This document describes experiments using the fruit fly Drosophila melanogaster to study the effects of knocking down alanyl-tRNA synthetase, a protein involved in neurological diseases, through RNA interference. Two crosses were performed - one with whole body knockdown using an Actin promoter, and one with nervous system specific knockdown using a C155 promoter. For whole body knockdown, all flies died before eclosion, with ~4.5% pupal lethality and ~95.4% larval/embryonic lethality, indicating significant developmental effects. Nervous system specific knockdown also resulted in pre-eclosion lethality, but to a lesser degree. The results suggest alany

1. A full body and nervous system
specific knockdown of Alanyl-tRNA
synthetase in Drosophila
melanogaster
AJ Sacks
University of Miami Miller School of Medicine
Department of Cellular and Molecular Pharmacology

2. Drosophila melanogaster
• Large population
numbers- stronger
statistically
• Short lifespan- can
observe inheritance of
many generations
• High homology of genes
~60%
• Genetic manipulation
Carolina Drosophila Manual

3. Background
• 6% Americans have undiagnosed diseases (many neurological)
– Sequence genome- elucidate potential causes
– Mutations identified- (alanyl t-RNA synthetase)
Alanyl t-RNA synthetase
Molecular Biology for Masters
Dr. G. R. Kantharaj
Binds appropriate tRNA
molecule to the amino acid
alanine in tRNA charging
http://www.scripps.edu/news/scientificreports/sr
2004/mb04schimmel.html
http://mol-biol4masters.masters.grkraj.org/html/Ribose_Nucleic_Acid3B-Aminoacyl_tRNA_Synthases.htm

4. Purpose
• To observe the effects of the protein
knockdown of alanyl tRNA synthethase in the
whole body and also only in the nervous
system
– Loss of function, development, lifespan

5. Nature Reviews
The art and design of genetic screens:
Drosophila melanogaster
Daniel St Johnston
Gal-4 UAS System
Father- has gene for Gal 4 protein
after tissue specific promoter region
Mother- has UAS located right before the
gene of interest (RNAi)
Parents will mate and
produce F1 generation
F1 generation expresses
Gal 4 protein (show on
left) which binds to the
Upstream Activation
Sequence (UAS) and
drives the expression of
the RNAi (shown on
right)
F1
Actin or
C155 promoter
region
http://www.nature.com/nrg/journal/v3/n3/box/nrg751_BX2.html
Gene for
RNAi

6. Tissue Specific Promoter regions
• Actin- whole body
– Actin- cytoskeleton protein involved in the
structure and function of microfilaments
– Found in all eukaryotic cells
• C155- nervous system
– Enhancer trap insertion into the elav locus on the
X chromosome of Drosophila
– Only in nervous system cells

7. CROSS 1: Whole Body
y-w-
Cross 1:
;
[
]
,
Whole Body
;+ X
.
; + ; RNAi
Legend
+ =wild type
y-= yellow body
w-=white eyes
Cyo= curly wings
F1 possibilities:
;
;
.
;
;
Both will express Gal 4 which will drive the expression of the RNAi
;
,
;
;
.
,
;
Both Will NOT express RNAi because no Gal 4 protein, instead will have curly wings

8. CROSS 2- Nervous System
Cross 2:
C155-gal4, w- ; UAS-Dcr 2 ; + X
+ ; + ; RNAi
F1 Possibilities:
,
;
;
,
.
;
;
All flies will express RNAi because they all have Gal 4 protein to
drive its expression
Legend
+ =wild type
y-= yellow body
w-=white eyes
Cyo= curly wings

9. Methods
• Setting the Crosses:
– 11 flies per vial (6 females, 5 males)
• Allowed to mate for 2 to 3 days
• Transfer parents to new vial
• Parents in new vial left to lay eggs for 2 days (then parents
transferred out)
• Culturing: in vials with the standard cornmealagar-molasses-yeast medium at the bottom and a
cotton swab at the top, 25°C
• Pupae counting- at the end of all eclosion the
number of pupae were counted and recorded

10. Results:
Eclosion:
Cross Set: 10/01/2013
CROSS 1: Whole body
Date:
10/13 10/14 10/15 10/16 10/17 10/18 10/19 10/20 10/21
Actin,
RNAi
0
0
0
-
0
-
-
-
0
Actin,
RNAi
0
0
0
-
0
-
-
-
0
Cyo,
RNAi
21
21
23
-
4
-
-
-
1
Cyo,
RNAi
17
19
25
-
17
-
-
-
3
CROSS 2: Nervous System
Date:
10/13 10/14 10/15 10/16 10/17 10/18 10/19 10/20 10/21
C155
RNAi
0
0
0
-
0
-
-
-
0
C155
RNAi
0
0
0
-
0
-
-
-
0

11. Cross 1: Actin-Gal 4 X RNAi
• 154 empty pupae (154 flies to eclose)
– All flies to eclose were curly= 154 curly flies eclosed
– 1:1 ratio inheritance 154 knockdown flies SHOULD have eclosed
• 7 pupae with uneclosed flies
– Must have been caused by knockdown
because pf the established 100% eclosion
rate of curly flies
• 7/154= 4.5% pupae lethal
• 147/154= 95.4% larvae or
embryonic lethal
LEGEND
1: eclosed pupa
2: uneclosed pupa
3: larva
http://www.killi.co.uk/graphics/fruitfly10.jpg

12. Discussion
• All flies with the fully body knockdown died before
eclosion- significant lethality to knockdown, especially early
in development
– Pupa lethality rate- 4.5%
– larva or embryonic lethality rate 95.4%
• All flies with nervous system specific knockdown died
before eclosion
– More pupae had formed on the vials of nervous system
knockdown
– Less severe as full body
• Charcot-Marie-Tooth disease
– Inherited neurological disorder
– Peripheral neuropathy
• CMT caused by mutations in genes for proteins of myelin sheath

13. Charcot Marie Tooth Disease
• Recent study (McLaughlin HM et al.) associated point
mutation in gene for alanyl-tRNA synthethase
– P. Arg329His reduces enzyme activity
• Several mutations in genes of aminoacyl-tRNA synthethase
have been implicated in CMT
• Another study (Motley et al.) revealed that it is not the loss
of function of GARS but instead the toxicity of the mutant
tRNA synthethase

14. Next Step
• Human “rescue”
• Insert the human homolog into the flies
simultaneously with knockdown of the protein
• Tell whether the protein is conserved between
flies and humans
– Whether we can successfully express this human
protein in flies

15. •
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Acknowledgements and References
Dr. Grace Zhai, and her lab at University of Miami, especially Chris Bello
Ms. Jennifer Gordinier
References:
Zhao Z, Hashiguchi A, Hu J, Sakiyama Y, Okamoto Y, Tokunaga S, Zhu L, Shen H,
Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal
hereditary motor neuropathy. Neurology
McLaughlin HM, Sakaguchi R, Giblin W; NISC Comparative Sequencing Program, Wilson
TE, Biesecker L, Lupski JR, Talbot K, Vance JM, Züchner S, Lee YC, Kennerson M, Hou YM,
Nicholson G, Antonellis A. 2012 A recurrent loss-of-function alanyl-tRNA synthetase
(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N).
Duffy B. Joseph, 2002 GAL4 System in Drosophila: A Fly Geneticist’s Swiss Army Knife,
Department of Biology, Indiana University, Bloomington, Indiana
National Institute of Neurological Disorders and Stroke:
http://www.ninds.nih.gov/disorders/charcot_marie_tooth/detail_charcot_marie_tooth
.htm
Madame Curie BioScience Database, Lluis Ribas de Pouplana, Karin Musier-Forsyth and
Paul Schimmel, Alanyl tRNA synthetase
Raymond O. Flagg, Carolina Drosophila Manual, 2005 Carolina Biological Supply
Company
Motley WW, Seburn KL, Nawaz MH, Miers KE, Cheng J, et al. (2011) Charcot-MarieTooth–Linked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type
GARS Levels. PLoS Genet 7(12): e1002399. doi:10.1371/journal.pgen.1002399