Seventh Progress Report

Sept. 2014-Feb. 2015

Role of hsr in polyQ pathogenesis and
development of Drosophila melanogaster

Supervisor
Prof. S. C. Lakhotia
Cytogenetics Lab.
Department of Zoology
B.H.U, Varanasi

Presented by

Mansoor Mustafa
Enrolment No. : 342034

Previous observation

Poly-Q mediated eye degeneration was

suppressed by expressing -RNAi in the
eyes even in hsr null background

Three possible reasons for

this observation
1. off-target
2. accumulation of both the strands of
280 bp sequence
3. presence of extra 84 bp in the
transgene

Work done
searched the possible off-targets due to 280
bp as well as the extra 84 bp sequence and
checked their expression using RNAseq
checked the presence of sense and antisense strand of 280 bp sequence using RTPCR and RISH
searched for the RBP which could bind to the
sense as well as anti-sense strand of 280 bp
and the extra 84 bp sequence
searched for the possible targets of mir-4951

Off-targets due to 280 bp

sequence

gene

transcript

status

wild type

Act5C-Gal4>RNAi

fs(1)h
fs(1)h
fs(1)h
CG5118
CG11382
CG17272
chinmo
Dak1
CG17650
Lam
rdx
rdx
rdx
CG32369
CG4793
Ect3
mnb
Pten
Msr-110
CG10420

NM_206647
NM_206646
NM_167144
NM_134737
NM_130539
NM_142660
NM_134766
NM_143199
NM_134777
NM_057268
NM_169566
NM_169565
NM_142068
NM_168232
NM_165139
NM_141885
NM_170659
NM_058075
NM_139716
NM_143099

OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK

0.014404
0.002736
0.000982
15.9829
0.761161
82.361
0.436316
82.9232
6.84314
21.5911
3.06753
0.150113
6.7461
0.776646
1.39011
4.09616
2.98397
1.01133
48.2248
5.97988

1.62576
8.00069
4.99418
11.9631
0.884521
21.0983
4.06407
38.4438
0
60.1367
20.8688
1.50598
0.071505
16.2752
0.857721
10.8157
10.9527
0
129.741
4.18111

FOLD
CHANGE
112.8718
2924.539
5087.676
0.748494
1.162068
0.256169
9.314511
0.463607
0
2.785254
6.803128
10.03231
0.010599
20.95575
0.617017
2.640449
3.670513
0
2.690338
0.699196

Off-targets due to extra 84 bp

sequence
gene

transcript

status

wild type

Act5C-Gal4>RNAi

FOLD
CHANGE

CG13295

NM_139764

OK

7.14147

3.19756

0.447745

Gug

NM_206303

OK

1.37E-05

4.28995

313758

Gug

NM_079249

OK

3.90E-05

9.02609

231605.1

Gug

NM_176300

OK

0.738119

Gug

NM_145106

OK

0.001386

2.62825

1895.942

CG42492

NM_132009

OK

0.028296

11.9332

421.7319

MEP-1

NM_167959

OK

2.1485

14.8874

6.929206

MEP-1

NM_139476

OK

0.480022

0.00179752

0.003745

MEP-1

NM_167958

OK

0.198195

4.4329

22.36636

MEP-1

NM_167960

OK

1.02188

14.4169

14.10821

CG8414

NM_137240

OK

0.97

3.51638

3.625134

CG15247

NM_132356

OK

0.830694

0.871668

1.049325

CG31635

NM_164696

OK

3.52E-05

0.726835

20649.43

Result
no clear pattern in the expression
level of the predicted off-targets was
observed
280 bp as well as extra 84 bp
sequence may not have off-target

Sense strand exists in Act5CGal4>RNAi2.1 ; hsr66/ hsr66

Anti-sense strand exists in Act5CGal4>RNAi2.1 ; hsr66/ hsr66

RT-PCR to check the presence of both the strands

in Act5C-Gal4>RNAi2.1 ; hsr66/ hsr66

Result
Both sense and anti-sense strand of
280 bp accumulate in Act5CGal4>RNAi2.1 ; hsr66/ hsr66 3rd
instar larvae
These strand might be binding and
sequestering some of the RBP
leading to the observed phenotype

RBP predicted to bind the sense

strand of 280 bp sequence
Rbp9
fne

RNA-binding protein 9
found in neurons

oogenesis, germ cell proliferation

regulation of RNA metabolic process
compound eye morphogenesis, female gonad
development, mitotic nuclear division, neurogenesis,
heterogeneous nuclear
Hrb87F
ribonucleoprotein at 87F
regulation of alternative mRNA splicing via spliceosome,
response to heat, response to starvation
compound eye morphogenesis, female gonad
development, mitotic nuclear division, neurogenesis,
regulation of alternative mRNA splicing via spliceosome,
heterogeneous nuclear
Hrb98DE
ribonucleoprotein at 98DE
response to heat, response to starvation, negative
regulation of RNA splicing, oogenesis, positive regulation of
translation
spliceosomal protein on the mitotic spindle organization, neurogenesis, mRNA splicing,
Spx
X
via spliceosome
synaptic growth at neuromuscular junction, transcription
caz
cabeza
initiation from RNA polymerase II promoter
Eukaryotic initiation factor
eIF-4B
translational initiation
4B
mRNA splice site selection, mitotic G2 DNA damage
SF2
SF2
checkpoint
qkr54B quaking related 54B
RNA binding
qkr58E-1 quaking related 58E-1
mRNA splicing, via spliceosome, neurogenesis
qkr58E-2 quaking related 58E-2
RNA binding

RBP predicted to bind the antisense strand of 280 bp sequence

pAbp
pum

pumilio

mbl

positive regulation of translation, negative

regulation of neuron death, regulation of
compound eye photoreceptor development
translation repressor activity, regulation of
synaptic transmission, regulation of synaptic
growth at neuromuscular junction
regulation of alternative mRNA splicing via
spliceosome, regulation of gene expression,
rhabdomere development, apoptotic process,
eye development

eIF-4B

Eukaryotic initiation
factor 4B

SF2

SF2

Rbp9
fne
qkr54B
qkr58E1
qkr58E2

RNA-binding protein 9
found in neurons
quaking related 54B

mRNA splice site selection, mitotic G2 DNA

damage checkpoint
oogenesis, germ cell proliferation
regulation of RNA metabolic process
RNA binding

quaking related 58E-1

mRNA splicing, via spliceosome, neurogenesis

quaking related 58E-2

RNA binding

translational initiation

RBP predicted to bind the extra 84

bp sequence
CG32062

Ataxin-2 binding protein 1

nervous system development,

positive regulation of
transcription

YT521-B

YT521-B

RNA metabolic process

SF2

SF2

mRNA splice site selection,

mitotic G2 DNA damage
checkpoint

YT521-B

YT521-B

RNA metabolic process

Result
Apart from sense strand, anti-sense
strand of 280 bp and both the
strands of 84 bp extra sequence
have potential to bind with RBP
They might sequester some of these
RBP leading to the observed
phenotype

Transcripts of hsr

Target
4951

prediction

of

mir-

Targets of miR-4951 3p if the seed sequence lies at 5' end of

micro-RNA
gene

transcript

status

wild type Act5C-Gal4>RNAi FOLD CHANGE

CG12581 NM_141178

OK

8.50242

19.4441

2.286890085

CG12581 NM_164313

OK

0.000384

5.60839

14607.19839

CG12054 NM_143596

OK

1.65736

14.5307

8.767377033

CG31869 NM_001144343 OK

0.971586

4.64697

4.782870482

CG31869 NM_001144342 NOTEST

0.000209

0.000414704

1.98782487

CG31869 NM_164944

0.773159

4.71281

6.095524983

OK

crb

NM_001043286 NOTEST

#DIV/0!

crb

NM_001275980 NOTEST

#DIV/0!

crb

NM_079756

OK

0.167212

15.6817

93.78334091

crb

NM_001260355 OK

0.311191

15.9943

51.39705197

22.0471

43.1973

1.959318913

Pmm45A NM_136609

OK

Targets of miR-4951 3p if the seed sequence lies at 3' end of

micro-RNA
gene

transcript

status

wild type Act5C-Gal4>RNAi FOLD CHANGE

spn-F

NM_143601

OK

24.1558

13.1456 0.544200565

CCHa1

NM_00110431
OK
4

2.86745

1.88069 0.655875429

Result
mir-4951 3p with its seed sequence
lying at its 5 end is the mature micro
rna of mir-4951

Future plans
RISH with sense and anti-sense
probe of very high efficiency
cloning 280 bp in pUAST vector in
both the orientation
RNA pull down with both the strands
of 280 bp sequence

Thank You