Identification and Characterization of a Novel Non-Coding RNA Involved in Sperm Maturation
et al. (2011) Identification and Characterization of a Novel Non-Coding RNA Involved in Sperm Maturation. PLoS
ONE 6(10): e26053. doi:10.1371/journal.pone.0026053
Identification and Characterization of a Novel Non- Coding RNA Involved in Sperm Maturation
Lian Zhang 0
Min-Jie Ni 0
Zhi-Hong Hu 0
Qiang Liu 0
Mo-Fang Liu 0
Min-hua Lu 0
Jin-Song Zhang 0
Li Zhang 0
Juan Mata, University of Cambridge, United Kingdom
0 1 Shanghai Key Laboratory of Molecular Andrology, State Key Laboratory of Molecular Biology , Shanghai , China , 2 Core Facility for Non-Coding RNA, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences , Shanghai , China , 3 Shanghai Institute of Planned Parenthood Research , Shanghai , China
A long and ever-expanding roster of small (,20-30 nucleotides) RNAs has emerged during the last decade, and most can be subsumed under the three main headings of microRNAs(miRNAs), Piwi-interacting RNAs(piRNAs), and short interferingRNAs(siRNAs). Among the three categories, miRNAs is the most quickly expanded group. The most recent number of identified miRNAs is 16,772 (Sanger miRbase, April 2011). However, there are insufficient publications on their primary forms, and no tissue-specific small RNAs precursors have been reported in the epididymis. Here, we report the identification in rats of an epididymis-specific, chimeric, noncoding RNA that is spliced from two different chromosomes (chromosomes 5 and 19), which we named HongrES2. HongrES2 is a 1.6 kb mRNA-like precursor that gives rise to a new microRNA-like small RNA (mil-HongrES2) in rat epididymis. The generation of mil-HongrES2 is stimulated during epididymitis. An epididymis-specific carboxylesterase named CES7 had 100% cDNA sequence homology at the 39end with HongrES2 and its protein product could be downregulated by HongrES2 via mil-HongrES2. This was confirmed in vivo by initiating milHongrES2 over-expression in rats and observing an effect on sperm capacitation.
Funding: This work was supported by grants from the National Basic Science Research and Development Project of China [2006CB504002] and the Chinese
Academy of Sciences Knowledge Innovation Program [2006CB944002]. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript
Competing Interests: The authors have declared that no competing interests exist.
Cells face a wide range of threats and regulatory
demands.Among the many tools available to meet these challenges is a
collection of pathways that use small (,2030 nucleotides) RNAs
to recognize target nucleic acids and present them to specific
effector complexes that generally inhibit gene expression . In
animals, most of these RNAs could be sorted into three main
groups, namely microRNAs (miRNAs), Piwi-interacting RNAs
(piRNAs), and short interfering RNAs (siRNAs). Although they
share some common features, each RNA category can differ from
the others in various ways, including length, precursor structure,
cofactor requirement, modification state, sequence bias, and
regulatory function, and the differences can themselves vary
between species .
The recently established and most quickly expanding subgroup
of regulatory RNAs, the microRNAs (miRNAs) is composed of
1825 nucleotide long molecules that control the expression of
their target genes via antisense base pairing [3,4]. The genes
encoding miRNAs are initially transcribed as long, primary
microRNAs (pri-miRNAs), which vary in length from hundreds
to thousands of nucleotides. The pri-miRNAs are then sequentially
processed by two RNase-III enzymes, Drosha and Dicer, into a
stem-loop pre-miRNA, generating an imperfect double-stranded
RNA (dsRNA) duplex that contains both the mature miRNA
strand and its complementary strand (miRNA*) . For
posttranscriptional gene silencing, the mature miRNA strand is loaded
into the effector complex, the RNA-induced silencing complex
(RISC), and RISC guides messenger RNAs (mRNAs) to their
complementary sequences. Mammalian miRNAs have been
shown to be differentially expressed in specific cell types, tissues,
and embryonic stem cells. Distinct miRNA expression profiles
have also been associated with different diseases and diverse
developmental and physiological processes [7,8,9].
To avoid designating siRNAs or fragments of other RNAs as
miRNAs, miRNAs are identified using a combination of criteria
for both their expression and biogenesis. Expression criteria
include detection of a distinct ,22-nt RNA transcript by
hybridization to a size-fractionated RNA sample via the northern
blotting method (expression criterion A ), and identification of the
,22-nt sequence in a library of complementary DNAs (cDNAs)
made from size-fractionated RNA (expression criterion B). Other
criteria include prediction of a potential fold-back precursor
structure that contains the ,22-nt miRNA sequence within one
arm of the hairpin.(biogenesis criterion C) , phylogenetic
conservation of the ,22-nt miRNA sequence and its predicted
fold-back precursor secondary structure (biogenesis criterion D),
and detection of increased precursor accumulation in organisms
with reduced Dicer function (biogenesis criterion E).
Ideally, a miRNA is identified if an asymmetric ,22-nt product
accumulates (in vivo), and is processed from a phylogenetically
conserved hairpin precursor by Dicer (A + D + E). However,
among these criterion, criterion A is the most important, so that in
the absence of processing data, A + D is sufficient. Therefore, a
candidate gene can still be annotated as an miRNA gene if they
are in the line with .A + C, B + D or D + E .
Mammalian spermatogonia undergo mitosis, meiosis, and some
morphological changes, becoming fully differentiated, but not
mature, sperm in the testis. Most of the sperm maturation occurs
in the epididymis [12,13], so it is an ideal research target organ
that could yield better understanding of the molecular mechanism
of sperm maturation and provide new ideas for the design of male
fertility-control drugs, personalized infertility diagnosis, and
treatments and evaluations of sperm health. However, few studies
on smRNAs in the epididymis, have been reported [14,15,16].
The Mammalian Gene Collection(MGC)and FLJ Human
cDNA Database together reported a total of 24,409 cDNAs with
full-open reading frames (ORFs) from more than 200 cDNA
libraries. This number of genes is close to that predicted by the
International Human Genome Sequencing Consortium in 2004
(20,00025,000). However, an epididymis cDNA library was not
present in the sequencing target list.
To identify genes that are important in the rat epididymis, a rat
epididymis cDNA library was screened and a 1.3-kb expressed
sequence tag (EST ) was found and named HongrES2.
HongrES2 was subsequently identified as a novel, 1.6-kb,
epididymis-specific, mRNA-like, chimeric noncoding RNA that
produces a microRNA like small RNA (mil-HongrES2).
MilHongrES2 down-regulates CES7 gene expression and is involved
in the sperm epididymal maturation process.
Inter-chromosomal chimeric RNAs have been reported in a
variety of organisms. These encode proteins [18,19] that are
present in abnormal cells, such as cancer cells. Sklars group
reported a neoplastic gene fusion that mimics the trans-splicing of
RNAs in normal human endometrial stromal cells [19,20].
Recently, a genome-wide screening of chimeric RNAs in budding
yeast, fruit fly, mouse, and human genomes identified thousands of
chimeric transcripts in all organisms except for yeast, in which
only five chimeric RNAs were observed . To the best of our
knowledge, the 1.6-kb chimeric transcript (HongrES2) identified in
the present study is the first reported epididymis-specific full-length
non-coding RNA derived from two chromosomes.
Materials and Methods
Healthy male Sprague Dawley (SD) rats were purchased from
the Animal Center of the Chinese Academy of Sciences (Shanghai,
China). They were housed for 710 days in the animal housing at
our institute before manipulation. Food and water were freely
available throughout the experiments. The protocol conforms to
internationally accepted guidelines for the humane care and use of
laboratory animals. All research involving animals were conducted
according to the approval of the Institute Animal Care Committee
of Shanghai Institute of Biochemistry and Cell Biology. The
approved permit number for this study is SYXK2007-0017.
Total RNA and small RNA isolation and northern blot
Tissue samples were obtained from male rats after they were
sacrificed, and the samples were immediately frozen in liquid
nitrogen. Total RNA was extracted for northern blot analysis,
which was performed according to a previously described
procedure . Twenty micrograms of total RNA from each
sample was loaded into each lane. The probe was a 32P-labeled
504-bp cDNA fragment (seqNo 861-1364-nt) of rat HongrES2 that
was cut from the vector (T-easy-H2). An 18S rRNA hybridization
signal was used as a loading control.
The Ambion mirVanaTM miRNA Isolation Kit was used for
small RNA isolation, according to the manufacturers protocol.
Small RNAs (30 mg per sample, ,200 bp), including miRNAs,
were separated on a 12% acrylamide/8M urea denaturing
polyacrylamide gel before being transferred to an Ambion
BrightStarPlus Nylon membrane.
Hybridization was carried out using a 24 bp LNA probe
purchased from Exiqon (Woburn, MA, USA). Northern blot
analysis was performed according to a protocol published on the
Exiqon website (http://www.exiqon.com). All of the Northern blot
analyses were carried out at 65uC under high stringency
RACE analysis, PCR performance ,ORF identification and
secondary structure analysis
The BD SMARTTM RACE cDNA Amplification and the
Ambion First Choice RLM-RACE kits were employed for first
and second rounds of 59RACE, respectively, according to the
manufacturers protocols, two arounds of PCR amlifications were
used, and 40 cycles of amplification were used in each round.
Amplification of the 39 end (39 RACE) was performed by the
Ambion FirstChoice RLM-RACE kit. The Amplified fragments
were cloned into pGEM-T-Easy (Promega) for sequence analysis.
RT-PCR for the 1258 bp fragment of HongrES2 cDNA in
Figure 1D was amplified with nested PCR strategy (two arounds
with different upper primer and the same lower primer). The
727bp and 504-bp fragments of HongrES2 cDNA in Figure 1D and
Figure 2B was gained without nested PCR. The Takara Ex -Taq
PCR reaction system was applied, the annealing temperature was
55uC, and the amplification was carried out under 40 cycles.
ORF searching of the full-length sequence of HongrES2 was
carried out using the ORF Finder Program (http://www.ncbi.
nlm.nih.gov/gorf/gorf.html). The secondary structure of the
sequence, beginning from seqNo 1365 and extending to seqNo
1588, was predicted by the mfold server (http://mfold.rna.albany.
edu/?q = mfold/download-mfold).
For mRNA quantification,a total of 2 ug of RNA prepared as
described above was used for reverse transcription, which was
performed with a ReverTra Ace-a-TM kit (Toyobo Co., Osaka,
Japan) according to the manufacturers instructions. Real-time PCR
was performed in a total volume of 20 ul of reaction mixture
containing 10 ul of SYBR Green Real-time PCR Master Mix
(Toyobo).Amplification of gene of interest cDNA was normalized to
that of Gapdh. Results are calculated by two standard curve methods
and expressed as the fold-increase of gene of interest cDNA compared
with the control. For small RNA quantification an improved methods
were carried out according to the previous publication .
The gene of interet (mil-HongrES2)was defined as 1 U,and the
other was normalized to it
Tissue section preparation and in situ hybridization
Adult rat tissue slides were prepared as described previously
. In situ hybridization was carried out according to previously
described methods . DIG-labeled antisense and sense RNA
probes were transcribed by T7 and SP6 RNA polymerase
(Promega) from the pGEM T-easy-H2 vector and the
pGEM-Teasy-bin1b vector, and NBT/BICP (Roche) was used as the AP
substrate to generate a purple signal. The FITC-labeled antisense
probe was transcribed by SP6 RNA polymerase from the
pGEMT-easy-CES7 vector, and INT/BCIP (Roche) was used as the AP
substrate to give a brown signal.
Transfection and luciferase assay
Transfections were performed using Lipofectamine 2000
(Invitrogen), according to the manufacturers protocol. For
examination of the reduction of CES7 protein expression, the
pcmv-tag4a-CES7 and Pcmv-tag4a-H2 (pcmv-tag4a-H2T or
pcmv-tag4a/mock) constructs (1:3.5) were co-transfected into
PC1 cells  cultured in 6-well plates. For examination of
Renilla luciferase activity variation, pRL-TK vectors were
constructed with the 39 end of CES7, pGL-3 vectors were used
as an internal control and the HongrES2 expression vector (1:0.1:2)
was co-transfected into cells cultured in 96-well plates. The relative
luciferase activity was assayed by Berthold multilabel reader
Mithras LB940 after 48 hours of culture. The vector for the
analysis of luciferase activity was pRL-TK (Promega), with the
wild-type 271 bp fragment of the CES7 39 end (seqNo1858-2129)
sequence or its target site mutant 39 end fragment obtained by
Protein extracts and Western blot analysis
Total protein extracts of the cultured cells after transfection
were prepared using RIPA buffer with protease inhibitors. Total
protein extracts for each cell sample (30 mg/lane) were separated
onto 12% SDS-PAGE gels and transferred to polyvinylidene
difluoride membranes (Amersham Pharmacia Biotech). A
polyclonal antiserum against the recombinant rat CES7 protein was
used as the primary antibody (dilution 1:10,000). The secondary
antibody was a goat horseradish peroxidase (HRP)-conjugated
anti-rabbit immunoglobulin G (IgG; dilution 1:20,000;
CalBiochem). Peroxidase activity was detected with a chemiluminescence
substrate (Western Blot Chemiluminescence Reagent Plus;
Amersham Pharmacia Biotech). The vector used for the
expression of the HongrES2 RNA and CES7 protein in PC1 cells
contained the full-length sequence (pcmv-tag4a). A mouse
monoclonal antibody specific for Dicer (cat. No. ab14601) was
purchased from Abcam and used at a 1:500 dilution. A polyclonal
antibody against FLAG was purchased from Sigma (F7425) and
used at a 1:300 dilution. All quantifications were carried out using
PC1 cells were co-transfected with the HongrES2 expression
plasmid and a FLAG-tagged hAgo2 expression plasmid (a gift
from Li-Gang Wu, SIBS) using Lipofectamine 2000 (Invitrogen).
Forty-eight hours after transfection, cells were collected by
scraping and resuspended in 500 mL lysis buffer (50 mM Tris
[pH 7.5], 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 0.5%
Nonidet P-40, and 1 mM dithiothreitol). Lysates were cleared by
centrifugation at 16,0006 g for 10 minutes. One third of each
cleared lysate was added to 1 mL Trizol and saved for assaying the
total input. The remaining lysates were mixed with 15 ml
antiFLAG rabbit polyclonal antibody-conjugated M2 beads (Sigma
cat.# F7425) and rotated at 4uC overnight. The beads were then
sedimented by centrifugation and washed 3 times in lysis buffer.
Eighty percent of the beads were resuspended in 1 mL Trizol for
RNA isolation, and 20% of the beads were resuspended in 20 mL
2X complete Laemmli buffer for protein analysis .
Two siRNA sequences targeting the ORF of the mouse Dicer1
gene were designed using published sequences [27,28]. The
siRNAs were transfected using Lipofectamine 2000 at a level of
6 mg siRNA per dish (100-mm dish). The cells were collected after
48 hours for subsequent analysis.
Infection SD rat model construction
Pathogenic bacteria were obtained from human patients with
epididymitis and 3 adult SD rats were inoculated with cultured
bacteria in their cauda tubules. NaCl solution (0.9%) was used as a
negative control and was injected into the cauda tubules in 3
additional adult SD rats. The volume injected per animal was
25 mL, and the amount had an optical density of AOD600 = 3.0.
The Staphylococcus and Morganella strains were cultured about
eight hours and their optical density was examined and adjusted
equally to AOD600 = 1.0 (1OD bacteria = 2.56108 cfu/ml)by the
bacteria culture solution(LB solution) before they were injected
into the rat epididymis. The scrotum was not cut open when the
rats were injected. The treated animals were fed for 4 days before
being sacrificed. The pathogenic bacteria of human patients with
epididymitis were obtained from Shanghai Jiao Tong University
School of Medicine. Informed consent was obtained from all
participants, this consent was written, and the study was approved
by the Institutional Ethics Board of School of Medicine, Shanghai
Jiao Tong University.
Construction of the small RNA library
Rat epididymis microRNA libraries were constructed to find
novel microRNAs expressed in this male organ, and the caput
sublibrary was used. The basic protocol is described in the Cloning
of Small RNA Molecules published by Current Protocols in Molecular
Biology (2003) 26.4.1-26.4.8. The total RNA of each tissue was
extracted using the Trizol reagent (Invitrogen). The fraction of 18
25 nt small RNAs was excised from a 12% acrylamide/8 M urea
denaturing polyacrylamide gel and purified to construct the small
RNA library. Adapters for the 39 and 59 ends were ligated onto the
RNA, and the following steps were carried out according to the
method of Cloning of Small RNA Molecules of Current Protocols
in Molecular Biology [29,30].
Mil-HongrES2 over-expression SD rat model construction
An miRNA analog (agomir) of mil-HongrES2 was purchased
from GuangZhou RiboBio.Co., Ltd , and injected into the
cauda epididymis of adult male rats (450 g500 g) at a dose of
4 nmol per side. A scrambled miRNA agomir was used as the
negative control. The animals were sacrificed 3 days after injection
for further analysis.
Evaluation of sperm capacitation
We applied the protocol of protein tyrosine phosphorylation
assessment as used in previous studies. The spermatozoa from the
cauda epididymis of the mil-HongrES2 over-expression rats and the
control rats were released into the capacitation medium (94.6 mM
NaCl, 25 mM KCl, 1.71 mM CaCl2, 1.19 mM MgSO4,
1.19 mM KH2PO4, 25 mM NaHCO3, 5.56 mM glucose,
10.76 mM sodium lactate, 0.5 mM sodium pyruvate, 0.002%
phenol red, 4 mg/ml bovine serum albumin; 50 mg/ml
streptomycin sulfate, and 75 mg/ml potassium penicillin, pH 7.4,
osmolarity ,310 mosmol/kg). The sperm pellet was suspended
in SDS sample buffer, and total sperm protein was separated by
SDS-PAGE with 8% Tris-glycine gels. Tyrosine-phosphorylated
proteins from the spermatozoa were detected by western blotting
with mouse monoclonal anti-phosphotyrosine4G@
antibody(Millipore,catNo 05-321) at a diluton of 1:10,000, and the a-tubulin
was used as the internal control.
The sequences of primers, probes, and siRNAs used are listed in
Table S1. The vectors used are listed in Table S2.
Cloning of a novel mRNA-like transcript (HongrES2)
To identify the homologous gene of monkey CES7 in rat, a rat
epididymis cDNA was screened , using a 163 bp probe of a mouse
sequence that was homologous to the 39 end fragment of the
monkey CES7 gene ( the probe sequence see Figure S1). Among
the plaques we obtained, except for the rat homologous CES7
gene EST, which has already benn reported [32,33], another two
new gene ESTs were found as by-products and named HongrES1
EST and HongrES2 EST. Over the past several years our lab has
sequentially reported the identification and biological function of
the new gene HongrES1 which was a new member of serpin family
in rat epididymis  .
The present paper focused on the identification and
characterization of the remaining 1.3 kb new gene HongrES2 EST
(Figure 1A). Northern blot analysis with a region of this HongrES2
EST (504-bp) as a probe (probe1 in Figure1C, lower panel)
detected an mRNA (approximately 1.6 kb) in the rat epididymis
(Figure. 1B, left panel).
Two additional cDNA fragments, 159-bp and 169-bp in size,
were obtained after two rounds of 59RACE. No additional
sequences were obtained with a third round of 59RACE. The
newly achieved 328-bp cDNA by 59 RACE was continued on
genome and combined with the 328 bp additional cDNA
fragment, the full-length HongrES2 cDNA was 1588-bp, which
was roughly equal in size to the mRNA detected in the northern
The ORF Finder Program was used to identify the potential
ORF in HongrES2 cDNA. Unexpectedly, many stop codons were
spread uniformly throughout the sequences and no ideal ORFs
could be found. Only three short fragments were spread along the
entire transcripts with average length of about 144-bp (Figure. 1E,
upper panel). An added poly(A) signal (AATAAA) was found at the
39 end of HongrES2 cDNA, because HongrES2 was screened from a
cDNA library constructed with an oligo dT primer(Figure. 1E,
lower panel). Using an Ambion First Choice RLM-RACE kit,
which can be used to identify full-length mRNAs with a 59 cap, the
59 terminal sequence of HongrES2 cDNA was successfully
obtained. Thus, HongrES2 was found to be an mRNA-like,
noncoding RNA with a 59 cap and 39 poly(A) tail.
With further genomic sequence analyses, this transcript was
unexpectedly found to be chimeirc, that is derived from two
different chromosomes: chromosome 5 for 11379 nt of the
cDNA, and chromosome 19 for 13731588 nt of the cDNA. The
two chromosome shared seven nucleotides (CTGCTTT) , which
may mark a potential splicing site. The genome location of the 59
fragment for 11379 nt of the HongrES2 cDNA was in the
chromosomal region 5q2223 and the 39fragment for 1373
1588 nt in the region 19p1314 (Figure. 1C, upper panel). Using
Blast analysis and comparing the sequences of the HongrES2 and
rat CES7 cDNA carefully, the two genes were found to share one
common 39 end, which is a 216-bp cDNA fragment from
chromosome 19 (Figure. 1C lower pannel). The fragments on
each chromosome in this chimeric transcript were contiguous
without introns. Using the Ambion FirstChoice RLM-RACE kit,
the HongrES2 39 end sequence was validated by 39RACE spanning
two sides of the junction portion, which suggests that the 59 and 39
ends of HongrES2 were in the same transcript (Figure 1A) and
ensuring that this was not a cloning artifact. Meanwhile, reverse
transcription polymerase chain reation (RT-PCR) of rat cauda
epididymis RNA and two different primer pairs spanning the
junction portion was carried out to check the corresponding
chimeric transcripts (Figure. 1D). The 1258-bp band in lane1 was
cloned and sequenced to confirm that it was exactly the HongrES2
sequence. Northern blot analysis was performed with another
probe (probe2 in Figure. 1C, lower panel) containing the 39end
region (from chromosome 19 ) of this HongrES2 EST to further
check that the cloned cDNA of HongrES2 contains sequences from
two separate chromosomes and were not generated because of an
artifact during PCR. Two bands were detected(Figure 1B middle
panel). The lower band has the same size as the band obtained by
probe1 in rat epididymis and the higher band (approximately
2 kb) was set as the signal of rat CES7 mRNA, because when the
rat CES7 specific probe (probe3 in Figure. 1C lower panel)
containing only the 59end region of CES7 cDNA was used only
one band of approximately 2-kb was detected in rat epididymis
(Figure 1B right panel).
These results showed that HongrES2 was a mRNA-like chimeric
noncoding RNA. Its full-length cDNA sequence was submitted to
GenBank (Accession No FJ201982) and was presented in
Figure 1E, lower panel.
HongrES2 RNA is expressed spatially and temporally in
According to Northern blot analysis, HongrES2 RNA was
predominantly expressed in the adult epididymis and was not
detected in eleven other tissues tested (Figure 2A). HongrES2 RNA
was detected in the cauda region of the epididymis when probe1
was used in the Northern blot analysis of rat epididymis total RNA
(Figure 2B, upper panel). However, HongrES2 transcripts could be
detected in both the caput and corpus region, under the detection
sensitivity of RT-PCR.. Real-time PCR analysis revealed that
HongrES2 RNA was predominantly expressed in the cauda region,
about 70% and 85% amount off in the corpus and caput regions
respectively.(Figure 2B, lower panel). These implied that although
HongrES2 RNA was expressed in the caput, corpus, and cauda
regions of rat epididymis, its expression level and/or degradation
rate (RNA stability) might not be uniform throughout the
epididymis tubules, which was exhibiting spatial proneness. In
situ hybridization of HongrES2 revealed that the signal was mainly
localized in the distal cauda region,a weak signal in the initial
segment of the caput epididymis was detected with longer staining
time.In situ hybridization of CES7 indicated intense signals
throughout the whole rat epididymis from distal caput to proximal
cauda region,which was quite differet from the expression pattern
of HongrES2 (Figure. 2C).
Northern blot analysis was performed to determine the onset of
HongrES2 RNA expression. HongrES2 RNA began to be detected
approximately at 30 days of age. It increased gradually and
remained at a stable level until the animal was 450 days age, when
the expression level was reduced slightly (Figure. 2D). The
expression was also up-regulated by androgen, which was
determined in castrated animals (Figure. 2E).
HongrES2 RNA is the precursor of a new miRNA-like small
RNA( mil-HongrES2) in rat epididymis
HongrES2 was investigated as RNA-coding transcript because no
ideal ORFs could be found in the cDNA. In situ hybridization of
the HongrES2 RNA was performed. Small nuclear RNA (SnRNA)
U6, a known nuclear RNA, was used as a positive control, and a
known cytoplasm localization gene, Bin1b was used as another
control probe[35,36]. Comparaing the expression pattern of
HongrES2 with those of snRNA6 and Bin1b RNA, the signal
localization of HongrES2 was found to be more similar with the
SnRNA6 than the Bin1b.This suggested that HongrES2 RNA
might be prone to gathering around the nuclear area. In previous
reports, pri-miRNAs were considered to have the nuclear
localization traits [37,38,39], suggesting that HongrES2 might be
a potential primary microRNA (Figure. 3A).
Further attempts to predict the secondary structure of the 39
end, 224 bp (seqNo 13651588) fragment of HongrES2 were
carried out using the mfold program. The fragment seqNo 1428
1538 from chromosome 19 (with purple label in Figure 3) was
predicted to fold into a stem-loop hairpin structure that lack
perfect Watson-Crick complementarity. The seqNo 15151537
(yellow box in Figure 3) indicated the guide strand of the predicted
24-nt, mature microRNA generated by HongrES2 (Figures. 3B and
3C). Northern blot analysis was used to investigate whether or not
HongrES2 could be processed into a mature miRNA in the
epididymis. Using an LNA probe with sequence complementary to
the predicted mature miRNA guide sequence, a faint but distinct
band of approximately 23 nt in the cauda and caput regions of the
epididymis was observed (Figure. 3D). Furthermore, an intense
signal of approximately 100 bp was also detected, demonstrating
the existence and accumulation of its pre-miRNA form.
To confirm the size and existence of this newly discovered small
RNA, independent Northern blots with the 24-nt DNA oligo
marker were performed (Figure S2). During the validation for the
expression of this new miRNA-like small RNA (mil-HongrES2) by
Northern blots, its 100- bp pre-miRNA-like band was quite intense
and stable, whereas the expression of the ,23-bp microRNA-like
small RNA (mil-HongrES2) in normal tissues was low and
sometimes difficult to be detected. Thus, a BlAST search was
performed against the Sanger miRbase to determine identified
miRNAs with sequence similar to that of mil-HongrES2; ron-mir-298
was the only one found to have about 50% homology with
milHongrES2 in sequence and a very low expression ratio (about 1024)
compared with the mir-29a signal in miRNA chips (data not
shown) in rat epididymis (Figure 3D). Hence, the ,23-nt band
could not possibly be generated because of cross hybridization to
other small RNAs, including the ron-mir-298, under high stringent
hybridization conditions at 65uC with LNA probes having less
than 50% sequence complementary.
To study the small RNAs existing in this special male organ
which might have important functions during sperm maturation, a
small RNA (1825 nt) library of rat epididymis was constructed by
JS Zhang (paper in revise). However, the mil-HongrES2 sequence
was not screened by PCR amplification using 39 and 59 adaptor
primers. Considering the low expression demonstrated by the
Northern blots, it was expected that small RNAs with low copy
number similar to mil-HongrES2 would not be included in such a
small-sized library, that totally no more than fifty different small
RNA sequences were achieved including the known and new
miRNAs, siRNAs, and some piRNA like small RNAs.
Thus, the selective primer of mil-HongrES2 (GSP) was used as an
upper primer and the 39 adaptor primer was selected as the lower
primer to bail the mil-HongrES2 sequence in this small RNAs
library (Figure 3E, upper panel).
The PCR band was cloned and sequenced. Four positive clones
were acquired and compared with the sequence predicted from
the stemloop structure, The 39 terminus of the mil-HongrES2
around the uracil (seqNo1535 of HongrES2) was found to possibly
have had some variations. One of the four clones(clone4) was an
18-bp core sequence of mil-HongrES2
(59AGGAGGGCTGGTCCATTA 39 ) inserted right between the upper and lower primers,
meaning that not only a single small RNA was ligated into the
59linker adaptor and 39linker adaptor when the library was
constructed. This situation was comparatively rare but quite
possible. This sequence, screened independently of the upper
primer, ascertained the 59terminal of mil-HongrES2, which might
start from the adenine (seqNo1515 of HongrES2) rather than the
predicted guanine. Real-time PCR was carried out to demonstrate
that the copy number of mil-HongrES2 was so low in the
constructed small RNA library that there was about only one
copy molecule among six thousand copies was produced
To confirm further that HongrES2 could be the primary
transcript of this novel ,23-nt miRNA- like small RNA, and to
ruled out the possibility that the low expression signal detected by
Northern blotting was due to cross hybridization, a plasmid
expressing the full-length HongrES2 RNA (pcmv-tag4-H2) was
transfected into the mouse epididymal cell line, PC1, which
did not express any endogenous mil-HongrES2 (Figure S2).
Fortyeight hours after transfection, the extra band corresponding to the
mil-HongrES2 was detected by Northern blot (Figure. 3F, upper
panel). Another strong band at ,50-bp position was found below
the ,100-bp pre-form. RT-PCR was performed with the poly(A)
tailing strategy to check the small RNAs generated in these
transfected cells (Figure 3F middle panel). The RT-PCR bands of
the ,100-bp and ,120-bp were cloned and sequenced. The
sequencing results showed that the smaller product was the 22-bp
fragment of the mil-HongrES2 sequence (2-bp swing at the
39terminal), and the larger product was the 45-bp fragment of
the constant HongrES2 sequence (seq. No 15141558) including
the 23-bp mil-HongrES2 and the 22 bp sequence behind it
(Figure 3F lower panel). We guess it probably be the
intermediated product detained during mil-HongrES2 maturation.
To determine whether or not this small RNA was generated
through a miRNA pathway-dependent process and RISC
association, the PC1 cells were co-transfected with the HongrES2
expression vector (plenti-H2-2) and a FLAG-tagged-Ago2
construct. Northern blot analysis showed that the pre- and mature
forms of HongrES2 were not only generated in the transfected cells,
but were also bound to the FLAG-Ago2 protein that was
immunoprecipitated by an anti-FLAG antibody conjugated with
Sigma M2 beads. The results indicated that Argonaute2 not only
bound this small RNA, but it was also probably involved in earlier
steps of its biosynthesis, which was consistent with the recently
reported dual role of the Argonaute proteins. A simultaneous
control experiment was carried out with the pri-mir-29a
expression vector instead of the HongrES2 expression vector to
determine the specificity of the IP experiments (Figure. 3G).
The dependence of the maturation of mil-HongrES2 on DCR
was investigated by co-transfecting the PC1 cells with the
HongrES2 expression vector (plenti-H2-2) and two different
siRNAs (siDCR1 and siDCR2) that target mouse Dicer1 mRNA.
Western blot analysis showed that both siRNAs depleted the Dicer
protein and siDCR1 was a little bit effective. The mil-HongrES2
expression in Dicer knockdown cells was tested by Northern
blotting. The results revealed that mil-HongrES2 was slightly
decreased in the Dicer-deficient cells. Meanwhile, no ,100-bp
pre-form acummulation was found in both of the Dicer
knowckdown cells, which was beyond our expectation. To confirm that
this phenomena was not due to the special cell line or the
transfections operation, an unrelated microRNA, mir-29a, was
tested as a control. Mir-29a was known to be endogenous in the
PC1 cells and the Northern blot analysis showed that its expression
was inhibited in both cells with siDCR RNAi as well, and its
premiRNA forms were accumulated. These findings indicated that
the biosynthesis of mil-HongrES2 might not directly relied on the
actions of Dicer as a prototypical microRNA (Figure. 3H).
Sequence analysis (BlAST search) was performed using the
mouse and human genome to determine whether or not the
milHongrES2 coding region showed sequence conservation between
these two species. Each species had one copy, on mouse
chromosome 8 and human chromosome 16, with 95% and 78%
identity, respectively (Figure 3I, upper panel). A gene homologous
to mil-HongrES2 was also detected in mouse (ICR) by Northern
blotting (Figure 3I, lower panel).
Inflammation accelerated the generation of mil-HongrES2
Interestingly, the mil-HongrES2 abundance was very low whereas
its pre form remained high.Comparing Figure 2B and Figure 3D,
the processing of mil-HongrES2 seemed to be controlled in a
regionspecific manner in the adult rat epididymis.This behavior was
somewhat simliar with the previous report where ubiquitously
expressed precursor miRNAs were processed to mature miRNA in
a tissue and /or cell specific manner, in the these studies, HeLa
cells expressed the miR-1382 precursor but not the mature
miR138 because of the presence of some unknown inhibitory
factor.This observation demonstrated that the regulation of
premiRNA/mature miRNA by Dicer was extremely
complicated[41,42,43] . Epididymitis was the most common disease in the
male reproductive system that severely affected the sperm quality,
the biogenesis characters of mil-HongrES2 mentioned above
suggested considering whether or not the generation of
milHongrES2 would be altered under a pathological context. The
cauda tubules on the right-side epididymis of three adult SD rats
were injected with cultured pathogenic bacteria from human
patients with epididymitis. A 0.9% saline solution was used as a
negative control on the left side of the organs without cut the
scrotum open. Four days after injection, the animals were
sacrificed. Northern blot analysis showed that the expression of
mil-HongrES2 in the epididymis was significantly enhanced, and the
expression of HongrES2 could hardly be detected in the infected
group (the tissues were pooled together). In the control group, the
expression of mil-HongrES2 was much lower than in the infected
group, and the HongrES2 RNA remained detectable by Northern
blot analysis (Figure. 4A and 4B). Other epididymal genes were
also tested:, unlike HongrES2, some of their expression were not
excessivly reduced by the bacteria infection (Figure S3).Real-time
PCR analysis indicated that the amount of Ncp2 mRNA was
generally unchanged in the infector group (Figure. 4A, lower
panel).These results suggest that the processing of HongrES2 and
the amount of mil-HongrES2 are controlled in the physiological
state and can be altered by pathological changes. Furthermore, a
sample of the pathogenic bacteria from human patients, which
were used to induce the over-expression of mil-HongrES2 was sent
for fractional cultivation. The results showed that the bacteria
were mainly composed of two types: Staphylococcus and Morganella.
These bacteria were cultured and injected into the cauda region of
rat epididymis respectively. Northern blot analysis showed that just
like the complex pathogenic infector from human patients
mentioned above, the pure cultured Morganella germ could also
induce the over expression of mil-HongrES2 in rat epididymis,
whereas the less harmful bacteria Staphylococcus could not
The epididymis protects the sperm from outside invasion of all
kinds of pathogens and epididymitis is a most common disease of
this organ. The over expression of the mil-HongrES2 under
epididymitis confirmed the existence of this newly defined small
RNA, and provided indications of its possible anti-infection
functions in vivo as well.
From the results above, this small RNA of mil-HongrES2 was
found to exhibited some expression and biogenesis features quite
similar to a microRNA, as well as a few properties different from a
typical miRNA. First, the expression of its primary form
(HongrES2) was more stable and higher than the usual
primiRNAs, which could hardly be detected and identified. Second,
the ,100-bp pre form size of mil-HongrES2 was much bigger than
that of typical pre-microRNAs which were usually about
60,80 bp in animals. Third, the extra band of the 45-bp
preform of mil-HongrES2 repeatedly appeared in the transfected PC1
cells and it occasionally appeared in the normal rat epididymis
tissues too. Fourth, it was not quite consistent with the biogenesis
criteria widely accepted as a microRNA that no detection of
increased precursor accumulation in cells with reduced Dicer
function, although recently it had been reported that some
microRNAs could be generated in the pathway independent of
According to the criteria for a new microRNA validation report
, the 23-bp small RNA(mil-HongrES2) was considered to be a
new miRNA-like small RNA, with the chimeric non-coding RNA
HongrES2 RNA as its primary transcript in rat epididymis.
The epididymis-specific gene, CES7 could be
downregulated by HongrES2 via mil-HongrES2
The sequence homology of HongrES2 and the rat CES7 gene
was presented in Figure 1C. This rare phenomenon of sharing the
common 39end 216-bp sequence caused us to consider the
relationship between these two genes. Non-coding RNAs
(ncRNAs), both small and large, have recently gained popularity
as versatile regulators of protein coding gene expression. [49,50].
The role of HongrES2 in regulating the expression of CES7 gene
was investigated by performing co-transfection experiments in
PC1 cells with the expression vector containing the full-length
CES7 (pcmv-tag4a-CES7) and the HongrES2 expression vector
(H2) as the tested group. In addition, cells co-transfected with the
CES7 expression vector and pcmv-tag4a vector (mock), and cells
co-transfected with the CES7 expression vector and the truncated
vector comprising the mil-HongrES2 coding region lacking the
HongrES2 39 end (H2T), were used as two different control
groups. The expression of the CES7 protein was reduced by the
HongrES2 expression vector (H2), as compared to the controls
(mock, H2T) (Figure. 5A). These experiments were repeated
independently several times, and the variation of the CES7 protein
was quantified (Figure S4).
Moreover, using the synthetic mimics of mil-HongrES2 (D+1)
instead of the primary precursor(HongrES2), the protein level of
CES7 was also found to be down-regulated compared with the
negative control mimics (Figure. 5B). These experiments were
repeated several times, and the variation of CES7 protein was
quantified (Figure S5). All of these observations indicated that
HongrES2 could down-regulate the expression of CES7 in vitro,
probably through the mil-HongrES2 function.
To determine whether HongrES2 directly down-regulats the
CES7 gene expression via mil-HongrES2 by sequence base pairing,
PRL-TK Renilla luciferase reporters containing either the CES7
wild-type 39UTR or the mil-HongrES2 base pairing site mutant
39UTR were constructed (Figure. 5C). A dual luciferase assay
showed that the relative luciferase activity dropped by 20%50%
when the wild-type reporter construct was co-transfected into PC1
cells with all of the regulators necessary to generate the
milHongrES2, such as the full-length HongrES2 expression vector
(H2), a 59 end, 591-bp truncated HongrES2 vector (H2-2), the
perfect mil-HongrES2 mimics duplex (D+1), and the imperfect
duplex (A+1) of the mil-HongrES2 mimics. However, the 39 end,
216-bp truncated HongrES2 vector (H2T), which cannot generate
mil-HongrES2, did not inhibit the activity. A reporter with
mutations in the predicted mil-HongrES2 target site was not
suppressed by the regulators, except for the perfect duplex mimics
(D+1), which retained an approximate 20% suppression effects
(Figure. 5D). Considering the sequence homology between CES7
and HongrES2 mentioned above (Figure. 1C), its passenger strand
would pair perfectly with the mil-HongrES2 coding region on the
39UTR, potentially explaining the knockdown effect.
The developmental changes in the rat CES7 mRNA and the
HongrES2 RNA expression levels during the rat lifespan were
compared by Northern blot analysis. Interestingly, the trend in
their expression patterns was quite different. CES7 mRNA
expression quickly increased at an early stage (before 90 days
old), and it plateaued to a stable level until two years of age,
Figure 6. Over-expression of mil-HongrES2 by injecting agomir into the epididymis to reduce the cauda sperm capacitation. (A)
MilHongrES2 over-expression by mil-HongrES2 mimics agomir down-regulation of CES7 protein expression. The two upper panels show the northern blot
analysis of mil-HongrES2 over-expression. C: control group. H2: over expression group. U6 was used as the internal loading control. The number
shows two or three different individuals. The two lower panels show the corresponding CES7 protein level in vivo, and a-tubulin was used as the
loading control. The experiments were carried out independently and the results of 3 replicates are shown and indicated by the replicate number (1st,
2nd or 3rd). (B) Quantification analysis of mil-HongrES2 over-expression and the down-regulation of CES7 protein from (A) (n = 7 ). The data are
expressed as the mean 6 SEM. **P,0.001. (C) The change in protein tyrosine phosphorylation in mil-HongrES2 over-expressing sperm after
incubation in the sperm culture medium. Total protein from the spermatozoa was collected for western blot analysis after 2 h, 3 h and 4 h of
incubation; a-tubulin was used as the loading control.
whereas the HongrES2 RNA expression level increased for a
longer time after initiation (until 270 days of age; Figure. 5F).
The wave trend of the HongrES2 RNA and CES7 mRNA
expression curve was temporally reciprocal. According to a
previous report, this phenomena was coincident with the
regulator-target pair relationship between HongrES2 and CES7
mentioned above. Thus, this hypothesis was formed:, When
the exprssion level of CES7 protein needs to be cut down in vivo,
not only CES7 mRNA expression is reduced by the organism,
but the expression of HongrES2 is increased to prohibit the
CES7 protein translation process at the post-transcription level
Mil-HongrES2 over-expression in the cauda epididymis
reduced the CES7 protein level and sperm capacitation
To investigate whether the amount of mil-HongrES2 was critical
to the epididymis micro-environment during sperm epididymal
maturation, mil-HongrES2 RNA was over expressed in vivo by
directly injecting the modified mil-HongrES2 agomir into the right
side of the cauda epididymis; a seed sequence mutant scramble
agomir was used as a negative control in the left side. Four days
later, the amount of CES7 protein was decreased, whereas the
amount of mil-HongrES2 expression was elevated in the
overexpression group (Figures. 6A and 6B). The CES7 gene is a newly
defined carboxylesterase with cholesterol esterase activity [32,33];
cholesterol is very important during the sperm capacitation
process . Tyrosine phosphorylation of sperm proteins has
been reported to be an indicator of capacitation signaling cascade
activation.[53,54,55]. Tyrosine phosphorylation of the sperm
proteins was then assayed to investigate whether or not sperm
capacitation would be affected when mil-HongrES2 was
overexpressed. Western blot analysis showed that tyrosine
phosphorylation of the sperm protein during capacitation was affected
(Figure. 6C). This result meant that the endogenously low
expression of mil-HongrES2 was important for sperm maturation
in the normal rat epididymis, and that HongrES2 RNA may play a
regulatory role in sperm maturation via mil-HongrES2 maturation.
In the present study, a new 1.6 kb mRNA-like chimeric
noncoding RNA named HongrES2 in adult rat epididymis was found
occasionally. Its 39terminal sequence was homologous with
another epididymis specific gene, CES7. The gene was further
identified and characterized to be the primary precursor of a
,23bp miRNA-like small RNA (mil-HongrES2), which could regulate
the expression of CES7 gene.
A relatively long, polyadenylated transcript encoded by the
Caenorhabditis elegans let-7 gene undergoes trans-splicing to the spliced
leader 1 (SL1) RNA to generate a mature microRNA . A newly
defined conserved ncRNA gene Dmr (Dmrt1-related gene) in
rodents was demonstrated to trans-splice with DMRT1
(Doublesexrelated transcription factor) recently. Dmr was demonstrated to
serve mainly as a 39 UTR, which promotes trans-splicing to produce
a novel chimeric transcript (Dmr-Dmrt1), down-regulating the
Dmrt1 protein expression . DMRT1, which was reported to
function in coordinating spermatogonial development and mitotic
amplification with meiosis, is considered as an important gene
involved in spermatogenesis. Therefore, the chimeric transcript
(Dmr-Dmrt1) was presumed to play a negative regulatory role in
male sexual regulation . These facts suggested the possibility
that both the conding gene and the noncoding gene could serve as a
39UTR to trans-splice to produce a novel chimeric transcript
(coding or noncoding) to increase the diversity of gene functions; the
mechanism for this, however, is not yet clear. Wangs group found
that approximately half of the chimeric RNAs have short
homologous sequences (SHSs) at the junction sites that are essential
for generating a novel kind of chimeric RNA. In the present study,
an SHS (CTGCTTT) was found between the two fragments from
different chromosomes. However, this sequence did not match any
of the previously reported consensus sequences . This peculiar
chimeric, non-coding RNA formed by the 216-bp 39fragment of the
HongES2 originating from the coding sequence, and the 1372-bp
59fragment of the HongES2 originating from non-coding sequence in
chromosome 5 combined with an SHS bridge, provides an ideal
model for gaining further understanding of the transcriptional
regulation of precursors of small RNAs.
Mil-HongrES2 was firstly hypothesized to be a microRNA
because of its length and the pre-miRNA like fold-back structure
(Figure 3B). However, the stem-loop structure did not have the
typical characteristic feature of a pre-miRNAs, as reported
previously, for which the guide strand should be located on the
stem region of about 3036 bp . Among the three main
categories of small RNAs (microRNAs, siRNAs and piRNAs) in
animals, mil-HongrES2 was most similar to the features of the
microRNAs, as siRNAs can be processed from a broader range of
duplex structures that are perfectly base paired, or nearly so, and
the structure of the piRNAs precursor were not well defined but
are apparently single-stranded.
Following the discovery of miRNAs, researchers have started
searching for their targets and uncovering the biological functions of
individual miRNAs[61,62]. However, less attention has been paid to
the sub-classification of these miRNAs. The most important criteria
for annotating a cloned sequence as a miRNA are their characteristic
length (_22 nucleotides) and a compact pre-miRNA fold-back
structure . Furthermore, miRNAs generally adhere to additional
properties, including precise 59end processing, asymmetric strand
accumulation, and sequence conservation. Surprisingly, not all
miRNA sequences comply with these criteria. A recent study
reported the miRNAs to be sub-divided into four categories, namely,
prototypical, repeat-clustered, repeat-derived, and
unclassified miRNAs . MiRNAs were considered prototypical if they
met defined criteria regarding their 59 end processing, lack of
repetitiveness, and cross-species conservation. Overall, 59% of all
miRNA genes were classified as prototypical. Repeat-clustered and
repeat-derived miRNAs originate from highly repetitive genomic
sequences, either clustered or dispersed. In contrast to prototypical
miRNAs, precursors of repeat-clustered miRNAs did not preserve the
strand asymmetry between sequence-related precursors. The
remaining 28% of the miRNA genes were termed unclassified, their
products showed irregularities in processing and/or unusual sequence
variations including deletions and variation in the seed sequence
between human and rodent orthologs . Only 17% of all miRNA
sequences identified by a recent deep-sequencing study were
prototypical . The remaining miRNAs, though clearly
originating from hairpin precursors, showed unusual maturation or sequence
conservation patterns. For many more recently reported miRNA
candidates, cloning evidence was not found. These small RNAs are
speculated to originate from dsRNA structures that only accidentally
enter the RNAi pathway, such as fold-back elements controlled by
dsRNA deaminases or the binding sites of RNAi unrelated
dsRNAbinding proteins. Thus, identification of mil-HongrES2 might also
indicate those potential unclassified miRNA candidates.
Transfection assays revealed that when the CES7 RNA, which had
the same 216-bp 39UTR but not the 59 1372-bp fragment of
HongrES2, was transfected into the PC1 cell line, no mil-HongrES2
signal was detected by Northern blot analysis. Dual luciferase assay
experiments with different types of constructs also implied that the
59RNA fragment derived from chromosome 5, might be important
for generating mil-HongrES2 (Figure S6A). In situ hybridization showed
that the CES7 mRNA was located in the cell cytoplasm, which is
different from the cellular localization trait of HongrES2 (Figure S6B).
The secondary structure of the 223-bp of the CES7 39end (with 216
bp identical to HongrES2) was also carried out by mfold; and none of
the predicted structures showed that the mil-HongrES2 sequence could
be the arm of a possible stem-loop (Figure S7). Sequences and
structures flanking the miRNA hairpin affect its maturation . The
same mechanism might explain the observations above.
In summary, beginning from a new gene EST(HongrES2 EST) the
full length of a new gene (HongrES2) was obtained. Subsequent
analyses of its biological properties revealed it to be the precursor of a
miRNA-like small RNA (mil-HongrES2), which could down-regulated
the expression of CES7 both in vitro and in vivo. However, these findings
are still preliminary. The over-expression of mil-HongrES2 in the rat
epididymis implied that it functions in sperm maturation. Therefore,
further in vivo studies are necessary to explore the biological
significance of this ncRNA in the context of sperm maturation.
Figure S2 Northern blot analysis detected the
milHongrES2 expression in rat epdidydimis. PC1 cells and
rat liver RNA were used as negative control. The 24 bp DNA
oligo had complimentary sequence to the LNA mil-HongrES2
probe was used as positive control and a precise size marker. CD
total: ttl RNA of rat cauda; Cd small:small RNA of rat cauda; cell:
ttl RNA of PC1 cells; Liver: ttl RNA of rat liver. marker: Ambion
small RNA marker(10 nt-100 nt).
Figure S4 CES7 protein level was reduced by HongrES2
in PC1 cells. (A) Western blot of CES7 proteins after
cotransfection into PC1 cell with CES7 and different
constructs.Mock: pcmv-tag4 plasmid without insertions. H2:
Pcmv-tag4aH2. H2T: Pcmv-tag4a-H2T. 3rd panel was the raw data of
Figure. 5C. (B) Quantification analysis of the CES7 protein
expression of the western blot in A. Data were expressed as the
Figure S5 CES7 protein level was reduced by
milHongrES2 mimics. (A) Western blot of CES7 proteins after
co-transfection into PC1 cells with CES7 expression vector and
different dsRNA regulators. Nosi1 and Nosi2: negative control of
irrelevant dsRNAs. A+1: the imperfect duplex of mil-HongrES2
mimics; D+1: the perfect duplex of mil-HongrES2 mimics. 3rd
panel was the raw data of figure5D. (B) Quantification of the
CES7 protein expression of the western blot in A(1st,2nd ,3rd ).
Data were expressed as the means6SEM
Figure S6 CES7 mRNA could not play the role of the
precursor of mil-HongrES2. (A) Dual luciferase assay activity.
CES7: pcmv-tag4a-CES7; control: pcmv-tag4a mock plasmid. (B)
In situ hybridization of CES7 mRNA. The signal was brown and
was stained in the cytoplasm of epididymal epithelium.
We are grateful to Ai-Hua Liu for preparing all of the slides used for the in
situ hybridization experiments. We are also thankful for all of the advice
from Dr. Dang-Sheng Li, Dr. Li-Gang Wu, Dr. Shuang Zhao, Dr. Ya
Zhao, Dr. Meng-gui Wang, Dr. Guo-Chen Yao and Dr. Jia Sheng. We are
grateful to Yinusa Raji for the writing assistance.
Conceived and designed the experiments: Y-LZ. Performed the
experiments: M-JN Z-HH M-hL J-SZ LZ. Analyzed the data: Y-LZ M-JN QL
M-FL. Contributed reagents/materials/analysis tools: M-JN. Wrote the
manuscript: Y-LZ M-JN QL M-FL. Principal investigator: Y-LZ. Did the
majority of the experiments: M-JN. Cloned the HongrES2 EST fragment
and CES7 cDNA and defined their epididymis-specificity: Z-HH. The
work stuff of professor Muo-Fang Liu: M-hL. Created the rat epididymis
small RNA library: J-SZ. Castrated rat epididymis RNA gel and prepared
the polyclonal anti-serum of rat CES7: LZ. Created the rat epididymis
cDNA library and cloned the monkey CES7 cDNA and taught ZH Hu
how to screen rat cDNAs from the cDNA library: QL. Gave a lot of
suggestions and methodology help: M-FL.
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