Deep annotation of mouse iso-miR and iso-moR
Hongjun Zhou1, Mary Luz Arcila1, Zhonghan Li2, Eun Joo Lee1, Christine Henzler1,
Jingya Liu2, Tariq M. Rana2and Kenneth S. Kosik1,*
1Neuroscience Research Institute and Department of Cellular Molecular and Developmental Biology,
University of California, Santa Barbara, CA 93106 and2Program for RNA Biology, Sanford-Burnham
Medical Research Institute, La Jolla, CA 92037, USA
Received January 9, 2012; Revised March 2, 2012; Accepted March 5, 2012
With a dataset of more than 600 million small RNAs
deeply sequenced from mouse hippocampal and
reprogramming to induced pluripotent stem cells,
we annotated the stem–loop precursors of the
known miRNAs to identify isomoRs (miRNA-offset
RNAs), loops, non-preferred strands, and guide
strands. Products from both strands were readily
detectable for most miRNAs. Changes in the
dominant isomiR occurred among the cell types,
as did switches of the preferred strand. The
terminal nucleotide of the dominant isomiR aligned
well with the dominant off-set sequence suggesting
that Drosha cleavage generates most miRNA reads
without terminal modification. Among the terminal
modifications detected, most were non-templated
mono- or di-nucleotide additions to the 30-end.
Based on the relative enrichment or depletion of
specific nucleotide additions in an Ago-IP fraction
there may be differential effects of these modifica-
tions on RISC loading. Sequence variation of the two
strands at their cleavage sites suggested higher
fidelity of Drosha than Dicer. These studies demon-
strated multiple patterns of miRNA processing and
considerable versatility in miRNA target selection.
MicroRNAs (miRNAs) are ?21nt non-coding RNAs that
serve as key post-transcriptional regulators of gene expres-
sion in the specialized cells of multi-cellular organisms
(1–3). In animals, miRNAs are typically transcribed
from the genomes as primary (pri-miRNA) transcripts
by RNA polymerase II (4), and less often as mirtrons
from spliceosome-mediated processing (5,6). The pri-
miRNAs fold into hairpins and are cleaved at their base
into pre-miRNAs by the RNase III family member,
Drosha (7–9). Pre-miRNAs are ?70nt stem-loop struc-
tures, which are exported to the cytoplasm (10–12),
where another RNase III family member, Dicer, cleaves
the pre-miRNA loop to make a 21-bp duplex (9,13,14)
that associates tightly with Argonaute (Ago) proteins
(15,16). miR-451 is processed in a Dicer-independent al-
ternative pathway in which the pre-miRNAs are cleaved
by AGO2 and then are processed to maturity by
exonucleolytic cleavage (17–19). Generally, one strand of
the duplex is preferentially selected for incorporation into
the RNA-induced silencing complex (RISC) (14,20) where
it represses gene expression through interaction with the
30-untranslated regions (30-UTRs) of mRNAs [reviewed in
(1,3)] and the other strand is released and degraded
(21,22). The preferred strand has been called the guide
strand or the mature miRNA and non-preferred strand
has been called the passenger or the star strand
(miRNA*). In general, the retained strand is the one
with a less stably base-paired 50-end in the miRNA/
miRNA* duplex (2,20,21,23). The passenger strand may
also be incorporated into a functional RISC (24–27).
Depending upon the depth of sequencing low copy
number by-products of miRNA processing can be
detected (28–31). Importantly, the complex processing of
miRNAs greatly expands the targeting potential due to
the utilization of both strands of the miRNA:miRNA*
duplex, length heterogeneity, sequential Dicer cleavage
and RNA editing.
We collected small RNA deep sequencing data from
two large groups of cell and tissue types and annotated
each known mouse miRNA to reveal general patterns of
miRNA biogenesis and expression. Based on the large
scale and depth of the integrated data, we were able to
collect phased reads and annotate extensively the stem-
loop precursors of each known mouse miRNA expressed
*To whom correspondence should be addressed. Tel: +1 805 893 5222; Fax: +1 805 893 2005; Email: firstname.lastname@example.org
Nucleic Acids Research, 2012, Vol. 40, No. 13Published online 19 March 2012
? The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
pre-miRNA fragments suggests a high differential stability
of the fragment categories—5p strand, 3p strand, loop
regions, moRs, as well as extensive isoform variation
that may contribute to functional versatility of miRNA
these samples.Thesnapshot quantificationof
MATERIALS AND METHODS
Small RNA samples and datasets
The small RNA samples and datasets are listed in
Supplementary Table S1. Six hippocampus samples were
used for total RNA isolation with mirVanaTMmiRNA
Isolation kit (Ambion, Austin, TX) following the manu-
facturer’s protocol. Four samples came from four differ-
ent mice and were used to isolate small RNAs (designated
samples fromthe same
immunoprecipitated with three different Ago antibodies,
AGO1, AGO2 and 28A [generously donated by Z.
Mourelatos (32) designated Hippocampus-AGO-IP data]
by usingDynabeads protein G, kit (Invitrogen #100.03D).
Eight samples came from mouse cells at various stages of
reprogramming with four factors (33,34) (designated
Group II: Reprogramming-total RNA samples). These
samples included uninfected Oct4-GFP MEFs (mouse em-
bryonic fibroblasts), Thy1- cells harvested from Day 5
post-transduction cultures, SSEA1+ cells [identified by
colonies purified by cell sorting, and induced pluripotent
stem cell lines (ipS cells). Another three sets of cells were
mouse embryonic cells (mES) and two partially induced
pluripotent stem cell lines (piPS_4 and piPS_5). The
libraries were prepared with the SOLiDTMSmall RNA
Expression Kit and sequenced by SOLiDTMsequencer
version 3 and 4. The samples were sequenced at a length
of 25 bp for some datasets and then sequenced at 35 bp.
Small RNA mapping analysis
Using the SOLiDTMsmall RNA Analysis pipeline (http://
solidsoftwaretools.com), the raw data was filtered for
tRNA, rRNA and adaptor sequences and then aligned
against miRNA precursors and the mouse genome. To
identify the adaptor sequences at the 30-end of reads, the
first 18nt with a maximum of two or three mismatches
were aligned to find the candidate matches. Candidate
matches were then extended to identify the best alignments
and locate adaptors, producing high quality data for the
exact size of the small RNAs (Supplementary Figure S1).
Reads were first mapped to known mouse miRNAs
(miRBse version 16), and then reads that did not align
were mapped against the mouse genome for further
analysis. A sequencing error rate of <1% was calculated
by determining the percentage of tags that did not match
the first 10nt of the mature miRNA.
The coordinates of known miRNA loci from miRBase
were used to make a miRNA precursor database from the
UCSC genome browser, and 50nt were added at both
ends to identify moR sequences. Reads were aligned
against the precursor database to obtain matches for 5p
strands, 3p strands, moRs and loop regions. Only perfect
matches were used for further analysis. The most fre-
quently sequenced isomiRs of both arms were chosen as
a reference to classify other isomiRs. IsomiRs with no
more than a 3nt difference at either end from the most
Supplementary Datasets (Supplementary Datasets S1, S2
and S3). moRs were identified by surveying clustered reads
outside the dominant isomiRs; a gap of 3nt at most
between moRs and dominant isomiRs was allowed.
Only very rarely did reads span the cleavage sites of the
hairpin. Most of the analysis was done with in-house
written Perl scripts. Z-tests were used to determine the
significance of isomiR variation.
Non-templated nucleotides at the termini of mature
miRNAs were identified by checking sequences before
adaptors that did not exactly match the genome. The
accuracy of adaptor identification was very important
Therefore, only data with 35-bp reads, where much of
the adaptor was sequenced, were used.
Deep sequencing of mouse small RNAs
A total of 34 datasets from 17 samples and more than 626
million (M) reads were analyzed (see Materials and
Methods section and Supplementary Table S1). The
188M reads matched known mouse miRNA precursors
from miRBase (Figure 1) and an additional 128M
matched exons, introns and intergenic regions in the
mouse genome. Reads that matched multiple miRNAs
were rejected and only perfect matches were kept for
downstream analysis. miRBase version 16 contains 582
identified 526 miRNA species in our samples (416 for
Group I and 489 for Group II) by requiringdetection of
both strands with at leastten reads on the non-preferred
Figure 1. Total phased reads of miRNA/miRNA*, moRs and loops.
Nucleic Acids Research, 2012,Vol.40, No. 135865
strand. Those miRNAs not validated in our samples may
be expressed in tissues not used in our analysis. miRNA
expression in Group I and Group II have distinct and
(Supplementary Figure S2, Supplementary Tables S2
and S3). The miRNAs in AGO-IP data from the mouse
hippocampus (Supplementary Table S4) correlated very
well with total hippocampus small RNA profiling data,
whereas groups I and II miRNAs were not significantly
correlated. With a cutoff of >0.1% of miRNA percent in
the sample, half of the expressed known miRNAs
overlapped between Group I and II.
Abundance of 5p and 3p strands
Of those reads that perfectly matched miRNA precursors,
180M reads corresponded to the more abundant strands
and 8M reads to the less abundant strands from each
duplex. The preferred strand was considered the one
with more reads. Strands were also named 5p or 3p
without regard to the preferred strand. Mature miRNAs
can be processed from either arm of the pre-miRNA
hairpins, but in most cases, the discrimination of the
preferred strand was very high. Although a bias toward
preferential stabilization of the 50-arm in mammalian
species has been reported (35,36), our data show more
equal strand stabilization (Supplementary Figure S3) as
seen in Caenorhabditiselegans (37).
The products of the preferred strand were 25 times that
of the non-preferred strand in Group I and 18 times in
Group II cells. We calculated the strand ratio for the 416
miRNAs with more than 50 reads on both arms. Sixty-five
miRNAs had preferred strand reads that were < 2-fold
higher than the non-preferred strand including some
highly expressed miRNAs such as miR-292, miR-126,
let-7d and miR-22. A total of 200 miRNAs had
>10-fold difference between the two strands of the
hairpin structures. The expression of 5p and 3p strands
did not correlate well (R2=0.093) and large variations
In the AGO-IP samples, the preferred strand was 39
times as abundant as the opposite strand. The correlations
of the preferred/non-preferred ratio in the hippocampus
between the AGO-IP samples and the total small RNAs
data (Supplementary Figure S5) indicated that most
miRNAs had higher ratios in the AGO-IP samples.
Thus preferred strands were relatively more enriched in
the AGO-IP sample, a finding, which supports the
Nevertheless, non-preferred strands were often highly rep-
resented and may either associate with AGO, or alterna-
tively, even with relatively high reads, fail to enter the
RISC. If they are captured by a RISC, they could play a
physiological role. A total of 90% of the miRNAs, which
aligned to miRBase version 16 had more than 10 reads
from the non-preferred strand and 71% of them had more
than 50 reads from the non-preferred strand. miR-7a,
miR-9, miR-129, miR-136, miR-132 miR-126 and let-7d
in the mouse hippocampus (Supplementary Table S2), and
miR-24, miR-199, miR-130 and the miRNA cluster on
miR-294 and miR-295 in Group II cells all had both
strands highly represented (Supplementary Table S3).
Characteristically, miRNAs are differentially expressed
among tissue types. Most miRNAs showed a consistent
strand preference across the cell and tissue types. The ratio
of levels of 50- to 30-arms was well correlated between
groups I and II (Figure 2). However, some miRNAs
switched the preferred strand when comparing the two
sample groups (Table 1). Most strand switches occurred
among those miRNAs with read counts that did not differ
greatly between the arms in the two sets. For example,
hippocampal miR-22 had 50-read counts of 69071 and
30-read counts of 49670; however, in Group II cells the
reads were 41172 and 60029 respectively. On the other
hand, major strand switching occurred in the case of
miR-1-2, which had 8368 and 22642 reads on 50-and 30-
arms in hippocampus and the corresponding reads in
Group II cells were 10356 and 903 (Table 1).
moRs and miRNA loop sequences
For each miRNA in our samples we determined the reads
for each of the five phased products of miRNA processing:
50-and 30-moRs, loops, 50-and 30-miRNA strands. The sum
of the lengths of the 50-and 30-miRNA strand and the loop
sharply peaked in the range 55–65nt (Supplementary
Figure S6) and corresponded to the length distribution
of the sequences in miRBase. A total of 65000 reads
matched moRs sequences, and 20000 reads matched
loop sequences within miRNA precursors (Figure 1).
Relative to total small RNA profiling samples, reads
from the mature miRNAs dominated (Figure 1).
Reads of moRs were very common for most of the
miRNAs (Figure 3, Supplementary Tables S2, S3, S4,
Supplementary Datasets S1, S2 and S3). Although more
highly expressed miRNAs tended to have higher loop and
moR reads, their levels were not strictly correlated with
the expression level of mature miRNAs. A total of 77% of
known miRNAs had >10 moR reads. Relatively fewer
R2 = 0.823
-15 -10-505 10 15
g2(5p/3p) in R
log2(5p/3p) in Hippocampus-total RNA
Figure 2. Correlation of 5p expression and 3p expression between
Group I and II. The ratio of expressed 50-strand and 30-strand for
each miRNA in each group were calculated and the log values plotted.
5866Nucleic Acids Research, 2012,Vol.40, No. 13
moR reads were sequenced in Ago-IP samples, compared
to data from total small RNA profiling (Figure 1). Reads
of 50-moR reads exceeded 30-moRs by>3-fold in Group I
and 5-fold in Group II samples. 50-moRs were also
reportedtobe more abundant
Drosophila (30), and therefore moR processing may be
a highly conserved feature of the miRNA biogenesis
pathway. The levels of 50-moRs and 30-moRs did not cor-
relate (R2=0.025) suggesting some degree of independ-
ence in their generation. In Group II cells, miR-27b,
miR-503, miR-21, miR-24 and miR-16 had the most
abundant 50-moRs, while miR-294 and miR-30e had the
most 30-moRs. In the hippocampus, miR-134 and
miR-503 had abundant 50-moRs (Supplementary Table
S2, Supplementary Datasets S1, S2 and S3). Generally,
the dominant moRs aligned perfectly with the dominant
isomiRs (e.g. for a 30-miR, the last base of the dominant
isomiR was the base preceding the first base of the
products without terminal modifications predominate
among the phased fragments (Figure 3, Supplementary
Datasets S1, S2 and S3). These findings also suggest that
Drosha generates the ends of the 50-and 30-moRs that are
adjacent to the pre-miR stem.
Although >90% of mouse miRNA precursors found in
miRBase version 16 (31) had detectable loop regions, only
15% of the miRNAs had loop read numbers that exceeded
10. Therefore, loop sequences were the most unstable
among all of the phased fragments. For comparably ex-
pressed mature miRNAs, the loop levels could differ
greatly in different tissues. For example, some miRNAs,
highly expressed in Group II had thousands of loop reads,
e.g. such as miR-34a (2927 loop reads, 0.26% of the
dominant strand), miR-106b (2359 loop reads, 5.34% of
miRNAs from the dominant strand), miR-182 (2285 loop
reads, 0.21% of the dominant strand) and miR-27a (2189
loop reads, 0.57% the dominant strand), whereas in the
mouse hippocampus these miRs were also highly ex-
pressed, but had less than 10 loop reads (Supplementary
Tables S2, S3, Supplementary Datasets S1 and S2). In
mouse hippocampus, miRNAs with the most abundant
loops were miR-138-2 (1018 loop reads, 0.06% of the
dominant strand), let-7c-1 (745 loop reads, 0.21% of the
dominant strand) and miR-219-2 (380 loop reads, 0.43%
of the dominant strand).
Variation in canonical miRNA isomiRs
Deep sequencing revealed variation in the Drosha and
Dicer cleavage sites, which generate pre-miRNAs and
RNA duplexes. These products are termed isomiRs
(28,38). Some highly expressed miRNA genes had over a
hundred isomiRs. The dominant isomiR was considered
the one with the most reads. IsomiRs in our data set that
exceed the length of the dominant isomiR are unlikely to
be explained by the addition of non-templated nucleotides
that fortuitously match the genome sequence (39–41).
IsomiRs that are shorter than the dominant isomiR may
arise in part due to exonucleolytic cleavage, as occurs in
plants (42), but not, as yet, observed in animals (43). The
variation is unlikely due to degradation or sequencing
error (44,45), because species that were shorter than the
dominant isomiR were approximately as frequent as those
that were longer (39) and their numbers exceeded the
rate of sequencing error. When considering the most
abundant isomiRs, they differed by only 1 or 2nt at the
50-or 30-end of their sequences. Consistent with a previous
proposal (46), the distribution of isomiRs in the cell is
probably not random. However in our samples, the
typical size distribution of isomiRs was nearly normal,
(Figure 4A). When the isomiRs were grouped by only
their 50-ends or by their 30-ends, they remained normally
distributed (data not shown). This pattern was more
obvious for highly expressed miRNAs. Our data showed
that both strands exhibited higher 50-fidelity, which agrees
with previous data in several other species (30,36).
Figure 4B shows the distributions of all isomiRs
grouped by their 50-ends and 30-ends, rather than by the
preferred strand. We further observed that both the
50- and 30-ends of the preferred strands showed a higher
percentage of dominant isomiRs, which indicated less
Table 1. miRNAs that switched their dominant strands
miRNAs Group I, Hippocampus-total RNAHippocampus-AGO-IP Group II, Reprogramming-total RNA
5p 3p Log(5/3)5p3p Log(5/3) 5p 3pLog(5/3)
Nucleic Acids Research, 2012,Vol.40, No. 13 5867
variation than occurs on the non-preferred strand
Much of the isomiR variability can be explained by
variability in Dicer and Drosha cleavage positions.
Variation in the termini of isomiRs produced by Dicer
and Drosha reflect the fidelity of Dicer and Drosha
(Figure 5) quantified as the percent of a set of isomiRs
represented by the dominant isomiR. We grouped all the
isomiRs based on their locations on the precursor hairpins
as isomiRs derived from either the 50- or 30-arms, and
compared the sequence variation of the enzyme cleavage
positions. Because variation differed greatly at the 50-ends
compared to the 30-ends of isomiRs, in order to compare
directly the fidelity of Drosha to Dicer, we set up com-
parisons for both ends produced by the two enzymes. We
found that the dominant isomiRs were 95.2% of the total
Figure 3. Phased read distributions of (A) miR-125b and (B) miR-191.
5868 Nucleic Acids Research, 2012,Vol.40, No. 13
Percent of is
preferred strand 5'end
non-preferred strand 5'end
preferred strand 3'end
non-preferred strand 3'end
d t d 5'd
Distance to the dominant isomiRs
Figure 4. Distributions of isomiRs. (A) miR-124 isomiRs. IsomiRs were listed according their 50-ends, and for each group with the same 50-end, up
to five most abundant isomiR with different 30-ends are shown. (B) The distributions of all isomiRs grouped either by their 50-end or 30-end for both
strands. IsomiRs were grouped by their distance of 50-end or 30-end compared to the dominant ones (X-axis). At most 3nt variations at either end
Distance to the dominant isoforms
Figure 5. End variations of isomiRs at Drosha and Dicer cleavage sites. Two comparisons were performed: one was based on variations at the
50-end of isomiRs and the other was based on variation at the 30-ends. Both comparisons showed Drosha had a higher chance to match the sequences
with the dominant isomiRs.
Nucleic Acids Research, 2012,Vol.40, No. 135869
isomiRs at the 50-end of the 5p strand (Drosha cleavage),
compared to 91.3% the 50-end of the 3p strand (Dicer1
cleavage) (Z-value=1004.4 andP-value<0.0001). The
dominant isomiRs were 60.5% of the total isomiRs at
the 30-end of the 3p strand (Drosha cleavage) compared
to 56.2% at the 30-end of the 5p strand (Dicer1 cleavage)
(Z-value=592.0 and P-value<0.0001). Overall, for both
50- and 30-ends of isomiRs, the variations on Drosha
cleavage positions were less than Dicer (Figure 5), a
finding, which implies a higher fidelity of Drosha than
Dicer. As a result, more variation in the cleavage sites
occurs near the loop than at the base of the stem in the
pre-miRNA hairpins. Although Dicer functions as a
precise ‘measuring stick’ from the Drosha cleavage site,
several possible RNA folding structures with variation
in the nucleotide at the terminus of the stem could serve
as possible Dicer substrates. An earlier report in mouse
suggested greater fidelity of Dicer (36); however, those
authors restricted their analysis to cases in which Dicer
produced the 50-terminus of the miRNA and was
determined by the percent of offset reads. A study in
C. elegans (37) showed approximately equal fidelity of
Dicer and Drosha after they removed a single miRNA
(miR-58) with an abundance that greatly exceeded all
the other miRNAs.
Weighted average size of nucleotide variation
Although most miRNAs showed typical symmetrical dis-
tributions of isomiRs, the proportion of sub-dominant
isomiRs could vary greatly for different miRNA loci.
Alternative miRNA isomiRs expressed at sufficiently
high levels could direct the repression of a distinct target
set (36). Therefore, we identified miRNAs with relatively
abundant secondary isomiRs and miRNAs with differen-
tially expressed isomiRs in different samples. As shown
previously (37,46), the extent of variation at the ends of
isomiRs varied among different miRNAs. Some miRNAs
had high variation at their sequence ends with nearly
equal proportions of their isomiRs. For example, the
dominant isomiR of miR-1982 was only 49.55% of the
total reads and the secondary isomiR, which differed by
2nt, accounted for 45.67% of total reads. To measure the
isomiR variation, we calculated the weighted average size
of nucleotide variation (WAZNV) according to the
dominant sequences as:
where piwas the percentage of isomer i in total observed
isomiRs and diwas the relative distance of ends of isomiR
i compared to the dominant one. WAZNV can be
calculated based on either the 50- or 30-end for both the
preferred and non-preferred strands. Because the miRNA
targeting depends heavily on the 50-end, we mainly
focused on WAZNVs based on the 50-end of miRNAs.
WAZNV measured the variation among isomiRs. The
top 10 sequences, whether preferred or non-preferred
strands, with high isomiR variation based on all data
sets are shown in Figure 6A. At the top of the list is
miR-485-3p, which had shown significant variation in
the literature (47–49). In our dataset, the dominant
isomiR (isomiR I) was on the 30-arm, which accounted
48.9% of miR-485-3p reads. The secondary and tertiary
isomiRs accounted 28.1% (isomiR II) and 19.7% (isomiR
III) of total reads, which shifted the mature sequence 2
and 3 nt in the 50-direction. Although the isomiR distri-
bution around the dominant isomiR is usually normal,
some isomiR distributions shift the secondary and
tertiary isomiRs 2 and 3nt from the dominant isomiR
By comparing isomiR variation between different
samples, WAZNV can detect differentially expressed
isomiRs. By defining the position of dominant isomiRs
within the whole data set, we calculated the WAZNV of
miRNAs in both Group I and II samples as well as the
AGO-IP sample separately. Switching of dominant
isomiRs was uncommon between hippocampus and
Group II cells (Supplementary Datasets S1 and S2), and
also very rare between AGO-IP and total small RNA
samples. However, we identified several cases that
miRNAs switched the dominant isomiRs between hippo-
campus and Groups II cells such as miR-485-3p, or
(Table 2 and Figure 7). In addition to miR-485-3p
(Figure 7C) and mu-miR-137-3p (Figure 7D) also
switched dominant isomiRs between Group I and II
cells. miR-125b-2-3p (Figure 7E) was highly expressed
in hippocampus, in which the dominant isomiR ac-
counted for 78.9% of total reads; in contrast, the second-
ary isomiR, which shifted 2nt in the 50-direction
compared to the dominant isomiR, accounted for 40.0%
of the total 3261 reads of miR-125b-2-3p in Group II cells.
A few cases showed large differences between AGO-IP
and total small RNA samples. miR-690 had 1010 reads,
3162 reads and 2196 in AGO-IP, Group I and Group II
cells respectively, but its dominant isomiRs in AGO-IP
samples shifted 2nt in the 30-direction compared to the
location of dominant isomiR in the other two groups
which showed identical distributions of isomiRs (Figure
7F). The dominant isomiR of miR-34a-3p in AGO-IP
samples shift 1nt compared the dominant ones in the
other two samples. These examples could indicate an
isomiR preference of the AGO complex to maintains
greater uniformity in the mature miRNA that is guided
to the mRNA.
Non-templated nucleotide addition
Sequence alterations of miRNAs can occur by addition of
non-templated nucleotides to miRNA termini (38,40,49).
Most miRNAs in our data set did not have non-templated
nucleotide modifications. Of the total reads, 16.00%,
14.10% and 12.67% were extended by 1nt in the
Ago-IP, Group I, and Group II samples, respectively. Of
the total reads, 7.81%, 6.85% and 3.18% were extended
by 2nt in the Ago-IP, Group I and Group II samples,
respectively. The nucleotides most frequently added to
murine miRNAs were U and A (Figure 8 and Table 3).
However, in the Ago-IP sample from hippocampus the
addition of C was most common; C also showed a small
increase in the Group I samples compared to the Group II
5870 Nucleic Acids Research, 2012,Vol.40, No. 13
samples. Di-nucleotide extensions not infrequently con-
sisted of two different nucleotides (Supplementary Table
S5). Half ofthe miR-143-3p reads in our samples were
extended similarly to the very high proportion of
extended reads previously described for this miRNA
(36). Among highly expressed miRNAs, 32% of the
miR-124 reads were extended and 33% of the miR-24
reads were extended (Supplementary Table S6).
Nucleotide addition can occur on the mature miRNA
(50) or on the precursor species (43,51). For our entire
data set nucleotide addition on the 50-end of both the 5p
(0.32%) and 3p (0.31%) strand was infrequent. The
pattern of non-templated extension differs between
Group I hippocampal tissue and Group II cultured cells
undergoing re-programming (Figure 8). Group II cells
also have a greater tendency toward 30-mono-uridylation
Percent of isomP
miR 377 3p-
miR 302c 5p miR-302c-5p
Distance to the dominant isomiRs
5’ donimate isomiRs
48.9% dominant isomiRs, 28.1% second, 19.7 third ones
Figure 6. miRNAs with high isomiR variation. (A) Distributions of preferred and non-preferred isomiRs with high variation grouped by their
50-ends. These miRNAs have second isomiRs with significant abundance, which usually shifted 2 or 3nt’s compared to the dominant isomiR. (B) The
distributions of three principle isomiRs of miR-495-3p positioned on the hairpin structure of miR-495 precursors. The dominant isomiR switched in
Group II cells.
Table 2. Top 10 miRNAs with large isomiR variation
miRNAs Group I, Hippocampus-total RNAHippocampus-AGO-IP Group II, Reprogramming-total RNA
Nucleic Acids Research, 2012,Vol.40, No. 13 5871
on the 3p arm relative to the 5p arm in Group II compared
to Group I (Table 3, 4.88%/3.88% in Group I and 8.21%/
1.84% in Group II). In general, most of the nucleotide
additions occurred on the 30-end with 19.85% on the 3p
strand and 16.07% on the 5p strand. Whether the bias
toward the 30-end of the 3p strand represents nucleotide
addition on the pre-miRNA or a bias to the 3p strand
after Dicer cleavage cannot be discerned from these
data. A further breakdown by the specific nucleotide
added shows addition of A, U and C, but very low
values of G as previously noted (46). The specific
extended nucleotide at the 30-ends shows the expected
preference for mono-uridylation on the 3p arm, i.e. the
extreme end of the precursor; however, the 5p arm has a
rcent of isomiR
-3 -2 -10123
Distance to the dominant isomiRs
Hippocampus-total RNA Reprogramming-total RNA
Figure 7. Distributions of isomiRs in different groups of samples for miRNAs with isomiR variation. (A) miR-485-3p which switched the dominant
isomiR in Group II samples. (B) miR-543-5p; (C) miR-592-5p; (D) miR-137-3p; (E) miR-125b-2-3p which did not switch the dominant isomiR, but
the second isomiR increased in Group II; (F) miR-690-3p which showed the difference between AGO-IP and total small RNA samples.
Untemplated nucleotide addition
Rate of modi
Figure 8. Untemplated nucleotide addition rate at the 30-ends of
Table 3. Non-templated nucleotide addition at the 30-ends of
miRNA/miRNA* between 5p and 3p
5872Nucleic Acids Research, 2012,Vol.40, No. 13
bias toward the addition of adenine (Table 3). One
possible reason for this asymmetry is a nucleotide
specific tendency to extend the pre-miRNA versus the
mature miRNA. There is a hint in the data that the
choice of U or A may have an effect on promoting or
hindering RISC entry. Comparing total miRNAs from
the hippocampus (Group I) to miRNAs Ago IP’ed from
the hippocampus there is a relative increase in mono U
extension and a relative decrease in mono A extension in
the Ago-IP fraction.
Several studies have used deep sequencing for detailed
miRNA annotations in a range of model organisms
including D. melanogaster (30), C. elegans (37) and mouse
(29,36). By sampling diverse tissues and cells and
sequencing at a great depth, several novel observations
emerge here and other findings were confirmed. More
than 90% of mouse miRNAs had more than 10 reads
from both arms of the precursor, and therefore both
strands could potentially play a biological role in target
repression. Taking advantage of the tight association
between miRNAs and AGO, we compared total small
RNA (Group I) and a pan-Ago IP both from the mouse
hippocampus to profile miRNAs which entered RISC.
These data showed fewer non-preferred strands present in
the RISCeven incases with highreadcounts. Nevertheless,
not infrequently, the non-preferred strand also associated
with AGO, and therefore possibly with the RISC. It seems
likely that in some cases, a pool of pre-miRNAs sort their
preferred and non-preferred strands into different RISCs
without degradation of one of the strands. Although
usually the preferred strand is readily recognizable by a
many-fold enrichment, some miRNAs show quite similar
read counts from both the preferred and non-preferred
strands. 10% of miRNA loci showed a comparable expres-
sion of both strands with <2-fold difference.
Data from several large-scale miRNA sequencing
studies have demonstrated that the arm from which
the preferred miRNA is processed can switch in different
tissues and at different developmental times (36,38,52–55).
Among a variety of human samples, Cloonan et al. (56)
found that 12.9% of miRNAs switched the dominant
strand in at least one tissue. Utilization of opposite
strands for target binding would result in significant
changes inthe miRNA
miRNA loci with comparable expression levels of both
arms show switching of the preferred strand in different
tissues, and in these cases both strands may be used in
different proportions rather than a more dramatic switch
in the target field if one or the other strand were used
exclusively. When strand choice is closer to equilibrium
different tissues appear to tilt the balance toward one
strand or the other. The use of both strands among a
sizable fraction of miRNAs is consistent with the demon-
stration of selection at the 50-end of both strands (57).
Other reports proposed that star sequences would not be
excluded from functional complexes because they are
present in substantial levels or sorted to different
Argonaute complexes, in which the dominant arm
directs translational repression (by means of Ago1) and
the miR* sequence directs transcriptional degradation by
means of Ago2 (25–27,36,38,58–63). Star sequences, such
as miRNAs like miR-19* (64) and miR-223* (65), can
Drosophila and mammals (25–27,54,66).
Although isomiRs are frequently observed, their origin
has been attributed to sequencing or alignment artifacts
(67–69). However, non-random features of their distribu-
tions suggest otherwise (29,70) and recently it was sug-
gested that isomiRs with different repertoires of mRNA
targets would distribute the ‘off-target’ hits while still tar-
non-random features of isomiRs is variation in the choice
of the dominant isomiR. Furthermore, a potentially dis-
ruptive shift of the dominant isomiR by 2nt (36) was
observed. In particular, variation at 50-end of the mature
isomiR will presumably have greater effects on targets
(30,36). Secondary isomiRs could be explained by differen-
tial processing of the two paralogous hairpins (36,38), or
alternative Drosha and/or Dicer1 cleavages (30). Finally,
miRNAs can be modified by the non-templated addition of
nucleotides almost always at the 30-end. Reports in the lit-
erature vary on the prevalence of non-templated nucleotide
described in mammals (36,49), worms and flies (38,40). A
bias toward mono-uridylation
mono-adenylation among miRNAs in Ago IP fractions
compared to the total small RNA fraction suggests that
these two additions may serve to promote or hinder asso-
ciation with the RISC. The methods here preclude the dis-
covery of long 30-terminus poly(U)-tailing and those
reports that do use deep sequencing to annotate miRNAs
also do not report extensive poly(U)-tailing (46).
The origin of moRs is unclear. They may arise by
exonucleolytic activity on precursor transcripts (38,73) or
double-stranded cleavage of extended hairpin regions on
pri-miRNA transcripts via secondary DROSHA process-
ing (30,74). However, the broad length distributions of
pre-miRNAs are inconsistent with a DROSHA-based
cleavage mechanism (72). However, our data show that
the ends of both the dominant 50-and the 30-moRs fit very
well with the dominant termini of the 3p and 5p strands
arising from Drosha cleavage. It therefore seems likely that
at least one end of the moR sequences is a Drosha product.
on target networksin
(56). Among the
and away from
The sequencing data reported in this study can be
obtained from the Sequence Read Archive (SRA) at
NCBI under accession number SRP010127, SRP010168,
SRP010169 and SRP010170.
Supplementary Data are available at NAR Online:
Supplementary Tables 1–6, Supplementary Figures 1–6
and Supplemental Datasets 1–3.
Nucleic Acids Research, 2012,Vol.40, No. 135873
The authors wish to the Kosik lab members for helpful
California Institute for Regenerative Medicine (CIRM)
(to K.S.K.); National Institutes of Health (to T.M.R.); a
postdoctoral fellowship from California Institute for
Regenerative Medicine to E.J.L. Funding for open
accesss charge: California Institute for Regenerative
Medicine and the NIH.
Conflict of interest statement. None declared.
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