piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
Gianfrancesco F (2011) piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human
Melatonin Receptor 1A Gene. PLoS ONE 6(7): e22727. doi:10.1371/journal.pone.0022727
piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
Teresa Esposito 0
Sara Magliocca 0
Daniela Formicola 0
Fernando Gianfrancesco 0
John J. Rossi, Beckman Research Institute of the City of Hope, United States of America
0 Institute of Genetics and Biophysics Adriano Buzzati-Traverso, National Research Council of Italy , Naples , Italy
Piwi-associated RNAs (piRNAs) are a distinct class of 24- to 30-nucleotide-long RNAs produced by a Dicer-independent mechanism, and are associated with Piwi-class Argonaute proteins. In contrast to the several hundred species of microRNAs (miRNAs) identified thus far, piRNAs consist of more than 30,000 different species in humans. Studies in flies, fish and mice implicate these piRNAs in regulating germ line development, the silencing of selfish DNA elements, and maintaining germ line DNA integrity. Most piRNAs map to unique sites in the human genome, including intergenic, intronic, and exonic sequences. However, the role of piRNAs in humans remains to be elucidated. Here, we uncover an unexpected function of the piRNA pathway in humans. We show for the first time, that the piRNA_015520, located in intron 1 of the human Melatonin receptor 1A (MTNR1A) gene, is expressed in adult human tissues (testes and brain) and in the human cell line HEK 293. Although the role of piR_015520 expression in brain tissue remains unknown, the testes-specific expression is consistent with previous findings in several species. Surprisingly, in contrast to the mechanism known for miRNA-mediated modulation of gene expression, piRNA_015520 negatively regulates MTNR1A gene expression by binding to its genomic region. This finding suggests that changes in individual piRNA levels could influence both autoregulatory gene expression and the expression of the gene in which the piRNA is located. These findings offer a new perspective for piRNAs functioning as gene regulators in humans.
Funding: The work was supported in part by grants from the Italian Institute of Genetics and Biophysics, National Research Council of Italy. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.
Competing Interests: The authors have declared that no competing interests exist.
Small non-coding RNAs have emerged as potent regulators of
gene expression at both the transcriptional and
post-transcriptional levels . Recently, small RNAs that interact with Piwi
proteins have been discovered in the mammalian germ line and in
Drosophila. These Piwi-interacting RNAs (piRNAs) represent a
distinct small RNA pathway and differ from miRNAs in several
ways . In flies, piRNA mutations lead to the overexpression and
mobilisation of retrotransposons, which results in DNA lesions that
cause germ line DNA damage . The biogenesis and
mechanism of action of piRNAs is not well understood. For
example, it is not known whether piRNAs primarily control
chromatin organisation, gene transcription, RNA stability or RNA
translation. Moreover, proteins involved in piRNA production
have been implicated in the control of gene expression in somatic
cells and in learning and memory . These data suggest that
piRNAs might impact a broad range of biological processes.
Studies in mice showed that piRNA-encoding regions are
distributed over most chromosomes and range in size from 0.9 to
127 kb. Although piRNAs map exclusively to one chromosomal
strand in many regions, some regions encode piRNAs in both
orientations. In mammals, piRNAs predominantly map to a single
genomic locus, whereas in flies they map to repetitive sites such as
transposable elements. Betel et al. describes that 25% of piRNA
clusters have 59 and 39 ends that coincide, indicating that they are
not random degradation products of long transcripts . Because
no stem and loop regions have been identified for piRNAs, it is
possible that long-range dsRNA structure or sequence-specific
protein machinery is involved in guiding the maturation process
In a recent study, we identified a genetic link between variants
of intron 1 of the melatonin receptor 1A (MTNR1A) gene and
calcium nephrolithiasis . In this study we conducted a
bioinformatic analysis of this 22 kb genomic region in order to
identify possible regulatory elements. From this analysis, we
identified the piR_015520-encoding region in intron 1 of the
MTNR1A gene. Interestingly we demonstrate that the piRNA gene
is expressed in human tissues and we show that this small RNA
molecule is able to repress the expression of the melatonin receptor
Results and Discussion
Bioinformatics analysis and genotyping
The human MTNR1A gene is located on the chromosomal
region 4q35.2, it spans a genomic region of 21.913 bases and is
split in two exons.
The 21.913 bases were interrogated with repeat-masker (http://
www.repeatmasker.org/cgi-bin/WEBRepeatMasker) to eliminate
Alu and Line repetitive elements, and then the output sequence
was targeted against both the NCBI non redundant (NR) and NT
databases using BLASTN as tool (http://blast.ncbi.nlm.nih.gov/
Blast.cgi). We identified a piRNA-encoding region, a new class of
small RNA molecules (PIWI RNA, hsa_piR_015520) in intron 1
of the MTNR1A gene, occurring 14 kb downstream of the ATG
Transcripts encoded by piR_015520 belong to a newly
identified class of small RNA molecules called piRNAs. The
piR_015520 locus is organised in a cluster of 30 bp units and
contains directed and overlapping repeat segments of 7 bp at the
59 and 39 ends (Fig. 1a). To determine if the piR_015520 genomic
region is polymorphic in humans, we designed primers outside the
genomic cluster and amplified DNA from 100 unrelated subjects.
At least four different bands were detected and sequenced,
showing that the genomic cluster could contain 12, 14, 16, and 18
repeats (Fig. 1b). Although our previous results indicate an
association between allelic variants of MTNR1A and recurrent
calcium nephrolithiasis, we confirm here that this association is not
driven by the piR_015520 polymorphism.
piRNA expression profile
Two real-time PCR-based piRNA expression assays were
designed to detect the specific expression of the mature form of
piR_015520 in adult human brain and testes (Fig. 2a).
miRNA can be localised in the intronic regions of host genes.
Interestingly, it has been demonstrated that many intronic
miRNAs and their host genes are co-regulated and co-transcribed
from a common promoter . To explore the possibility that
expression of piR_015520 was related to the host gene, we
determined the expression profile of the MTNR1A gene using
realtime PCR and the same RNA panel used for piRNA expression
analysis. Significant expression of the MTNR1A gene was found in
several tissues. Further, MTNR1A exhibited a different expression
pattern compared to piR_015520, suggesting that these two genes
use different promoter regions (Fig. 2b).
Many miRNAs are aberrantly expressed in various pathologies
including cancer and regulate tumor- and metastasis-associated
genes . We therefore investigated the expression of
piR_015520 in various cell lines: human breast adenocarcinoma
cell line (MCF7), human gastric cancer cell line (MNK-28), human
epithelial carcinoma cell line (HeLa), human neuroblastoma cell
line (SH5Y5Y) and human embryonic kidney cell line (HEK 293).
In particular, piR_015520 showed a very low level of expression
only in the HEK 293 cell line (CT = 48, undetectable on agarose
gel, Fig. 2a).
MTNR1A gene expression regulation
In Drosophila, piRNAs could silence gene expression by
promoting heterochromatin assembly, which could directly
Figure 1. Genomic structure of piRNA_015520. a, Physical location and genomic structure of the MTNR1A gene and the piRNA_015520. The
30 bp sequence of the mature piRNA is shown, with the directed repeat segments of 7 bp at the 59 and 39 end depicted in superscript. b, Genotyping
of the piRNA_015520 genomic region from Caucasian DNA samples.
suppress transcription . We performed transfection
experiments to test the ability of piRNA-015520 to bind its
genomic region, thereby affecting gene regulation of the MTNR1A
The piRNA genomic cluster, which spans about 500 bp and
includes 16 repeats, was amplified from genomic DNA and ligated
into a linearised pRL-CMV vector (Promega) at the 39 end of the
Renilla luciferase reporter gene. The pRL-PIWI plasmid was
transfected into HEK 293 cells, along with increasing
concentrations (50 nM, 100 nM, 200 nM, 300 nM) of a chemically
synthesised piRNA-015520 mimic, which mimics the function of
the same piRNA. Addition of the piRNA mimic was able to
promote the repression of Renilla luciferase activity in a
concentration-dependent manner (Fig. 3a). To test the
endogenous regulation of the MTNR1A gene by the piRNA transcript, we
transfected HEK 293 cells with increasing concentrations (50 nM,
100 nM, 200 nM) of the piRNA mimic and tested the expression
levels of the endogenous MTNR1A gene using real-time PCR. We
observed a repression of melatonin receptor 1A gene expression in
a concentration-dependent manner (Fig. 3b).
These data demonstrate that there is an effective, specific, and
functional interaction between a piRNA and its genomic region,
suggesting that changes in piRNA levels may effectively modulate
its expression and the expression of the gene in which is located.
Repression of the MTNR1A gene by increasing concentrations
of piRNA transcript was also confirmed by Western blot analysis
using an anti-MTNR1A antibody (Novus Biological) (Fig. 3c).
piRNA-RNA protein interaction
Mature piRNAs are double stranded RNAs of 2631
nucleotides that form RNA-protein complexes through
interactions with Piwi proteins. To demonstrate an interaction between
piR_015520 and RNA-binding proteins, we performed an
electrophoretic mobility shift assay (EMSA) using a radiolabelled
piRNA mimic probe.
Two distinct complexes were revealed upon incubation of the
piRNA mimic probe with cytoplasmic extract from unstimulated
HEK 293 cells (Fig. 4). The binding specificity was demonstrated
in a competition assay using a 100-fold excess of unlabelled probe.
The unlabelled RNA probe was able to fully compete with the
probe, whereas the 30 bp dsDNA probe was not, demonstrating
that the binding was specific to RNA-binding proteins. Moreover,
to determine if the Piwi protein was one of the factors bound to the
complexes, an anti-Piwi antibody was used for a supershift assay.
No supershift was detected upon addition of the Piwi antibody,
suggesting that other RNA-binding proteins not yet identified
could bind the piRNA. In conclusion, we have identified a
piRNA-encoding region (piR_015520) in the first intron of the
human melatonin receptor 1A (MTNR1A) gene. We demonstrate
that the human gene encoding the small RNA molecule is
expressed in human tissues, specifically in brain and testes. To our
knowledge, this is the first demonstration that a human piRNA is
expressed in tissues distinct from the testes. Interestingly, we
detected piRNA expression in the brain where deregulation of the
MT1 and MT2 receptors occurs in neurodegenerative diseases
such as Alzheimers disease. Specifically, reduced levels of the
MT2 receptor-subtype and enhanced MT1 receptor expression
have been described . Further, elevated expression of the MT1
receptor was found in malignant human breast epithelia compared
to normal breast epithelia and stroma .
To date, we do not know whether our data for piR_015520
represents a single case or a more general phenomenon. However,
if this is also true for a different piRNA, then it is important to take
it into account. In fact, piRNAs consist of more than 30,000
different species, in contrast to only several hundred species of
miRNAs  . Most piRNAs map to the genome in clusters of 20
to 90 kbs in a strand-specific manner, with each cluster likely
representing a long single-stranded RNA precursor, or more often,
two non-overlapping and divergently transcribed precursors [12
13]. Our EMSA shows that the piRNA is able to interact with
RNA-binding proteins present in both the nucleus and cytoplasm.
However, mouse and Drosophila Piwi-class Argonaute proteins
are also present in the cytoplasm, and
piRNAPiwi-classArgonaute complexes could silence gene expression by targeting
the destruction of mature mRNA following exit from the nucleus.
It is also possible that piRNA-Argonaute complexes function in
both the nucleus and cytoplasm during the development of
complex multicellular organisms.
Thus, piRNAs may have a strong impact on gene expression, by
affecting epigenetic programming, transposition, and
post-transcriptional regulation. This study therefore offers a new
perspective for piRNAs functioning as gene regulators, and suggests a role
for piRNAs in regulating physiological and/or pathological
Materials and Methods
This research was reviewed and approved by the University of
Naples Human Research Ethics Committee and all subjects
participating in the study gave written informed consent.
Genomic DNA was isolated from whole blood by a standard
salting out procedure. The piRNA genomic region genotyped in
this study was amplified using the following procedure: an initial
denaturation step of 180 sec. at 94uC, followed by 35 cycles of
30 sec. at 94uC, 30 sec. at melting temperature 58uC, 30 sec. at
72uC, and a terminal extension of 10 min. at 72uC.
Primer sequences used were the following: the sense primer
sequence was 59- CCCTTAGTACTTTGCAGCAA -39, and the
antisense sequence was 59- TCTGTTTGATGCTGTGATGG
39. PCR products 450-600 bp in length were analysed by
electrophoresis on 1.5% agarose gels.
Samples were ExoSap-digested (Amersham) and sequenced
using the Big Dye Terminator Ready Reaction Kit (Applied
Biosystems). Sequencing reactions were performed on a 9700
Thermal Cycler (Applied Biosystems) for 25 cycles of 95uC for
10 sec., TM for 5 sec. and 60uC for 2 min. After the sequencing,
each reaction was column-purified (Amersham) to remove excess
dye terminators, and was subsequently run on the ABI prism 3700
Genetic Analyser (Applied Biosystems). Sequences were analysed
using multiple alignments of sequences with the program
Autoassembler (Applied Biosystems).
Expression profile of piR_015520
Total RNAs from human adult tissues were purchased from
Stratagene. Two different assays were used to show the expression
profile of the piR_015520 transcript. In the first assay, 1 mg of total
RNA was reverse transcribed with the miScript Reverse
Transcription Kit according to the manufacturers instructions
(Qiagen). During the reverse transcription step, miRNAs were
polyadenylated with poly(A) polymerase. Reverse transcriptase
was used to convert RNA (including precursor miRNA, mature
miRNA, other small noncoding RNA, and mRNA) to cDNA using
both oligo-dT and random primers. qPCR reactions were
performed in triplicate using an oligo-dT primer with a universal
tag sequence on the 59 end, together with the piRNA-specific
primer. qPCR reactions were prepared using the miScript SYBR
Green PCR Kit (Qiagen) following the manufacturers directions.
The second assay used for expression profiling was performed
with the Custom TaqManH Small RNA Assays kit (Applied
Biosystems). Procedures are reported in the legend for Figure 2.
The Figure 2 legend also reports the procedures used to determine
MTNR1A gene expression levels.
Figure 4. Electrophoretic mobility shift assays (EMSAs). Two distinct complexes (indicated with arrows) formed upon incubation of the piRNA
mimic probe with cytoplasmic extracts from HEK 293 cells (lanes 25). Low-intensity bands were revealed upon incubation of the piRNA mimic probe
with nuclear extracts (lanes 69). In competition experiments, a 100-fold molar excess of cold mimic-RNA (lanes 4 and 8) was able to fully compete
with the probe, whereas a 30 bp dsDNA could not (lanes 5 and 9), demonstrating that the binding was specific to RNA-binding proteins.
Oligonucleotides and plasmids
A chemically modified double-stranded piRNA mimic
(hsa_piR_015520) was purchased from Qiagen. The sequence of the
59-UGUCUGACUGAAGGACCAGGUGCUGUCUGU-39. The pRL-CMV plasmid coding for Renilla luciferase
(Promega) was modified by insertion of the piRNA genomic sequence
in the XbaI restriction site. The direction of the inserted 39 UTR
region was confirmed by PCR and sequencing after ligation. The
pGL3 plasmid encoding for firefly luciferase was used as a control.
Protein extraction and Western blot analysis
For Western blotting, cells were solubilised in lysis buffer
(50 mM HEPES pH 7.5, 150 mM NaCl, 4 mM EDTA, 10 mM
Na4PO7, 2 mM Na3VO4, 100 mM NaF, 10% glycerol, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 100 mg/mL
aprotinin, 1 mM leupeptin) for 60 min. at 4uC. Cell lysates were
clarified at 5,000 g for 15 min. Solubilised proteins were then
separated by SDS-PAGE and transferred onto Immobilon-P
membranes (0.45 m pore size; Millipore, Bedford, MA). The
membrane was probed with a 1:500 dilution of primary antibody
(anti-MTNR1A antibody) and subsequently probed with an
antigoat HRP-conjugated secondary antibody at a 1:2,000 dilution.
Signals were visualised using the Bio-Rad Chemidoc System.
Densitometry analysis was performed using Scion Image (Ver.
4.0.2; Scion Corporation, USA). The signals obtained for each
protein were normalised to beta-actin, and the mean6SE of three
independent experiments was plotted.
All antibodies were purchased from Novus Europe.
Immunoreaction signals were visualised with enhanced
chemiluminescence (ECL Plus, Amersham Biosciences).
Transfection and inhibition experiments
The day before transfection, HEK 293 cells were seeded in
24well plates with 500 ml of antibiotic-free medium and were grown
to 7090% confluence. The standard co-transfection mix was
prepared for triplicate samples by adding 75 ng of the pGL3
control plasmid, 30 ng pRL-PIWI (for details see legend to Fig. 3),
and with increasing concentrations of the piR_015520 mimic
(50 nM, 100 nM, 200 nM, 300 nM) in 150 ml of serum-free
DMEM; 3 ml of TransFast reagent (Promega) was added
separately in 150 ml serum-free DMEM. The two solutions were
mixed, incubated at room temperature for 20-30 min, and 100 ml
of the mixture was then added to each well. The final volume of
the medium plus the transfection mixture was 600 ml. Cells were
incubated with the transfection mixture for 4 hrs.; the medium
was then replaced with new fully-supplemented culturing medium.
Twenty-four hours after transfection, firefly and Renilla luciferase
activities were measured using a dual luciferase assay according to
the manufacturers instructions (Promega).
Electrophoretic mobility shift assays (EMSAs)
EMSAs were performed using a radiolabelled piRNA mimic
probe. Nuclear and cytoplasmic extracts from HEK 293 cells were
prepared as described by Granelli-Piperno et al. . Binding
reactions (20 ml) contained 32P-labeled mimic probe (105 cpm),
10 mg of cytoplasmic extracts (lanes 25) or 10 mg of nuclear
extracts (lanes 69), 2 mg poly dI-dC, 100 mM KCl, 10 mM
MgCl2, 20 mM HEPES pH 7.9, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 20% glycerol, and protease inhibitors. Reaction
mixtures were incubated for 30 min. at room temperature,
resolved on non-denaturing 5% polyacrylamide gels, dried, and
then exposed to autoradiography.
Conceived and designed the experiments: TE FG. Performed the
experiments: TE SM DF. Analyzed the data: TE SM. Contributed
reagents/materials/analysis tools: TE FG. Wrote the paper: TE FG.
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