmic reticulum
(ER).
• Reducing agents, such as DTT, interfere with disulfide bond formation while
tunicamysin interferes with glycosylation; both agents cause proteins to
misfold in the ER thus triggering the UPR.
• The product of the ire-1 gene is the sensor of misfolded proteins and when
activated removes an intron from the pre mRNA from the xbp-1 gene.
• Active xbp-1 protein (from spliced mRNA) activates the genes that code for
ER chaperones, such as hsp-4.
Experimental Design
• Can we detect the UPR in the C. elegans?
• Can the UPR be induced by Tunicamycin which causes
ER stress?
• Can the UPR be induced in the SJ17 mutants
• Is there a survival phenotype for the SJ17
mutation?
• Are SJ17 worms hypersensitive to tunicamysin?
• Is the mutation located in ire-1 or xbp-1?
• Does electrophoresis of the RT-PCR product show that it
is xbp-1 mRNA is spliced or unspliced in mutants exposed
to ER stress
• If it is not spliced then the mutation is located in the
ire-1.
•If it is spliced then the mutation may lie in the xbp-1
gene.
Mutant C. elegans do not Induce GFP:Hsp-4
in Response to Tunicamycin
Untreated
Control
WILD TYPE
MUTANT
Tunicamysin
Treated
WILD TYPE
MUTANT
Survivorship of Wild Type C. elegans
150
Figure 1- untreated wild type
Total Counted
130
110
90
# Eggs
L1-L2
L3-L4
Dead
70
50
30
10
-10
Day 1
Day 2
Day 3
Day 4
Date Observed
150
Figure 2- treated wild type
Total Counted
130
110
90
# Eggs
L1-L2
L3-L4
Dead
70
50
30
10
-10
Day 1
Day 2
Day 3
Date Observed
Day 4
Survivorship for Mutant C.elegans
150
Figure 3. Untreated Mutant
Total Counted
130
110
90
# Eggs
L1-L2
L3-L4
Dead
70
50
30
10
-10
Day 1
Day 2
Day 3
Day 4
Date Observed
150
Figure 4. Treated Mutant
Total Counted
130
110
90
# Eggs
L1-L2
L3-L4
Dead
70
50
30
10
-10
Day 1
Day 2
Day 3
Date Observed
Day 4
RT-PCR Analysis of Xbp-1 messenger RNA
splicing
In order to determine whether the Xbp-1 mRNA has been spliced we must:
1. Extract total RNA from our worms.
2. Determine the quality and quantity of the RNA extracted.
3. Amplify the xbp-1 mRNA sequence present in the samples.
4. Examine the amplified RNA for spliced (small) and unspliced (large) xpb-1
message.
Figure 1:
Electrophoresis of the RNA that was
extracted from the C. elegans.
1
2
3
4
Figure 2:
Electrophoresis of the RT-PCR
product from the C. elegans
extracted RNA.
1
5
400 bp
Unspliced
Spliced
2
3
4
5
Table 1:
Lane identification key
Lane
1
2
3
4
5
Worm Strain
Wild Type
Mutant
Mutant
Mutant
Mutant
Treatment
Tunic.
Control
Control
Tunic.
Tunic.
200 bp
This Electrophoresis Result indicates that RNA has
been successfully extracted from our C. elegans.
Unspliced – 294 bp
Spliced – 271 bp
The Xbp-1 mRNA is spliced in both mutant and control animals exposed to Tunicamycin.
Summary
• We have shown that the UPR can be detected by
using a fluorescent reporter protein (hsp4). The ER
stressor, Tunicamycin, induces the GFP fusion hsp-4
product in wild type C. elegans, while in the SJ17
mutant, induction does not occur.
• We have also shown that the phenotype for the
mutant C. elegans (SJ17) exposed to tunicamysin, is
a severe delay in growth that can result in death.
• After careful analysis we have determined the
mutation in SJ17 to be located in the xbp-1 gene
because our RT-PCR analysis showed that splicing
of xbp-1 mRNA occurred and therefore the ire-1
gene product which is responsible for splicing is
functional.