Báo cáo khoa học: New human and mouse microRNA genes found by homology search pptx

New human and mouse microRNA genes found
by homology search
Michel J. Weber
Laboratoire de Biologie Mole
´
culaire Eucaryote, UMR5099, CNRS and Universite
´
Paul Sabatier, IFR109, Toulouse, France
MicroRNAs (miRNA) are  22 nucleotide-long RNAs
that function in translational repression by base pair-
ing with their target mRNA in a variety of pluricellu-
lar organisms. They originate from long precursors
(pri-miRNA) that, in animals, are cleaved by the Dro-
sha endonuclease in the nucleus [1] to give  70 nuc-
leotide-long miRNA precursors (pre-miRNAs) with a
characteristic hairpin structure. In the plants, excision
of pre-miRNAs is performed by DCL1, a Dicer homo-
logue [2,3]. Following the export of pre-miRNAs to
the cytoplasm by Exportin-5 [4,5], the loop region of
the hairpin is removed by the Dicer endonuclease to
produce a short double-stranded RNA (dsRNA), a
strand of which, corresponding to the mature miRNA,
is predominantly incorporated in the RNA-induced
silencing complex (RISC). The RISC complex either
inhibits translation elongation or triggers mRNA deg-
radation, depending upon the degree of complementar-
ity of the miRNA whith its target (for reviews, see
[6–8]).
Since the seminal identification of the first miRNAs,
Caenorhabditis elegans lin-4 and let-7, using genetic
approaches [9,10], hundreds of miRNAs have been

characterized experimentally using various cloning
strategies in plants, C. elegans, Drosophila melanogas-
ter, zebrafish, pufferfish, mouse, rat and human [8].
More recently, algorithms have been developed to
identify putative precursor miRNAs from sequenced
genomes [11,12]. These approaches predict that the
human genome might contain 200–255 miRNA genes
Keywords
antisense transcript; genomic localization;
human genome; microRNA; mouse
genome; mRNA degradation; RNA
interference
Correspondence
M. Weber, LBME, 118 route de Narbonne,
31062 Toulouse Cedex, France
Fax: +33 5 61 33 58 86
Tel: +33 5 61 33 59 56
E-mail:
(Received 4 June 2004, revised 24 August
2004, accepted 7 September 2004)
doi:10.1111/j.1432-1033.2004.04389.x
Conservation of microRNAs (miRNAs) among species suggests that they
bear conserved biological functions. However, sequencing of new miRNAs
has not always been accompanied by a search for orthologues in other spe-
cies. I report herein the results of a systematic search for interspecies ortho-
logues of miRNA precursors, leading to the identification of 35 human and
45 mouse new putative miRNA genes. MicroRNA tracks were written to
visualize miRNAs in human and mouse genomes on the UCSC Genome
Browser. Based on their localization, miRNA precursors can be excised
either from introns or exons of mRNAs. When intronic miRNAs are anti-

sense to the apparent host gene, they appear to originate from ill-character-
ized antisense transcription units. Exonic miRNAs are, in general,
nonprotein-coding, poorly conserved genes in sense orientation. In three
cases, the excision of an miRNA from a protein-coding mRNA might lead
to the degradation of the rest of the transcript. Moreover, three new exam-
ples of miRNAs fully complementary to an mRNA are reported. Among
these, miR135a might control the stability and⁄ or translation of an alter-
native form of the glycerate kinase mRNA by RNA interference. I also dis-
cuss the presence of human miRNAs in introns of paralogous genes and in
miRNA clusters.
Abbreviations
miRNA, microRNA; pre-miRNAs, miRNA precursors; RISC, RNA-induced silencing complex; snoRNA, small nucleolar RNA.
FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 59
[13]. However, a comprehensive list of these sequences
is still not available.
It was recognized that many miRNAs are evolu-
tionarily conserved, some of them from worm to
human [14]. MiRNA genes have been characterized
experimentally from a variety of organisms and
might have orthologues in other species, suggesting a
powerful method to predict the existence of new
miRNA genes. I report here on the results of a sys-
tematic search for potential human orthologues of
mouse miRNAs deposited in the miRNA Registry
[15] and, vice-versa, of potential mouse orthologues
of known human miRNAs. In addition, I searched
for human orthologues of recently identified rat
miRNAs [16]. This led me to identify potential
orthologues of miRNAs that were described previ-
ously in one species, but not in the other. After

inclusion of these new data, the total number of
human and mouse miRNAs deposited at the miRNA
Registry now approaches the theoretical number of
255 predicted by Lim et al. [13].
Using this information, I wrote custom tracks that
allow for the localization of the miRNA genes in the
human and mouse genomes. These are now available
on the UCSC Genome Browser [17]. Using this tool, I
systematically determined the position and orientation
of miRNA genes relative to known transcriptional
units, examined the conservation of miRNA gene local-
ization between the human and mouse genomes, and
made a comprehensive list of miRNA clusters. This
search led to several testable hypotheses concerning
the transcription of miRNA genes, and to the predic-
tion of new mRNA targets.
Results and Discussion
New potential human and mouse microRNA
precursors
The entire set of human and mouse precursor and
mature miRNA sequences from the miRNA Registry
(version 2.2) was submitted to a BLAT search against
the human genome. The results (in BED format) were
exported to excel
TM
to generate a table with 2873 ent-
ries. A similar BLAT search was performed against the
mouse genome, generating an excel
TM
table of a sim-

ilar size. These tables were then ranked according to
chromosome number and chromosome position and
filtered for perfect and near-perfect matches. The cor-
responding sequences were subsequently examined for
a potential hairpin structure with mfold, and the
results were compared to those of known miRNAs
from the miRNA Registry.
I only considered, as valid candidates, those poten-
tial miRNA precursors that conformed to the empir-
ical criteria proposed by Ambros et al. [18] in
particular, a hairpin structure of the lowest free
energy, as predicted by mfold , and a minimum of 16
nucleotides of the mature miRNA engaged in Watson–
Crick or G ⁄ U base pairings (criterion C). The method
used for searching the new miRNAs ensured their phy-
logenetic conservation (criterion D). Moreover, the
detection of the  22-nucleotide mature forms by Nor-
thern blot (criterion A) and ⁄ or their identification in a
cDNA library (criterion B) were checked for the
sequences deposited in the Rfam2.2 miRNA Registry
(Wellcome Trust Sanger Institute) for at least one
species. In most cases, sequences of the new mature
miRNAs were perfectly conserved between human and
mouse, but differed by one nucleotide in few cases (see
for example, mir-155). Further validation of these can-
didates was performed on the basis of additional cri-
teria, such as conservation of the host gene (see below)
or position relative to known miRNA clusters.
This led to the identification of 60 new potential
miRNA precursors (15 for human and 45 for mouse)

that were made available before publication in Rfam
version 3 of the miRNA Registry of the Wellcome
Trust Sanger Institute (Table S1). Moreover, with the
collaboration of S. Griffiths-Jones (Wellcome Trust
Sanger Institute), the names of several miRNAs depos-
ited at the miRNA Registry have been changed, so that
orthologous precursors, based on conservation of both
sequences and synteny, have similar names in both
human and mouse. This was particularly important
when a mature miRNA had multiple, closely related,
precursors (see for example mmu-mir-9-2, mmu-mir-
138-12 and mmu-mir-199a-1).
Moreover, the coordinates of both new and previ-
ously described miRNA precursors were used to write
custom tracks that allowed their localization in the
human and mouse genomes on the UCSC Genome
Browser. A similar track was also written for the gen-
ome of C. elegans.
New human miRNA precursors predicted
by homology with rat microRNAs
I searched for potential human orthologues of recently
cloned rat miRNAs [16]. The best BLAT hits were
examined for conservation of both synteny in human
and rodent genomes and the presence of a stem-loop
structure. This allowed one to propose 20 new human
microRNA precursors (Table 1).
miR-151 was identified in the mouse [19], and miR-
151* in the rat [16]. In both cases, the same predicted
New human and mouse microRNA gene M. J. Weber
60 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS

Name
hsa-miR-322
*hsa-miR-323
*hsa-miR-324-5p
*hsa-miR-324-3p
hsa-miR-325
*hsa-miR-326
*hsa-miR-328
hsa-miR-329
*hsa-miR-330
*hsa-miR-331
*hsa-miR-335
Mature miRNA Predicted precursor hairpin structure Note
Differs from rno-miR322
by four nucleotides (italic)
Same intron of the host
gene of hsa-mir-299 (AK021542)
Differs from rno-miR-325
by two nucleotides (italic)
Differs from rno-miR-326
by one nucleotide (italic)
Differs from rno-miR-329
by four nucleotides (italic)
Differs from rno-miR-330
by one nucleotide (italic)
Table 1. New human microRNAs predicted by homology with rat miR. The potential human orthologues of rat microRNA precursors described by Kim et al. [16] were localized in the
human genome by BLAT on the UCSC Genome Browser server [54,57] or by examination of conserved sequences in the human genome in the regions orthologous to those that host
the rat or mouse miRNAs. MiRNAs indicated by an asterisk have been deposited in the miRNA Registry (version 3.1).
M. J. Weber New human and mouse microRNA gene
FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 61

Name
*hsa-miR-337
*hsa-miR-338
*hsa-miR-339
*hsa-miR-340
*hsa-miR-342
hsa-miR-345
hsa-miR-346
*hsa-miR-135b
*hsa-miR-148b
*hsa-miR-151*
*hsa-miR-151
Mature miRNA Predicted precursor hairpin structure Note
Differs from rno-miR-337
by one nucleotide (italic)
Differs from rno-miR-345
by two nucleotides (italic)
Differs from rno-miR-346 by
three nucleotides (italic),
and from mmu-miR-346 by two
nucleotides
hsa-miR-151 differs from
rno-miR-151 by one nucleotide
(italic)
Table 1. (Continued).
New human and mouse microRNA gene M. J. Weber
62 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS
precursor encodes both mature miRNAs from its 5
0
(miR-151*) and 3

0
(miR-151) portions. This also holds
true for hsa-mir-151, although the mature form of
miR-151 differs from the rodent sequence by one nuc-
leotide. This conservation reinforces the hypothesis
that, in mammals, the same precursor gives rise to both
miR-151 and miR-151*. From the predicted hairpin
structure of the precursor, the energies of hybridization
of the four nucleotides located at the 5
0
end of miR-
151* and miR-151 are 1.7 and 0.1 kcalÆmol
)1
, respect-
ively. As ‘RISC assembly favors the siRNA strand
whose 5
0
end has a greater propensity to fray’ [20], it is
expected that miR-151* will be more abundant than
miR-151. Indeed, miR-151 was cloned only once
among 913 miR sequences [19]. Although, to my know-
ledge, neither miR-151, nor miR-151* were cloned in
human, it is unlikely that the single base substitution
compared to rodent sequence (at nt 10 of miR-151)
could alter the balance between the two miRs.
For certain rodent miRNA precursors, a BLAT
search in the human genome produced no matches.
However, a likely orthologue could be found by
exploring the most likely (i.e. of conserved synteny)
portion of human genome for conserved sequences;

e.g. mmu-mir-345 resides upstream of the AK047628
RefSeq gene. Its human orthologue was found
upstream of C14orf69, the best BLAT hit for
AK047628. Such identification was made straightfor-
ward by the examination of the ‘Human ⁄ Chimp ⁄
Mouse ⁄ Rat ⁄ Chicken Multiz Alignments & PhyloHMM
Cons track’ of the human UCSC Genome Browser.
Similarly, hsa-mir-329 and -322 were identified on
the basis of their conserved stem-loop structure and
conserved position relative to other miRNAs or Ref-
Seq genes. However, the presumptive mature miRNAs
hsa-miR-329 and -322 differ from their mouse and rat
orthologues by four nucleotides (Fig. 1A,B). Most of
these changes retained base-pairing in the precursor
miRNA by forming G ⁄ U interactions or resided in un-
paired positions (data not shown). Consequently, the
folding free energy calculated using mfold was either
little affected (mir-329) or even decreased in the case
Fig. 1. Alignment of microRNA sequences from mammalian genomes. (A–C) Alignment of the sequences from mammalian mir-329
(A), mir-322 (B) and mir-346 (C). Abbreviations: hsa, Homo sapiens; mmu, Mus musculus; rno, Rattus norvegicus; pan, Pan troglodytes.
(D) Alignment of the sequences of rodent mir-350 with the corresponding sequences from human and chimpanzee genomes. Rodent
sequences were retrieved from The miRNA Registry. Human sequences were retrieved from the human genome sequence by examination
of highly conserved regions in the syntenic segments. The sequence of pan-mir-350 was obtained by BLATing the human sequence against
the chimpanzee genome. The sequences of mir-329 and mir-298 are 100% conserved between human and chimpanzee. The sequences of
mature microRNAs are boxed. The antisense miR boxes indicate the portion of the hairpin precursor structure that is base-paired with the
miR, as predicted by
MFOLD.
M. J. Weber New human and mouse microRNA gene
FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 63
of mir-322 (DG ¼ )46.6 for human and )41.0 kcalÆ

mol
)1
for mouse).
The same conclusion holds for hsa-mir-346, where
the mature miR sequence differed from those of mouse
and rat by two and three nucleotides, respectively
(Fig. 1C). In this case, the folding free energy of the
human and rat precursors remained comparable ()46.4
and )50.6 kcalÆmol
)1
, respectively).
Mutations in mature miARNs, particularly in their
3
0
portion, are compatible with their function as trans-
lational repressors [21]. The same study, however,
revealed that G:U wobble pairing in the 5
0
region of
the miRNA had detrimental effects that could not be
predicted on the basis of changes in the free energy of
annealing with the target mRNA. Therefore, hsa-mir-
329, hsa-mir-322 and hsa-mir-346 require further
experimental validation to be considered as bona fide
miRNA precursors.
It was however, surprising that, in the predicted
hairpin structures of mir-329, -322 and -346, the anti-
sense sequence of the miR was more conserved than
the miR itself (Fig. 1A–C). Significantly, a mutation in
the antisense sequence of miR-322 was accompanied by

a compensatory change in the miR sequence, so that a
G ⁄ C base pair in the mouse precursor was replaced by
an A ⁄ U in the human one. The single change in the
antisense sequence of miR-329 occurred at an unpaired
position in both human and rodent precursor hairpin
structures. As it is difficult to conceive that evolution-
ary pressure might be higher on the antisense than on
the sense strand [8], this may suggest that the antisense
strand was cloned accidentally in certain cases [16].
In a few cases, the homology search allowed local-
ization of human sequences similar to some rodent
miRNA precursors but that had accumulated deleteri-
ous mutations. For example, the human orthologous
sequence of rodent mir-350 could be localized in an
intron of the KARP-1-binding protein (KAB) gene.
However, a nine base-pair deletion in the human and
chimpanzee genomes removed the first seven nucleo-
tides of the mature microRNA (Fig. 1D). It this case,
it is noteworthy that the antisense strand accumulated
mutations, possibly due to a lack of selective pressure
after the inactivation of mir-350 by the deletion in the
mature miRNA sequence.
Human and mouse miRNA precursors reside
in conserved regions of synteny
Using the UCSC Genome Browser, I examined
whether orthologous human and mouse miRNA
precursors reside in conserved synteny regions. This
proved to be the case for miRNAs located in known
coding genes (see below). Many human miRNAs are
located in introns of noncoding mRNAs. In general in

these cases, the mRNA was not or poorly conserved
in the mouse genome. When human miRNAs were in
nonconserved genes, or outside characterized genes, I
examined the known flanking genes. In all cases but
three, human and mouse miRNAs were found to
reside in conserved synteny regions. The three appar-
ent discrepancies in the position of a miRNA in the
human and mouse genomes were hsa-mir-9-3, hsa-mir-
339 and hsa-mir-326. As detailed in Appendix S1,
these cases most probably originate from errors in the
present assembly of the mouse genome. Orthologous
human and mouse miRNAs thus reside either in
introns of orthologous genes, and ⁄ or in conserved
synteny with surrounding genes.
Human miRNA precursors that reside in introns
of known genes
Eighty one human miRNA precursors were found to
be located in an intron of a known gene, or of a gene
defined by a complete cDNA sequence, in the sense
orientation (Table S2). It is however, important to
note that human miRNAs that were classified as
located outside of known genes might in fact reside in
still uncharacterized splicing variants. For example,
hsa-mir-10b is located 972 nucleotides upstream of the
HOXD4 gene (NM_014621). However, mmu-mir-10b
resides in an intron of a long form of the mouse
Hoxd4 pre-mRNA (NM_010469), but this alternative
form has no documented human orthologue.
According to current models, intronic miRNA pre-
cursors that have the same orientation as their host

gene might be produced upon cleavage of the spliced
intron by the Drosha endonuclease. In certain cases,
the miRNA sequence is included in intronless ESTs
that are often members of a cluster of overlapping,
also intronless, ESTs. It is striking that, often, no such
EST cluster was found in the other introns of the
miRNA host gene (e.g. hsa-mir-103–2 and hsa-mir-98)
or only in adjacent introns. Due to the uncertainty in
the orientation of intronless ESTs, it was not possible
to assess the orientation of the clusters relative to that
of the host gene, and that of the microRNA. Never-
theless, this suggests that introns that host microRNAs
might be particularly stable. Alternatively, certain of
these intronic miRNAs might be produced by unchar-
acterized transcription units embedded in the same
orientation in the apparent host gene. However, this
possibility appears unlikely, as to my knowledge, there
is only two examples of such a situation in the human
genome [22,23].
New human and mouse microRNA gene M. J. Weber
64 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS
In addition, 17 miRNAs were located in an intron
of a known gene, but in the antisense orientation
(Table S3). Among these, 10 were in a miRNA cluster
(see below). To determine how these miRNAs might
be generated, their genomic context was carefully
explored. As shown in Table S3, several miRNAs in
this category are in fact in a transcription unit that has
an orientation opposite that of the apparent host gene.
In particular, hsa-mir-302 lies within an intron of the

HDCMA18P gene in the antisense orientation, but in
an intron of ESTs BG207228 and BU565001 in the
sense orientation (Fig. 2A). These two ESTs largely
overlap the HDCMA18P transcription unit in the
opposite orientation.
Similarly, hsa-let-7d resides in the gene defined by
the BC045813 mRNA in the opposite orientation
(Fig. 2B). In addition, it is located in a cluster of
 50 unspliced ESTs that spans about 2-kb and over-
laps the intronless BC064349 mRNA. The orientation
of this cluster is opposite that of the BC045813
mRNA, as shown by the polyA tails of the
BC064349 mRNA and of several ESTs (Fig. 2B).
These data thus strongly suggest that hsa-let-7d,
and possibly hsa-let-7f-1 and hsa-let-7a-1, are part of
an intronless transcript antisense to the BC045813
transcription unit.
A third example is hsa-mir-142 that lies within an
intron of the AK090885 gene in the antisense orienta-
tion, but also in the antisense, intronless and polyaden-
ylated AX721088 mRNA (1.6kb). Interestingly, a
natural chromosome translocation fuses the 5
0
portion
of hsa-mir-142 to a truncated c-Myc gene in aggressive
B-cell leukemia [19,24]. This translocation most prob-
ably fuses the AX721088 transcription unit to the
c-Myc gene.
In several cases, the miRNA was embedded in a
cluster of intronless ESTs, the orientation of which

could not be ascertained. This however, cannot be con-
sidered as indicative of an antisense transcript, as a
similar observation was made for some miRNAs that
reside in introns in the same orientation as the host
gene (see above).
Taken together, these observations suggest that
when miRNA genes are located in introns of known
genes in the antisense orientation, they might in fact
be part of transcription units in opposite orientation
to the presumptive host gene. In the case of hsa-mir-
302, the microRNA is clearly located in an intron of
Fig. 2. Antisense intronic miRNAs. (A) Localization of hsa-mir-302. This miRNA resides in an intron of the HDCMA18P gene in the antisense
orientation but in an intron of ESTs BG207228 and BU565001 in the sense orientation. In all figures, miRNA genes are colored in green
when they reside on the upper strand, and in magenta when on the lower strand. (B) Localization of hsa-let-7d. This miRNA resides in an
intron of pre-mRNAs BC045813 and BC036695 in the antisense orientation. The last exon of these two mRNAs overlaps the intronless
BC064349 mRNA in the antisense direction. The asterisks indicate the positions of the polyA tails of mRNAs and ESTs. The miRNA
hsa-let-7d resides in a cluster of several intronless ESTs, only some of which are shown. The orientation of this cluster is antisense to that
of the BC045813 mRNA, as indicated by the presence of a polyA tail in the sequence of mRNA BC0644349 and of two ESTs. The corres-
ponding transcription unit might also contain hsa-let-7a-1 and hsa-let-7f-1 in the sense orientation. Figures 2 and 3 are adapted from windows
of the UCSC Genome Browser.
M. J. Weber New human and mouse microRNA gene
FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 65
the antisense transcript (Fig. 2A). In other cases, like
hsa-mir-142, hsa-mir-133a-1 ⁄ mir-1-2 and possibly hsa-
let-7d ⁄ let
)1
-7f-1 ⁄ let
)1
-7a-1, the microRNA is located in
an exon of the antisense transcript.

Human miRNA precursors that overlap with
exons of known genes or ESTs
After intersecting the new human miRNA table of the
UCSC Genome Browser with the chrN_EST and
chrN_mRNA tables, I examined miRNAs that are
included in, or overlap with, exons. The miRNAs that
were only included in intronless ESTs were for the
most part not further examined due to the uncertainty
in EST orientation. In addition, the localization of sev-
eral miRNAs in an exon possibly corresponded to
intron retention events (hsa-mir-126, mir-25 and -mir-
224). Accordingly, these miRNAs were classified as
intronic.
Except for the four examples discussed below (hsa-
mir-135a-1, hsa-mir-99b, hsa-let-7e, hsa-mir-125a),
miRNAs that are embedded in, or overlap with exons
of known transcripts, are always in the same orienta-
tion. The corresponding genes were generally noncod-
ing, except for hsa-mir-198, which resides in the
3
0
-UTR of the FSTL1 (follistatin-like) gene, and for
hsa-mir-133a-2, located in the C20orf166 gene that
encodes a potential 117 amino acid protein. In addi-
tion, hsa-mir-21 probably resides in the sense orienta-
tion in an alternative form of the 3
0
-UTR of the
VMP1 gene, characterized by mRNA BC053563.
Accordingly, mmu-mir-21 is located in the 3

0
-UTR of
the mouse VMP1 ortholog, 4930579A11Rik, also in
the sense orientation.
These three cases are particularly intriguing, as exci-
sion of the miRNA precursor from the host mRNA by
the Drosha endonuclease would probably trigger the
degradation of the rest of the mRNA. A similar mech-
anism is documented in E. coli, where RNase III
cleaves its own mRNA at a stem-loop structure and
triggers its degradation [25]. Similarly, RNase III
cleaves the polycistronic metY-nusA-infB RNA, to
release the metY tRNA and initiate the decay of the
nusB-infB protein-coding mRNA [26]. Whether such a
mechanism also operates in higher eucaryotes remains
speculative. Of note, no rodent orthologue of hsa-mir-
198 could be found by a BLAT search in the mouse
and rat genomes.
Four miRNAs (hsa-mir-34b, hsa-mir-205, hsa-mir-
133a-2 and hsa-mir-99b) overlap a splicing site. In the
three first cases, it is not clear how the miRNA precur-
sor is produced. It might originate either from an
exon, or from an intron of uncharacterized alternative
forms of the host gene mRNA. The case of hsa-mir-
99b is further discussed below.
MiRNAs complementary to expressed sequences
and potential regulation of glycerate kinase gene
expression by miR-135a
The mouse mmu-mir-135a-1 miRNA resides in the
antisense orientation in an alternative, long form of

the 3
0
-UTR of the 6230410P16Rik gene, the ortho-
logue of the human GLYCTK (glycerate kinase) gene
(Fig. 3A). Therefore, mmu-miR-135a is perfectly com-
plementary to the alternative form of Glyctk mRNA
and might regulate its stability by an siRNA mechan-
ism. Interestingly, mmu-mir-135a-1 also resides in an
intron of the spliced AK051019 mRNA in the sense
orientation; the latter might thus be the actual miRNA
host gene. These two transcriptional units are largely
overlapping, so that several genomic segments are
exonic on both strands (Fig. 3A).
A similar situation holds for the human genome:
hsa-mir-135a-1 is located 741-base pairs downstream
of the GLYCTK gene, in the antisense orientation.
This miRNA is embedded in a cluster of 10 overlap-
ping intronless ESTs that might be part of a longer,
alternative form of the GLYCTK 3
0
-UTR (Fig. 3B).
This hypothesis is supported by the fact that several
ESTs of this cluster (AI493054, AI380271, AW204878,
AW207007 and BM555864) are polyadenylated. In
addition, hsa-mir-135a-1 resides, in the sense orienta-
tion, in an intron of EST AI936688. Based on these
observations, it is tempting to speculate that, in both
mouse and human, mir-135a-1 is produced from an
intron of the host gene (AK051019 and AI936688 in
mouse and human, respectively) and can direct the

degradation of a long form of the glycerate kinase
mRNA by an RNA interference mechanism. Accord-
ingly, a switch from GLYCTK to AI936688 gene tran-
scription would be accompanied by the production of
hsa-miR-135a, which could base-pair with pre-existing
GLYCTK long mRNAs and trigger their degradation.
This mechanism would thus block glycerate kinase
production at both the transcriptional and transla-
tional levels, while the shorter form of glycerate kinase
mRNA, which results from the use of an alternative
polyA site, would not be affected. In addition, hsa-
miR-135a could be produced from its second precur-
sor, hsa-mir-135a-2.
As noted above, hsa-let-7e and hsa-mir-125a are
located in the first exon of mRNA AK125996, in the
antisense orientation, whereas hsa-mir-99b overlaps
the splicing donor site (Fig. 3C). Interestingly, the first
exon of mRNA AK125996 overlaps that of the antisense
New human and mouse microRNA gene M. J. Weber
66 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS
mRNA AY358799 over 113 nucleotides. Although the
overlapping region is outside of the miRNA-containing
segment, it is tempting to speculate that these three
miRNAs reside, in the sense orientation, in a longer
form of the AY358799 mRNA, and might regulate the
translation and ⁄ or stability of the mRNA AK125996.
Fig. 3. Antisense exonic miRNAs. (A) Localization of mmu-mir-135a-1. This miRNA resides in the antisense orientation in an alternative lon-
ger form of the 3
0
-UTR of the mouse 6230410P16Rik mRNA. This gene is the orthologue of the human GLYCTK gene, as shown by BLATing

the sequence of the human protein (see BLAT track). The miRNA mmu-mir-135a-1 is also located in an intron of the antisense AK051019
mRNA, indicated in red. (B) Localization of hsa-mir-135a-1. This miRNA resides in a cluster of ESTs downstream of the GLYCTK gene, in the
opposite orientation. The asterisks indicate the localization of the polyA tail of some of these ESTs. In addition, hsa-mir-135a-1 resides in the
sense orientation in an intron of EST AI936688 (indicated in red). (C) Localization of hsa-mir-99b, hsa-let-7e and hsa-mir-125a. The latter two
miRNAs are in the first exon of the AK125996 mRNA, in the antisense orientation, whereas hsa-mir-99b overlaps the splicing donor site.
Note that the AK125996 mRNA overlaps in the antisense direction with the BC041134 and AY358799 mRNAs.
M. J. Weber New human and mouse microRNA gene
FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 67
Only two other vertebrate miRNAs have been previ-
ously shown to be fully complementary to a cellular
mRNA: mmu-mir-127 and mmu-mir-136 reside in the
intronless Rtl1 gene, in the opposite orientation.
Whereas the Rtl1 gene is paternally expressed, the two
miRNAs are only expressed from the maternal chro-
mosome [27]. This reciprocal imprinting suggests that
mmu-miR-127 and mmu-miR-136 regulate Rtl1 gene
expression by an siRNA mechanism. This situation
probably also holds for the human genome, as both
hsa-mir-127 and hsa-mir-136 reside in the inverse ori-
entation within the presumptive human orthologue of
Rtl1 (XM_352144).
Therefore, this study uncovers new cases where a
miRNA might regulate the stability of a cellular
mRNA through an siRNA mechanism. In all cases dis-
cussed above, the miRNA resides on the opposite
strand relative to its target mRNA. This situation dif-
fers from that of miR-196a, which is fully complement-
ary to the 3
0
-UTR of HOXB8 mRNAs, except for a

G ⁄ U wobble [28]. In that case, the miRNA gene resides
at distance from its target gene, although in the same
HOX locus.
MicroRNAs in gene families
As shown in Table 2, many related human precursor
miRNAs reside in corresponding introns of paralogous
genes. The mature miRNAs are either identical (miR-
15, miR-218), closely related (miR-199a and b,
miR26a and b), or display significant homology (hsa-
mir-148b and -152, hsa-mir-107, -103-1 and -103-2).
Therefore, the sequences of these intronic miRNAs
have been largely conserved after gene duplications,
raising the possibility that their function might have
been conserved as well. This also suggests that addi-
tional genes in the families shown in Table 2 might
contain still uncharacterized miRNAs. Intriguingly,
hsa-mir-211 and -204 are located within an intron of
the TRPM1 and TRPM3 genes, respectively, that
bracket paralogous exons. These localizations are con-
served in the mouse genome. However, these two miR-
NAs have no sequence similarity. In this case, it is
thus possible that the presence of miRNAs in the
introns of the TRPM1 and TRPM3 genes is posterior
to the expansion of the TRPM gene family. This
hypothesis is reinforced by the fact that the other
members of the family, TRPM2 and TRPM4 through
TRPM8, do not host known miRNAs. The large dif-
ference in the size of the introns of the TRPM1 and
TRPM3 genes that host mir-211 and mir-204 (3 and
44 kb, respectively) also suggests extensive rearrange-

ments posterior to gene duplication.
It is also intriguing to note that hsa-mir-199b,
-199a-1 and -199a-2 reside in introns of the DNM1,
DNM2 and DNM3 genes, respectively, but in the
opposite orientation (Table 2). As discussed above,
the DNM genes are thus probably not the actual
hosts for these miRNAs. Indeed, hsa-mir-199a-2 is
embedded in a large cluster of ESTs antisense to the
DNM3 gene that also contains hsa-mir-214 (Table
S3). Similarly, mmu-mir-199a-2 and mmu-mir-214
reside in the opposite orientation in an intron of the
dynamin 3 gene (9630020E24Rik) and are embedded
in a large cluster of antisense mRNAs and ESTs (not
shown). Although there is no clear evidence for tran-
scripts antisense to DNM1 and DNM2 genes, these
observations suggest that the conservation of highly
related miRNAs in the DNM gene family might result
from the expansion in the vertebrate genomes of
antisense transcripts in addition to the DNM genes
themselves.
Clusters of microRNAs
The existence of miRNA clusters has been already
noted [14,29,30], but a precise definition of a cluster
Table 2. MicroRNA that reside in introns of paralogous gene
families.
MicroRNA Host gene Function Notes
hsa-mir-199b DNM1 Dynamins
hsa-mir-199a-1 DNM2
hsa-mir-199a-2 DNM3
hsa-mir-26b CTDSP1 CTD RNA polII phosphatases

hsa-mir-26a-1 CTDSPL
hsa-mir-26a-2 CTDSP2
hsa-mir-153–1 PTPRN Protein tyrosine phosphatases
a
hsa-mir-153–2 PTPRN2
hsa-mir-218–1 SLIT2 Slit homologs, axonal guidance
b
hsa-mir-218–2 SLIT3
hsa-mir-107 PANK1 Pantothenate kinases
c
hsa-mir-103–2 PANK2
hsa-mir-103–1 PANK3
hsa-mir-211 TRPM1 Transient receptor potential
cation channels (melastatins)
d
hsa-mir-204 TRPM3
hsa-mir-148b COPZ1 Coatamer protein
complex, subunits zeta
e
hsa-mir-152 COPZ2
a
mmu-mir-153 is located in the Ptprn2 gene. No documented miRNA
resides in the mouse Ptprn gene.
b
No documented miRNA resides in
the human SLIT1 gene. In the October 2003 freeze of the mouse gen-
ome, the Slit2 gene, as evidenced by BLATing the human Slit2 protein,
comes in two parts, located on chr5_random (aa 133–538) and chr5 (aa
539–1529). Mmu-mir-218–1 resides in the chr5_random part.
c

hsa-mir-
103–2 and mir-103–1 are closely related to hsa-mir-107 (87 and 91%
identity, respectively).
d
hsa-mir-211 and )204 have no sequence homol-
ogy.
e
hsa-mir-148b and )152 display significant homology (77% identity
over 66 nucleotides).
New human and mouse microRNA gene M. J. Weber
68 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS
was not given. I suggest here three criteria: miRNAs in
a cluster are in the same orientation, and not separated
by a transcription unit or a miRNA in the opposite
orientation. This definition is clearly more restrictive
than that used by others [31]. Using such a definition,
hsa-mir-10a and hsa-mir-196–1, although 52 kb apart,
do not form a cluster, as they are separated by five
HoxB genes. Similarly, hsa-mir-181c was not included
in the cluster formed by hsa-mir-24-2, -27a and -23a,
as it is not in the same orientation. The condition that
clustered miRNAs have the same orientation might
imply that they originate from the nucleolytic degrada-
tion of a unique transcript, as previously shown in
HeLa cells for the hsa-miR-23 ⁄ 27 ⁄ 24-2 and hsa-miR-
17 ⁄ 18 ⁄ 19a ⁄ 20 ⁄ 19b-1 clusters [32]. A comprehensive list
of 37 human miRNA clusters is presented in Table S4.
All of them are conserved in the mouse genome,
although some mouse clusters contain additional
members, for which no human orthologue could be

detected (mmu-mir-300, -329 and -341).
Among the 37 human clusters, 19 are located in Ref-
Seq genes, known genes, or mRNAs from GenBank
while 18 lie outside of any characterized transcription
unit. In no case are clustered miRNAs located in differ-
ent introns of the same gene. This contrasts with intro-
nic snoRNAs, for which an intron always carries a
single snoRNA, but multiple introns of the same gene
can carry different, or highly related snoRNAs [33–35].
This apparent difference between miRNAs and snoR-
NAs might be in fact only provisional, as the maternally
expressed mouse Mirg gene may contain several
miRNAs in different exons [27]. Moreover, clustered
miRNAs are in three cases distributed between the
intronic and exonic parts of a gene. First, hsa-let-7c and
hsa-mir-125b-b2 are located in the same exon of mRNA
AK125996, whereas hsa-mir-99b resides on the intron–
exon junction, as discussed above (Fig. 3C). Second,
hsa-mir34b is located within an intron of mRNA
BC021736, whereas hsa-mir-34c resides in an adjacent
exon of the same transcript. Finally, hsa-mir-145 is
located within the intronless AK093957 mRNA, whereas
hsa-mir-143 resides outside of the mRNA. These special
cases will probably be more fully understood when
additional transcripts are available in the data bases.
The three miRNA clusters composed of hsa-mir-
125b-1, -let7a-2, -mir-100 (chr11), hsa-mir-125b-2, -let-
7c, -mir-99a (chr21) and hsa-mir-125a, -let-7e, -mir-99b
(chr19) are composed of related precursors (Table S4).
Interestingly, hsa-mir-125b-2 is the closest homologue

of C. elegans lin-4. The first two clusters have already
been described and are conserved in the mouse gen-
ome, while only one copy of the cluster resides in the
D. melanogaster and Anopheles gambiae genomes
[36,37]. The present identification of hsa-mir-99b thus
allows for the identification of an additional cluster
on human chromosome 19. In the human genome,
no sequence similarity was found in the mRNAs that
host the three clusters (AK091713, AK125996 and
AK095614, respectively). The chr11 and chr21 clusters
reside in an intron of their host gene, but no homolo-
gies in the two introns containing the clusters were
found outside of the miRNA sequences. In contrast,
two miRNAs of the chr19 cluster, hsa-mir-125a and
hsa-let-7e were in an exon of the AK125996 host gene
in the antisense orientation, whereas hsa-mir-99b
encompassed the intron–exon junction, as noted above
(Fig. 3C). Remarkably, the distances between the
miRNA in a given cluster are conserved between the
human and mouse genomes, but vary considerably
between clusters (Table S4). In contrast to the cases
shown in Table 2, these three clusters were thus prob-
ably not generated by the duplication of their host
gene. Functional studies are needed to better under-
stand the reasons why the miRNAs of the miR-
125 ⁄ lin-4, miR-100 ⁄ 99 and let-7 families reside in clus-
ters. Interestingly, miR-125, miR-100 and let-7 are co-
regulated during Drosophila pupal development, and
are expressed from a common precursor [36–38].
The newly described hsa-mir-299, -mir-323 and -mir-

329 miRNAs (Table 1) are part of a cluster that also
includes hsa-mir-134 and -mir-154 on chr14q32.31.
The mouse cluster contains in addition mmu-mir-300,
as well as several copies related to mmu-mir-134 ⁄ 154
[27]. In both human and mouse, this locus is subjected
to genomic imprinting [27,39]. Mmu-mir-154 (and
possibly other members of the cluster) resides in an
intron of the maternally expressed, noncoding Mirg gene
(AJ517757), which is poorly conserved in the human
genome. In this miRNA cluster, several miRNAs
are conserved between human and mouse: miR-323,
miR-134, miR-154, miR-323 and miR-299, while hsa-
miR-329 differs from its rodent orthologue by four nu-
cleotides (Table 1). Moreover, the human orthologue
of mmu-mir-300 could not be found by a BLAT
search. The reason for such a differential conservation
of miRNAs within this cluster will probably be under-
stood when their function is elucidated.
Prediction of miRNA candidates near known
miRNA genes
The analysis of highly conserved regions with a hairpin
structure near known miRNAs can reveal numerous
new miRNA candidates. For example, such a sequence
resides 94 base pairs from hsa-mir-144 (Figure S1A).
The orthologous mouse sequence is located 101 base
M. J. Weber New human and mouse microRNA gene
FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 69
pairs from mmu-mir-144. Two other candidates are
located near hsa-mir-195, in the intronless AK098506
mRNA (Figure S1B–C). This genomic region contains

additional discrete peaks of conservation between the
human, chimpanzee, mouse and rat genomes (Figure
S1D) and might thus contain additional candidates. As
pointed out in reference [8], there are more conserved
hairpin structures in the human genome than they are
miRNAs. However, restricting the infomatic search of
conserved hairpin structures to the vicinity of known
miRNAs will certainly unravel many interesting candi-
dates.
Conclusions
A systematic search of orthologues of known rodent
microRNA precursors in the human genome has led to
the identification of 35 new miRNA genes. Similarly,
42 new mouse miRNAs were found by orthology with
known human miRNA precursors. Added to those
deposited in the miRNA registry, about 200 miRNA
genes are now localized in both human and mouse
genomes, a number close to the total number (255)
predicted by Lim et al. [13].
Among the 196 human miRNA precursors analyzed,
98 were located in introns of known genes, 81 in the
sense orientation and 17 in the antisense orientation.
In the latter case, it is suggested that the miRNA in
fact belongs to an antisense transcription unit. Several
cases were presented where miRNA precursors are
totally included in a polyadenylated mRNA, some-
times as clusters. These data suggest that miRNA pre-
cursors can be generated by two mechanisms.
Precursors could be excised from introns, most
probably after splicing. As miRNA precursors are

excised by the Drosha endonuclease [1], their genera-
tion might occur independently of intron debranching.
This mechanism can also apply for certain miRNA
clusters. For example, the cluster of miR-17-18-
19a20-19-1 was detected by RT-PCR in a 0.4-kb
product [32]. This cluster resides in a 3.8-kb intron of
the BC040320 mRNA. The RT-PCR product might
thus originate either from the primary transcript before
splicing, or from a more stable product during the
degradation of the spliced intron. It remains to be
examined whether the cell ⁄ tissue specificity of the
expression of the host gene fully accounts for the spe-
cificity of miRNA expression [19,40,41]. Moreover, the
compilation of expression data might indicate whether
the host gene of a miRNA and candidate target genes
for the miRNA are expressed in the same cells.
Alternatively, miRNA precursors could also be gen-
erated from the exonic part of RNA polymerase II
transcripts. This might be the case for most miRNAs
classified as intergenic. It appears that such transcripts
are for the most part non protein-coding, often intro-
nless and poorly conserved and can carry a cluster of
miRNAs.
These two possible mechanisms present similarities
with those whereby snoRNAs are generated. In verte-
brates, snoRNAs (except for U3, U8 and U13) are
located in introns of host genes [42–44] and arise from
the exonucleolytic degradation of spliced, debranched
introns. A minor, splicing-independent pathway invol-
ving endonucleolytic cleavage within the intron has

also been identified in yeast and rat [33,45–48]. In
addition, in plants and certain yeasts, snoRNAs can be
produced from independent, polycistronic transcription
units. Differences exist, however, between miRNA and
snoRNA localizations. First, an intron always contains
a single snoRNA but can contain several miRNAs.
Second, a single gene can host several snoRNAs in
various introns, but a gene contains in general only
one intronic miRNA, or one cluster of miRNAs in a
single intron. The single known exception for this sec-
ond rule is the maternally expressed mouse Mirg gene,
which hosts several miRNA candidates in different
exons [27]. Whether this situation is restricted to loci
submitted to genomic imprinting deserves further
experiments.
The identification of miRNA targets in animals is
made difficult by the imperfect complementarity
between the miRNA and the target mRNAs [21,49–
51]. Therefore, only few miRNA targets have been
proposed in mammals [27,28,49]. Here, I provide three
new examples of a perfect complementarity between a
miRNA and a cellular mRNA: mmu-miR-135a, whose
sequence is fully complementary to a long form of the
3
0
-UTR of the mouse glycerate kinase gene, generated
by the use of an alternative polyA site. The same situ-
ation probably holds true in human. Similarly, hsa-let-
7e and hsa-miR-125a are perfectly complementary to
parts of an exon of the noncoding AK125996 mRNA.

The significance of this second case is however, uncer-
tain, as the AK125996 mRNA is only conserved in the
mouse genome in the portions that correspond to the
miRNA precursors.
A similar study was performed [52] and an analysis
of miRNAs hosted in the Mirg gene has also been
published [53].
Methods
The sequences of human and mouse mature (miR) and pre-
cursor (mir) miRNAs were obtained from The miRNA
Registry (version Rfam2.2), and the entire set of sequences
New human and mouse microRNA gene M. J. Weber
70 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS
was subjected to a BLAT search [54] in the human and
mouse genomes (UCSC version hg16, July 2003 and UCSC
version mm4, October 2003, respectively) on the UCSC
Genome Browser server. The BLAT results were exported
to two Excel
TM
tables, one for each genome, that were then
ordered according to chromosome number and BLAT hit
chromosomal position. This allowed for the determination
of the position of the closest match of a mouse miRNA in
the human genome, and vice-versa. Sequences correspond-
ing to potential new miRNA precursors were obtained from
the UCSC Genome Browser, and their hairpin structure
was assessed with the mfold program [55] (available
at />Multiple alignments were performed with the multalin
program [56] (available at />multalin/multalin.html).
Acknowledgements

I thank Sam Griffiths-Jones (The Wellcome Trust
Sanger Institute) for checking the new miRNAs before
their addition to The miRNA Registry, and for his
help in the submission of custom tracks to the UCSC
Genome Browser; Jim Kent, Donna Koralchik and Hi-
ram Clawson for integrating the microRNA tracks in
the UCSC Genome Browser. I thank Emmanuel Ka
¨
s
(CNRS, Toulouse, France) for helpful discussions and
Tamas Kiss and Herve
´
Seitz (CNRS, Toulouse,
France) for their critical reading of the manuscript.
This work was supported by grants from CNRS and
La Ligue Nationale contre le Cancer to Tamas Kiss.
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Supplementary material
The following material is available from http://www.
blackwellpublishing.com/products/journals/sup pmat/
EJB/EJB4389/EJB4389sm.htm
Appendix S1. Human and mouse miRNA precursors
reside in conserved regions of synteny.
Fig. S1. miRNAs gene candidates clustered with
known miRNAs genes.
Table S1. New microRNA precursors deposited in The

miRNA Registry (version 3.0), at the Wellcome Trust
Sanger Institute.
Table S2. Human miRNAs that reside in introns of
known genes in the sense orientation.
Table S3. Human miRNAs that reside in introns of
known genes in the reverse orientation.
Table S4. Clusters of human microRNAs.
M. J. Weber New human and mouse microRNA gene
FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 73