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Seventh Progress Report Sept. 2014-Feb. 2015
Seventh Progress Report Sept. 2014-Feb. 2015
Supervisor
Prof. S. C. Lakhotia
Cytogenetics Lab.
Department of Zoology
B.H.U, Varanasi
Presented by
Mansoor Mustafa
Enrolment No. : 342034
Previous observation
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
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
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
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
RNA-binding protein 9
found in neurons
pumilio
mbl
eIF-4B
Eukaryotic initiation
factor 4B
SF2
SF2
Rbp9
fne
qkr54B
qkr58E1
qkr58E2
RNA-binding protein 9
found in neurons
quaking related 54B
RNA binding
translational initiation
YT521-B
YT521-B
SF2
SF2
YT521-B
YT521-B
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-
transcript
status
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
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
transcript
status
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