Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, USA
Department of Microbiology, North Carolina State University, Raleigh, NC 27695, USA
Department of Pathology and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA
Abstract
Background
MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the
Results
We identified a total of 65 miRNAs in the
Conclusions
Background
Both
Of the over 3,000 mosquito species worldwide, microRNAs (miRNAs) have so far only been described in two species of African malaria mosquitoes,
miRNAs are a class of small, noncoding RNAs, from 1924 nt in length, that posttranscriptionally regulate gene expression by binding to complementary regions in, primarily, the 3' untranslated region (3' UTR) of target messenger RNAs. First identified in
Much of what we understand about insect miRNAs comes from studies in the fruit fly
Differential miRNA expression throughout the various stages of the
The process of miRNA biogenesis is conserved, initiating with the cleavage of long, endogenous nuclear primary miRNA transcripts, ranging from hundreds to thousands of nucleotides in length, into premiRNAs
Recent advances in mosquito genomics, such as the sequencing of the genomes of three mosquito species,
Results and Discussion
Deep sequencing of small RNAs
To identify miRNAs in
Deep sequencing of small RNA populations in
Deep sequencing of small RNA populations in
Most mosquito miRNAs are orthologs of known insect miRNAs
We aligned sequencing reads to known miRNAs and miRNA* strands present in miRBase v14. 1,541,048 reads from the
miRNAs identified in
C710
# miRNA
# miRNA*
Sequence
Length
aga
ame
dme
Ae. aegypti
Start
End
Strand
184
1487481
0
UGGACGGAGAACUGAUAAGGGC
22
Y
Y
Y
1.496
143378
143399
Minus
275
23841
78
UCAGGUACCUGAAGUAGCGC
20
Y
Y
Y
1.24
486591
486610
Plus
277
4453
7
UAAAUGCACUAUCUGGUACGAC
22
Y
Y
Y
1.265
508860
508881
Plus
9
4085
602
UCUUUGGUAUUCUAGCUGUAGA
22
Y
Y
Y
1.785
186231
186252
Plus
83p
3002

UAAUACUGUCAGGUAAAGAUGUC
23
Y
Y
Y
1.411
876091
876113
Plus
252.1
1608
13
UAAGUACUAGUGCCGCAGGAG
21
Y
Y
Y
1.56
1580060
1580080
Minus
bantam5p
1384

CCGGUUUUCAUUUUCGAUCUGAC
23
Y
Y
Y
1.49
157893
157915
Minus
71
1246
17
AGAAAGACAUGGGUAGUGAGAU
22
?
Y
?
1.268
889428
889449
Minus
85p
1244

CAUCUUACCGGGCAGCAUUAGA
22
Y
Y
Y
1.411
876052
876073
Plus
2761
1209
4
UAGGAACUUCAUACCGUGCUC
21
Y
Y
Y
1.5
2769510
2769530
Minus
2762


UAGGAACUUCAUACCGUGCUC
21
Y
Y
Y
1.134
39026
39046
Plus
3171
1118
0
UGAACACAGCUGGUGGUAUCU
21
Y
Y
Y
1.265
429503
429523
Plus
3172


UGAACACAGCUGGUGGUAUCU
21
Y
Y
Y
1.153
2154717
2154737
Minus
283
947
0
CAAUAUCAGCUGGUAAUUCUGGGC
24
Y
Y
Y
1.68
2729393
2729416
Minus
252.2
888

CUAAGUACUAGUGCCGCAGGAG
22
Y
Y
Y
1.56
1580060
1580081
Minus
let7
650
0
UGAGGUAGUUGGUUGUAUAGU
21
Y
Y
Y
1.43
1156331
1156351
Plus
2
708
1
UAUCACAGCCAGCUUUGAAGAGC
23
Y
Y
Y
1.268
888597
888619
Minus
998
561
0
UAGCACCAUGAGAUUCAGC
19
?
?
Y
1.744
322338
322356
Plus
92b
530
0
AAUUGCACUUGUCCCGGCCUG
21
Y
Y
Y
1.116
1319201
1319221
Plus
18893p
478

CACGUUACAGAUUGGGGUUUCC
22
Y
?
?
1.68
2720796
2720817
Minus
bantam3p
475

UGAGAUCAUUUUGAAAGCUGAU
22
Y
Y
Y
1.49
1579555
1579576
Minus
306
454
81
UCAGGUACUGAGUGACUCUCAG
22
Y
?
Y
1.785
213078
213099
Plus
281
398
1
AAGAGAGCUAUCCGUCGAC
19
Y
Y
Y
1.957
134462
134480
Plus
18895p
378

UAAUCUCAAAUUGUAACAGUGG
22
Y
?
?
1.68
2720896
2720917
Minus
980
309
6
UAGCUGCCUAGUGAAGGGC
19
?
?
Y
1.23
1043069
1043087
Plus
278
286
53
ACGGACGAUAGUCUUCAGCGGCC
23
Y
Y
Y
1.16
3596269
3596291
Plus
989
242
0
UGUGAUGUGACGUAGUGGUAC
21
Y
?
Y
1.115
804187
804207
Minus
14
247
0
UCAGUCUUUUUCUCUCUCCUAU
22
Y
Y
Y
1.249
1089019
1089040
Minus
11
222
31
CAUCACAGUCUGAGUUCUUGCU
22
Y
?
Y
1.744
322062
322083
Plus
190
210
0
AGAUAUGUUUGAUAUUCUUGGUUG
24
Y
Y
Y
1.195
82254
82277
Minus
1
169
0
UGGAAUGUAAAGAAGUAUGGAG
22
Y
Y
Y
1.812
291373
291394
Plus
34
147
1
UGGCAGUGUGGUUAGCUGGUUG
22
Y
?
Y
1.265
509536
509557
Plus
1890
123
0
UGAAAUCUUUGAUUAGGUCUGG
22
Y
?
?
1.204
1733356
1733377
Plus
988
118
24
CCCCUUGUUGCAAACCUCACGC
22
Y
?
Y
1.442
623057
623078
Minus
957
99
0
UGAAACCGUCCAAAACUGAGGC
22
Y
?
Y
1.7
464339
464360
Plus
305
96
4
AUUGUACUUCAUCAGGUGCUCUGG
24
Y
Y
Y
1.24
495601
495624
Plus
996
88
0
UGACUAGAUUACAUGCUCGUC
21
Y
Y
Y
1.437
567507
567527
Minus
87
79
0
GUGAGCAAAUUUUCAGGUGUGU
22
Y
Y
Y
1.36
1010846
1010867
Plus
12
68
3
UGAGUAUUACAUCAGGUACUGGU
23
Y
Y
Y
1.68
2720468
2720490
Minus
13
57
1
UAUCACAGCCAUUUUGACGAGUU
23
Y
Y
Y
1.268
888744
888766
Minus
92a
44
0
UAUUGCACUUGUCCCGGCC
19
Y
Y
Y
1.116
1267254
1267272
Plus
33
40
0
GUGCAUUGUAGUUGCAUUGCA
21
?
Y
Y
1.487
351656
351676
Plus
279
36
2
UGACUAGAUCCACACUCAUUAA
22
Y
Y
Y
1.437
572258
572279
Minus
79
33
6
GCUUUGGCGCUUUAGCUGUAUGA
23
Y
Y
Y
1.785
213277
213299
Plus
263
32
0
AAUGGCACUGGAAGAAUUCACGG
23
Y
Y
Y
1.981
164999
165021
Minus
7
32
0
UGGAAGACUAGUGAUUUUGUUGU
23
Y
Y
Y
1.1359
46041
46063
Plus
**2945
32
0
UGACUAGAGGCAGACUCGUUUA
22
Y
?
?
1.43
481083
481105
Plus
1005p
30

AACCCGUAGAUCCGAACUUGUG
22
Y
Y
Y
1.43
1142184
1142205
Plus
1003p
25

CAAGAACGGAUGUAUGGGAUUC
22
Y
Y
Y
1.43
1142224
1142245
Plus
970
21
0
UCAUAAGACACACGCGGCUAU
21
Y
?
Y
1.229
1045875
1045895
Plus
210.1
13
0
CUUGUGCGUGUGACAACGG
19
Y
Y
Y
1.512
515650
515668
Plus
999
14
0
UGUUAACUGUAAGACUGUGUCU
22
?
?
Y
1.100.
2315145
2315166
Plus
308
16
4
CGCGGUAUAUUCUUGUGGCUUG
22
Y
?
Y
1.107
508980
509001
Plus
125
7
3
UCCCUGAGACCCUAACUUGUGA
22
Y
Y
Y
1.43
1156615
1156636
Plus
210.2
6
0
UUGUGCGUGUGACAACGGCUAU
22
Y
Y
Y
1.512
515646
515667
Plus
307
6
0
CACAACCUCCUUGAGUGAGCGA
22
Y
?
Y
1.16
1859430
1859451
Minus
10001
6
0
AUAUUGUCCUGUCACAGCAGU
21
Y
Y
Y
1.187
325478
325498
Plus
10002


AUAUUGUCCUGUCACAGCAGU
21
Y
Y
Y
1.3798
224
244
Minus
375
4
0
UUUGUUCGUUUGGCUCGAGUUA
22
Y
Y
Y
1.309
1318752
1318773
Minus
3091
4

UCACUGGGCAAAGUUUGUCGCA
22
Y
?
Y
1.15
907938
907959
Plus
3092


UCACUGGGCAAAGUUUGUCGCA
22
Y
?
Y
1.602
94088
94109
Plus
932
3

UGCAAGCAAUGUGGAAGUGAAG
22
?
Y
Y
1.1064
154192
154213
Minus
315
2
0
UUUUGAUUGUUGCUCAGAAAGC
22
Y
Y
Y
1.612
104143
104164
Plus
927
1
0
CAAAGCGUUUGGAUUCUGAAAC
22
Y
Y
Y
1.26
2065461
2065482
Plus
**2943
1
0
UUAAGUAGGCACUUGCAGGC
20
Y
?
?
1.348
212450
212470
Plus
18911
predicted
UGAGGAGUUAAUUUGCGUGUUU
22
Y
?
?
1.199
1109750
1109771
Minus
18912
predicted
UGAGGAGUUAAUUUGCGUGUUU
22
Y
?
?
1.466
72802
72823
Plus
1175
predicted
AAGUGGAGUAGUGGUCUCAUCG
22
Y
?
?
1.125
1648037
1648058
Plus
1174
predicted
UCAGAUCUACUUAAUACCCAU
21
Y
?
?
1.125
1647921
1647941
Plus
993
predicted
UACCCUGUAGUUCCGGGCUUUU
22
Y
Y
Y
1.056
256798
256819
Plus
981
predicted
UUCGUUGUCGACGAAACCUGCA
22
Y
Y
Y
1.127
638380
638401
Minus
965
predicted
UAAGCGUAUAGCUUUUCCCAUU
22
Y
?
Y
1.51
2008701
2008722
Plus
316
predicted
UGUCUUUUUCCGCUUACUGCCG
22
?
Y
Y
1.289
891327
891348
Minus
285
predicted
UAGCACCAUUCGAAAUCAGUAC
22
?
?
Y
1.26
3339153
3339174
Minus
1371
predicted
UAUUGCUUGAGAAUACACGUAG
22
Y
Y
Y
1.1191
97844
97865
Plus
1372
predicted
UAUUGCUUGAGAAUACACGUAG
22
Y
Y
Y
1.137
260684
260705
Minus
133
predicted
UUGGUCCCCUUCAACCAGCUGU
22
Y
Y
Y
1.778
350306
350327
Minus
124
predicted
UAAGGCACGCGGUGAAUGC
19
Y
Y
Y
1.6
1664623
1664641
Minus
31
predicted
UGGCAAGAUGUUGGCAUAGCUGA
23
?
Y
Y
1.636
483257
483279
Minus
10
predicted
ACCCUGUAGAUCCGAAUUUGUU
22
Y
Y
Y
1.44
813557
813578
Plus
iab41
predicted
ACGUAUACUGAAUGUAUCCUGA
22
Y
Y
Y
1.708
50265
50286
Plus
iab42
predicted
ACGUAUACUGAAUGUAUCCUGA
22
Y
Y
Y
1.423
763526
763547
Minus
2940
125253
4125
UGGUUUAUCUUAUCUGUCGAGGC
23
?
?
?
1.222
643960
643982
Plus
2765
1162
0
UGGUAACUCCACCACCGUUGGC
22
Y
?
?
1.11
5248310
5248331
Plus
2951
1136
28
AAGAGCUCAGUACGCAGGGG
20
?
?
?
multiple
29411
9
0
UAGUACGGCUAGAACUCCACGG
22
?
?
?
1.385
413147
413168
Minus
29412


UAGUACGGCUAGAACUCCACGG
22
?
?
?
1.385
413451
413472
Minus
aga =
Top group: miRNAs and miRNA* strands identified by deep sequencing
Middle group: predicted miRNAs identified in
Bottom group: novel miRNAs identified in this study. miR2765 (not present in miRBase v14) has recently been identified in
** novel miRNAs (not present in miRBase v14) recently identified in
miRNAs identified in
Culex
# miRNA
# miRNA*
Sequence
Length
aga
ame
dme
supercontig
Start
End
Strand
184
107190
0
UGGACGGAGAACUGAUAAGGGC
22
Y
Y
Y
3.567
240312
240333
Minus
3171
71313
2
UGAACACAGCUGGUGGUAUCU
21
Y
Y
Y
3.36
1133209
1133229
Plus
3172


UGAACACAGCUGGUGGUAUCU
21
Y
Y
Y
3.36
1134875
1134895
Plus
277
58628
0
UAAAUGCACUAUCUGGUACGAC
22
Y
Y
Y
3.36
1153785
1153806
Plus
1
36084
0
UGGAAUGUAAAGAAGUAUGGAG
22
Y
Y
Y
3.78
246250
246271
Plus
989
23667
0
UGUGAUGUGACGUAGUGGUAC
21
Y
?
Y
3.315
321364
321384
Plus
275
13910
2
UCAGGUACCUGAAGUAGCGC
20
Y
Y
Y
3.291
329815
329834
Plus
957
11682
0
UGAAACCGUCCAAAACUGAGGC
22
Y
?
Y
3.787
29593
29614
Plus
83p
10950

UAAUACUGUCAGGUAAAGAUGU
22
Y
Y
Y
3.40
815865
815886
Minus
281
9322
95
AAGAGAGCUAUCCGUCGACAGU
22
Y
Y
Y
3.64
99744
99765
Plus
Let7
9266
5
UGAGGUAGUUGGUUGUAUAGU
21
Y
Y
Y
3.4
280610
280630
Plus
34
6301
3
UGGCAGUGUGGUUAGCUGGUU
21
Y
Y
Y
3.36
1154478
1154498
Plus
263
3749
2
AAUGGCACUGGAAGAAUUCACGG
23
Y
Y
Y
3.219
351848
351870
Minus
2521
3157
2
(C)UAAGUACUAGUGCCGCAGGAG
21
Y
Y
Y
3.1787
6836
6856
Minus
2522


(C)UAAGUACUAGUGCCGCAGGAG
21
Y
Y
Y
3.975
115594
115614
Plus
87
2364
0
GUGAGCAAAUUUUCAGGUGUGU
22
Y
Y
Y
3.431
379788
379809
Plus
71
2232
14
AGAAAGACAUGGGUAGUGAGAU
22
?
Y
?
3.366
117552
117573
Minus
bantam5p
1459

CCGGUUUUCAUUUUCGAUCUGAC
21
Y
Y
Y
3.65
199737
199759
Minus
9
1138
440
UCUUUGGUAUUCUAGCUGUAGA
22
Y
Y
Y
3.83
64733
64754
Plus
11
888
5
CAUCACAGUCUGAGUUCUUGCU
22
Y
?
Y
3.153
639669
639690
Minus
2761
860
2
UAGGAACUUCAUACCGUGCUCU
22
Y
Y
Y
3.136
340911
340932
Plus
2762


UAGGAACUUCAUACCGUGCUCU
22
Y
Y
Y
3.136
541192
541213
Plus
2763


UAGGAACUUCAUACCGUGCUCU
22
Y
Y
Y
3.2457
930
951
Plus
210.1
720
5
UUGUGCGUGUGACAACGGCUAU
22
Y
Y
Y
3.549
157657
157678
Minus
927
703
21
CAAAGCGUUUGGAUUCUGAAAC
22
Y
Y
Y
3.11
560282
560302
Plus
bantam3p
689

UGAGAUCAUUUUGAAAGCUGA
21
Y
Y
Y
3.65
199698
199718
Minus
85p
594

CAUCUUACCGGGCAGCAUUAGA
22
Y
Y
Y
3.40
815904
815925
Minus
2
547
2
UAUCACAGCCAGCUUUGAAGAGC
23
Y
Y
Y
3.366
116861
116883
Minus
998
434
0
UAGCACCAUGAGAUUCAGC
19
?
?
Y
3.153
639527
639545
Minus
210.2
405

CUUGUGCGUGUGACAACGGCUAU
23
Y
Y
Y
3.549
157657
157679
Minus
14
358
0
UCAGUCUUUUUCUCUCUCCUAU
22
Y
Y
Y
3.676
52251
52272
Minus
285
324
5
UAGCACCAUUCGAAAUCAGUAC
22
?
?
Y
3.98
262290
262311
Minus
1890
287
0
UGAAAUCUUUGAUUAGGUCUGG
22
Y
?
?
3.64
982786
982807
Minus
1901
231
0
AGAUAUGUUUGAUAUUCUUGGUUG
24
Y
Y
Y
3.181
347953
347976
Minus
1902


AGAUAUGUUUGAUAUUCUUGGUUG
24
Y
Y
Y
3.351
105098
105121
Minus
283
224
0
CAAUAUCAGCUGGUAAUUCUGGG
23
Y
Y
Y
3.57
559462
559484
Plus
7
192
0
UGGAAGACUAGUGAUUUUGUUGU
23
Y
Y
Y
3.1
3357390
3357412
Minus
100
170
43
AACCCGUAGAUCCGAACUUGUG
22
Y
Y
Y
3.4
271414
271435
Plus
1891
167
1
UGAGGAGUUAAUUUGCGUGUUU
22
Y
?
?
3.829
180383
180404
Minus
999
165
0
UGUUAACUGUAAGACUGUGUCU
22
?
?
Y
3.14
96917
96938
Plus
309
33
1
UCACUGGGCAUAGUUUGUCGCAU
23
Y
?
Y
3.145
66041
66063
Minus
375
144
0
UUUGUUCGUUUGGCUCGAGUUAC
23
Y
Y
Y
3.455
42584
42605
Plus
306
143
65
UCAGGUACUGAGUGACUCUCAG
22
Y
?
Y
3.83
80436
80457
Plus
125
140
7
UCCCUGAGACCCUAACUUGUGA
22
Y
Y
Y
3.4
280975
280996
Plus
315
131
0
UUUUGAUUGUUGCUCAGAAAGC
22
Y
Y
Y
3.438
61926
61947
Plus
124
105
0
UAAGGCACGCGGUGAAUGC
19
Y
Y
Y
3.8
2074772
2074790
Plus
92b
96
0
AAUUGCACUUGUCCCGGCCUG
21
Y
Y
Y
3.722
164913
164933
Minus
18895p
89

UAAUCUCAAAUUGUAACAGUGG
22
Y
?
?
3.57
562555
562576
Plus
9811
82
0
UUCGUUGUCGACGAAACCUGCA
22
Y
Y
Y
3.431
144482
144503
Plus
9812


UUCGUUGUCGACGAAACCUGCA
22
Y
Y
Y
3.431
151371
151392
Plus
12
80
2
UGAGUAUUACAUCAGGUACUGGU
23
Y
Y
Y
3.57
563009
563031
Plus
31
76
2
UGGCAAGAUGUUGGCAUAGCUGA
23
?
Y
Y
3.559
256577
256599
Minus
10
59
40
CAAAUUCGGUUCUAGAGAGGUUU
23
Y
Y
Y
3.12
96000
96022
Minus
1174
58
0
CUGGGUAUUUUAGAUCAUCGGC
22
Y
?
?
3.86
865901
865922
Plus
**2945
52
0
UGACUAGAGGCAGACUCGUUU
20
Y
?
?
3.4
184461
184481
Plus
1000
49
0
AUAUUGUCCUGUCACAGCAGU
21
Y
Y
Y
3.153
102853
102873
Minus
13
37
3
UAUCACAGCCAUUUUGACGAGU
22
Y
Y
Y
3.366
116994
117015
Minus
996
36
2
UGACUAGAUUACAUGCUCGU
20
Y
Y
Y
3.19
1437010
1437029
Minus
137
33
0
UAUUGCUUGAGAAUACACGUAG
22
Y
Y
Y
3.1714
27566
27587
Minus
133
32
0
UUGGUCCCCUUCAACCAGCUGU
22
Y
Y
Y
3.1189
55748
55769
Plus
1175
35
7
AAGUGGAGUAGUGGUCUCAUCG
22
Y
?
?
3.86
866116
866137
Plus
279
26
21
UGACUAGAUCCACACUCAUUAA
22
Y
Y
Y
3.19
1441123
1441144
Minus
92a
24

UAUUGCACUUGUCCCGGCCUAU
22
Y
Y
Y
3.722
174912
174933
Minus
9323p
22

UGCAAGCAAUGUGGAAGUGA
22
?
Y
Y
3.261
301413
301432
Minus
970
20
0
UCAUAAGACACACGCGGCUAU
21
Y
?
Y
3.495
35970
35990
Plus
316
18
0
UGUCUUUUUCCGCUUACUGCCG
22
?
Y
Y
3.496
152508
152529
Minus
305
17
1
AUUGUACUUCAUCAGGUGCUCU
22
Y
Y
Y
3.291
339134
339155
Plus
**2944a1
13
1
GAAGGAACUUCUGCUGUGAUC
21
Y
?
?
3.66
328681
328701
Minus
**2944a2


GAAGGAACUUCUGCUGUGAUC
21
Y
?
?
3.145
66240
66260
Minus
988
11
5
CCCUUGUUGCAAACCUCACGC
21
Y
?
Y
3.791
14331
14351
Plus
9325p
11

UCAAUUCCGUAGUGCAUUGCAG
22
?
Y
Y
3.261
301450
301471
Minus
18893p
7

CACGUUACAGAUUGGGGUUUCC
22
Y
?
?
3.57
562642
562663
Plus
993
4
1
UACCCUGUAGUUCCGGGCUUUU
22
Y
Y
Y
3.12
55487
55508
Plus
278
3
0
UCGGUGGGACUUUCGUCCGUUU
22
Y
Y
Y
3.16
1026212
1026233
Plus
965
2
0
UAAGCGUAUAGCUUUUCCCAUU
22
Y
?
Y
3.48
484177
484198
Plus
Iab4
2
1
ACGUAUACUGAAUGUAUCCUGA
22
Y
Y
Y
3.12
681163
681184
Plus
980
2
0
UAGCUGCCUAGUGAAGGGC
19
?
?
Y
3.263
352922
352940
Plus
308
3
1
CGCAGUAUAUUCUUGUGGCUUG
22
Y
?
Y
3.98
764133
764154
Plus
79
2
0
GCUUUGGCGCUUUAGCUGUAUGA
23
Y
Y
Y
3.83
80591
80613
Plus
**2943
1
0
UAAGUAGGCACUUGCAGGCAAAG
23
Y
?
?
3.121
94164
94186
Minus
**2944b1
1
0
GAAGGAACUCCCGGUGUGAUAU
22
Y
?
?
3.66
328838
328859
Minus
**2944b2


GAAGGAACUCCCGGUGUGAUAU
22
Y
?
?
3.145
66389
66410
Minus
33
predicted
GUGCAUUGUAGUUGCAUUGCA
21
?
Y
Y
3.1258
69381
69401
Minus
2951
162309
342
AAGAGCUCAGCACGCAGGGGUGGC
24
?
?
?
multiple
2952
2203

UAGUACGGCCAUGACUGAGGGC
22
?
?
?
3.5
753922
753943
Minus
29411
1221
3
UAGUACGGCUAGAACUCCACGG
22
?
?
?
3.5
753643
753664
Minus
29412

1
UAGUACGGCUAGAACUCCACGG
22
?
?
?
3.5
753797
753818
Minus
aga =
Top group: miRNAs and miRNA* strands identified by deep sequencing
Middle group: predicted miR33 was identified in C7/10 cells but absent from
Bottom group: novel miRNAs identified in this study.
** novel miRNAs (not present in miRBase v14) recently identified in
miRNA expression levels, based on the number of reads obtained, varied greatly, spanning over five orders of magnitude for
Mature miRNA species showed sequence lengths between 19 and 24 nt with a predominance of 22 nt and also exhibited strong bias for a 5' uracil (> 65% of all identified miRNAs) (Tables
Mosquito miRNAs are highly conserved
The
Several miRNA sequences mapped to multiple locations in the
With the exception of miR33, all
64 of the 75 miRNAs identified in
Sequence variation occurs predominantly at the 3' end of mature miRNAs
In each small RNA library, reads aligning to a given mature miRNA showed some degree of variability. Most variability occurred at the 3'ends of each mature miRNA, when compared to the 5' ends. Figure
At least two miRNAs, however, did not match this trend. For both miR210 and miR252, two dominant miRNA species were identified (Figure
miR252, which maps to two loci within the
The consequences of 5' variation can be severe, since an alteration to the 5' seed creates a new group of potential target mRNAs for a miRNA
Whereas some miRNAs exhibited sequence differences at the 5' or 3' ends, we also noted differences in the ratios of miRNA:miRNA* reads when examining the
We investigated the predicted miR8 premiRNA structures in
Whilst the total number of miRNA* strands accounted for a low percentage (<0.3%) of mapped reads in each small RNA library, some miRNA* strands were sequenced more frequently than individual miRNA species. For example, in total RNA from C7/10 cells, bantam3p was sequenced 475 times, and therefore accounts for a greater percentage of the small RNA population than those mature miRNAs sequenced less than 400 times. Likewise, miR281* in
Confirmation of mosquito miRNAs
We used primer extension analysis to confirm the expression of several of the miRNAs represented in our sequencing data. Five miRNAs, miR184, miR275, miR277, miR276, and miR92, were sequenced >500 times and were readily detectable in total RNA isolated from C7/10 cells (Figure
Primer extension analysis confirms miRNA expression
Primer extension analysis confirms miRNA expression. Total RNA was isolated from A) C7/10 cells or C7/10WNV312 cells persistently infected with WNV replicons and B, C)
Identification of novel mosquito miRNAs
To identify novel mosquito miRNAs, we used a combination of miRDeep
Four new
Identification of novel mosquito miRNAs
Identification of novel mosquito miRNAs. Primer extension analysis was used to confirm the expression of A) three novel
Novel mosquito miRNAs are clustered
Novel mosquito miRNAs are clustered. A) Predicted premiRNA stemloop structures for cqumiR29411, cqumiR29412, and cqumiR2952. B) Primer extension analysis confirms miR2941 expression in total RNA isolated from
miR2940, which lacks seed homology to any known miRNA, was amongst the most frequently recovered miRNAs present in the
Two novel
Whilst the majority of new miRNAs exhibited discrete lengths, as determined from both sequencing data and primer extension analysis (Figure
We queried three mosquito genomes (
Two additional miRNAs, aaemiR2941 and cqumiR2941, are also orthologs conserved in
Clusters of mosquito miRNA genes
The miR2941 cluster represents a novel miRNA cluster present in both
Culex miR989 and miR92 expression levels are altered during flavivirus infection
miRNAs are known to be important regulators of development. Additionally, miRNA expression profiles can be altered in response to environmental changes such as stress or infection. Four
We assayed miRNA expression in WNVreplicon C7/10 cells and WNVNY99 infected
Figure S1, miRNA quantification in primer extension experiments shown in Figure
Click here for file
Table S1, miRNA reads in
Click here for file
The targets of miR989 and miR92 in mosquitoes are not yet known; however, several studies have examined expression of these miRNAs during development. In
Conclusions
This study provides experimental evidence for over 65 conserved and seven novel miRNAs present in
Methods
Mosquitoes and Cell Lines
RNA extraction and Primer Extensions
Total RNA was prepared from ~100 whole mosquitoes and two 80% confluent T75 flasks of
Table S2, Oligonucleotides used in this study. Table of oligonucleotides used for primer extension and highthroughput sequencing.
Click here for file
Small RNA cloning
Thirty micrograms of total RNA were sizefractionated on a 15% TBEUrea polyacrylamide gel. Small RNA populations corresponding to 1828 nt in size were extracted, eluted, and ligated to a 3' linker using T4 RNA ligase (Epicentre). 3' ligation reactions were loaded directly onto a 10% TBEUrea polyacrylamide gel, and ligation products recovered by highsalt elution following electrophoresis. Next, a 5' linker was ligated, and products were used for SSII reverse transcription (Invitrogen). PCR reactions were carried out using the RT primer and 5' PCR primer. Linker and primer sequences are provided in Additional file
Bioinformatics
Sequencing reads were parsed using inhouse scripts according to the following criteria: a 5' and 3' linker match of at least 4 nt and an appropriate length (1828 nt).
To find miRNA orthologs, sequences were mapped to known miRNAs, miRNA star strands, and hairpins present in miRBase v14.0
Raw sequence data C710.fasta. FASTA file containing sequencing reads for C7/10
Click here for file
Raw sequence data Culex.fasta. FASTA file containing sequencing reads for
Click here for file
miRNA precursor sequences Aedes_precursors.fasta. FASTA file containing miRNA, miRNA*, and precursor sequences for
Click here for file
miRNA precursor sequences Culex_precursors.fasta. FASTA file containing miRNA, miRNA*, and precursor sequences for
Click here for file
Authors' contributions
RLS prepared the small RNA libraries, analyzed the data, and drafted the manuscript. DLV and SH reared the mosquitoes and performed the WNV infections. FS provided the C7/10 cells and generated the C7/10WNV replicon cells. BRC supervised the experiments and helped draft the manuscript. All authors read and commented on the final manuscript.
Acknowledgements
This work was supported by NIH grant U54AI057157 from the Southeastern Regional Center of Excellence for Emerging Infections and Biodefense to BRC, and by funds provided by the UTMB Department of Pathology to SH. DLV was supported by an NIH T32 grant (A107536). We thank Tonya Severson of the Duke Institute for Genome Sciences and Policy Core Sequencing Facility for assistance with highthroughput sequencing. We gratefully acknowledge the technical assistance of Jing H. Huang for the rearing of the