Processes 2018, 6,42
an expression analysis using our small RNA-Seq reads via BLAST+ [39]. Reads were aligned to known
miRNAs, limited to only the highest scoring alignment per read, and required to be 100% identical to
annotated miRNAs. Using these criteria, we identified 20 miRNAs for further evaluation based on
their relative high expressions (>50 reads per million) in both MCF-7 and MDA-MB-231 (Table 1).
Table 1. List of top 10 miRs where ADAR editing of mRNAs alters complementarity to miR seed
regions and either (A) creates novel target sites for regulation or (B) destroys predicted target sites.
In addition to altered edit complementarity, microRNAs included were also required to be present at
>50 reads per million in MCF-7 and MDA-MB-231 small RNA-Seq datasets.
miR
miRBase ID
Seed (RC)
Targets
(Edited)
Targets
(Unedited)
Expected
A
hsa-miR-513a-5p
MIMAT0002877
CCTGTGA
258
0
0.63
hsa-miR-450b-3p
MIMAT0004910
GATCCCA
252
4
0.79
hsa-miR-769-3p
MIMAT0003887
GATCCCA
252
4
0.79
hsa-miR-6089
MIMAT0023714
CGGCCTC
219
0
3.83
hsa-miR-4691-3p
MIMAT0019782
GTGGCTG
181
0
1.16
hsa-miR-3189-3p
MIMAT0015071
CCCAAGG
140
5
0.48
hsa-miR-140-3p
MIMAT0004597
CTGTGGT
139
0
1.11
hsa-miR-3065-3p
MIMAT0015378
GGTGCTG
118
0
0.5
hsa-miR-3940-3p
MIMAT0018356
CCGGGCT
111
0
0.72
hsa-miR-3680-3p
MIMAT0018107
ATGCAAA
108
2
0.82
B
hsa-miR-5089-5p
MIMAT0021081
AATCCCA
0
644
21.39
hsa-miR-6504-3p
MIMAT0025465
CTGTAAT
58
587
19.93
hsa-miR-6506-5p
MIMAT0025468
ATCCCAG
18
377
21.57
hsa-miR-619-5p
MIMAT0026622
ATCCCAG
18
377
21.57
hsa-miR-4775
MIMAT0019931
AAAATTA
0
351
19.37
hsa-miR-4735-5p
MIMAT0019860
AAATTAG
6
305
17.31
hsa-miR-6514-3p
MIMAT0025485
ACAGGCA
10
216
9.59
hsa-miR-4794
MIMAT0019967
TAGCCAG
10
173
8.05
hsa-miR-664a-5p
MIMAT0005948
TAGCCAG
10
173
8.05
hsa-miR-1273e
MIMAT0018079
TCAAGCA
2
169
5.22
3.3. MiR-140 Is Able to Target DFFA in MCF-7 but not MDA-MB-231
Next, after a thorough examination of the subset of miRNAs whose set of predicted target mRNAs
were significantly affected by deamination in our cell lines, we selected miR-140-3p for a detailed
experimental examination. Importantly, we found miR-140-3p was highly expressed in both cell lines
and, notably, its set of target mRNAs was found to be significantly altered by RNA editing in MCF-7
cells, but not in the MDA_MB-231 cells. Importantly, we found A-to-G mutations caused dramatic
changes to miR-140 ′ s set of predicted mRNA target sites in MCF-7s, with deamination events leading
to the creation of 91 new putative target sites in 34 mRNAs. Of note, through utilizing strategies
we previously employed to successfully identify sites created in a publicly-available set of >12,000
A-to-I human edit sites [37,41] (Figure 4), we identified a particularly interesting target site created
for miR-140-3p in MCF-7 cells—DNA fragmentation factor alpha (DFFA), also known as inhibitor
of caspase-activated DNase (ICAD) (Figure 5). As the principle function of DFFA is to trigger DNA
fragmentation during apoptosis, we hypothesized that the miRNA-mediated downregulation of this
gene specifically in MCF-7 cells might directly contribute to their characteristically lower rate of cellular
proliferation as compared to MDA-MB-231s.
136
an expression analysis using our small RNA-Seq reads via BLAST+ [39]. Reads were aligned to known
miRNAs, limited to only the highest scoring alignment per read, and required to be 100% identical to
annotated miRNAs. Using these criteria, we identified 20 miRNAs for further evaluation based on
their relative high expressions (>50 reads per million) in both MCF-7 and MDA-MB-231 (Table 1).
Table 1. List of top 10 miRs where ADAR editing of mRNAs alters complementarity to miR seed
regions and either (A) creates novel target sites for regulation or (B) destroys predicted target sites.
In addition to altered edit complementarity, microRNAs included were also required to be present at
>50 reads per million in MCF-7 and MDA-MB-231 small RNA-Seq datasets.
miR
miRBase ID
Seed (RC)
Targets
(Edited)
Targets
(Unedited)
Expected
A
hsa-miR-513a-5p
MIMAT0002877
CCTGTGA
258
0
0.63
hsa-miR-450b-3p
MIMAT0004910
GATCCCA
252
4
0.79
hsa-miR-769-3p
MIMAT0003887
GATCCCA
252
4
0.79
hsa-miR-6089
MIMAT0023714
CGGCCTC
219
0
3.83
hsa-miR-4691-3p
MIMAT0019782
GTGGCTG
181
0
1.16
hsa-miR-3189-3p
MIMAT0015071
CCCAAGG
140
5
0.48
hsa-miR-140-3p
MIMAT0004597
CTGTGGT
139
0
1.11
hsa-miR-3065-3p
MIMAT0015378
GGTGCTG
118
0
0.5
hsa-miR-3940-3p
MIMAT0018356
CCGGGCT
111
0
0.72
hsa-miR-3680-3p
MIMAT0018107
ATGCAAA
108
2
0.82
B
hsa-miR-5089-5p
MIMAT0021081
AATCCCA
0
644
21.39
hsa-miR-6504-3p
MIMAT0025465
CTGTAAT
58
587
19.93
hsa-miR-6506-5p
MIMAT0025468
ATCCCAG
18
377
21.57
hsa-miR-619-5p
MIMAT0026622
ATCCCAG
18
377
21.57
hsa-miR-4775
MIMAT0019931
AAAATTA
0
351
19.37
hsa-miR-4735-5p
MIMAT0019860
AAATTAG
6
305
17.31
hsa-miR-6514-3p
MIMAT0025485
ACAGGCA
10
216
9.59
hsa-miR-4794
MIMAT0019967
TAGCCAG
10
173
8.05
hsa-miR-664a-5p
MIMAT0005948
TAGCCAG
10
173
8.05
hsa-miR-1273e
MIMAT0018079
TCAAGCA
2
169
5.22
3.3. MiR-140 Is Able to Target DFFA in MCF-7 but not MDA-MB-231
Next, after a thorough examination of the subset of miRNAs whose set of predicted target mRNAs
were significantly affected by deamination in our cell lines, we selected miR-140-3p for a detailed
experimental examination. Importantly, we found miR-140-3p was highly expressed in both cell lines
and, notably, its set of target mRNAs was found to be significantly altered by RNA editing in MCF-7
cells, but not in the MDA_MB-231 cells. Importantly, we found A-to-G mutations caused dramatic
changes to miR-140 ′ s set of predicted mRNA target sites in MCF-7s, with deamination events leading
to the creation of 91 new putative target sites in 34 mRNAs. Of note, through utilizing strategies
we previously employed to successfully identify sites created in a publicly-available set of >12,000
A-to-I human edit sites [37,41] (Figure 4), we identified a particularly interesting target site created
for miR-140-3p in MCF-7 cells—DNA fragmentation factor alpha (DFFA), also known as inhibitor
of caspase-activated DNase (ICAD) (Figure 5). As the principle function of DFFA is to trigger DNA
fragmentation during apoptosis, we hypothesized that the miRNA-mediated downregulation of this
gene specifically in MCF-7 cells might directly contribute to their characteristically lower rate of cellular
proliferation as compared to MDA-MB-231s.
136