Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2007.00341.x |
Cloning and Characterization
of a Heterogeneous Nuclear Ribonucleoprotein Homolog from Pearl Oyster, Pinctada
fucata
Xunhao XIONG1, Qiaoli
FENG1,
Liping XIE1,2*,
and Rongqing ZHANG1,2*
1 Institute of Marine
Biotechnology, Department of Biological Science and Biotechnology, Tsinghua
University, Beijing 100084, China;
2 Protein
Science Laboratory of the Ministry of Education, Tsinghua University, Beijing
100084, China
Received: April 23,
2007
Accepted: May 29,
2007
This work was
supported by the grants from the National Natural Science Foundation of China
(30530600, 30371092 and 30221003)
*Corresponding
authors:
Liping XIE: Tel/Fax,
86-10-62772899; E-mail, [email protected]
Rongqing
ZHANG: E-mail, [email protected]
Abstract Heterogeneous nuclear ribonucleoproteins
(hnRNPs) have fundamental roles in the post-transcriptional control of gene expression.
Here, a cDNA encoding a presumed full-length RNA-binding protein was isolated
from pearl oyster (Pinctada fucata) using reverse
transcription-polymerase chain reaction with degenerate primers, and rapid
amplification of cDNA ends. The full-length cDNA consists of 2737 bp with an
open reading frame encoding a protein of 624 amino acids with a predicted
molecular weight of 69 kDa and isoelectric point of 8.7. The putative pearl
oyster RNA-binding protein presents a molecular organization close to the
hnRNPs, namely a relative acidic N-terminal followed by three RNA-recognition
motifs and a C-terminal that contains RG/RGG repeat motifs. When transfected in
HeLa cells, the Pf-HRPH (Pinctada fucata hnRNP homolog) gene
expression product was found only in nuclei, revealing that it is a nuclear
protein. The expression pattern was also investigated by quantitative real-time
polymerase chain reaction, which indicated that Pf-HRPH mRNA was
abundantly expressed in gonad, gill, and viscera. As far as we know, the
putative Pf-HRPH is the first hnRNP homolog cloned in mollusks. These
data are significant for further study of the multiple functions of RNA-binding
protein.
Key words RNA-recognition motif; heterogeneous nuclear
ribonucleoprotein; glycine-rich protein; Pinctada fucata
RNA-binding proteins are found abundantly from virus to mammalian
species, and are essential for post-transcriptional control of gene expression
by influencing the transport, stability, splicing, and editing of mRNA transcripts.
One class of such proteins contains the RNA recognition motif (RRM). The RRM,
also known as the RNA-binding domain or ribonucleoprotein (RNP) domain, was
first identified in the late 1980s when it was shown that mRNA precursors and
heterogeneous nuclear RNAs were always found in complex with proteins [1]. The
RRM comprises approximately 90 amino acid residues whose sequence is
evolutionarily conserved and also presents two characteristic motifs,
denominated RNP-1 and RNP-2. The RNP-1 motif is an octapeptide formed by the
sequence (K/R)G(F/Y)(G/A)FVX(F/Y), and its presence is a strong indicator for a
function in RNA recognition [2,3]. RNP-2, a less conserved motif than RNP-1, is
a hexapeptide. This six residue sequence located at the N-terminal of the
domain was defined as (I/V/L)(F/Y)(I/V/L)XNL [4]. Both RNP-1 and RNP-2
constitute the RNP consensus sequences that characterize these RRM-type
proteins.
Heterogeneous nuclear ribonucleoprotein (hnRNP), one of the most
important RNA-binding proteins, binds to mRNA precursors concomitant with
transcription and forms ribonucleoprotein complexes essential for
post-transcriptional events. The hnRNPs possess three types of RNA-binding
domains that directly interact with RNA, most likely in a sequence-specific
manner. The RRM is the most frequently found motif in hnRNPs [4]. Another type
of RNA-binding motif present in some hnRNP proteins is the RGG box,
characterized by several closely spaced RGG repeats [2,4], which may occur
either alone, as in hnRNP U [5], or in combination with one or more RNA-binding
domains, as observed in hnRNP A1 and D0 [6]. The third type of RNA-binding
domain is the K homology motif, first identified in hnRNP K, a stretch of
approximately 45 amino acids characterized by several highly conserved
non-polar amino acids [7,8]. The functions of hnRNP proteins range from mRNA
packaging and transport, to mRNA splicing and silencing. Further, indirectly,
their role has also been implicated in development, for example, oogenesis,
embryogenesis [9–11], and neural development [12,13].
Mollusks constitute a source of food all over the world and have an
important role in the functioning of the ecosystems they inhabit. Mollusk
farming is a fast-growing industry; in 2002 it represented 23.5% of total
aquaculture production with an annual yield of 10.7 million tons [14]. In
response to the importance of bivalves in marine ecosystems and aquaculture,
the study of bivalve genomics is beginning [15–19]. However, our knowledge
of the very basic physiological processes, such as the regulation of embryo
development or sexual maturation, is extremely poor in mollusks. As many
RNA-containing proteins play crucial roles in oogenesis and embryogenesis
[10,20–22], the study on cloning and expression of mollusk RRM-type genes
will be helpful to further understand the development processes at the
molecular level.
We report here the isolation of a pearl oyster cDNA encoding a 624
amino acid protein, the first mollusk hnRNP to be identified. Sequence and
phylogenetic analysis showed that the putative pearl oyster RNA-binding protein
shares the structural organization of hnRNPs. We also investigated the
expression pattern of Pf-HRPH mRNA and the subcellular location of its
expression product in mammalian cells.
Materials and Methods
Preparation of total RNA
Adult specimens of Pinctada fucata were sampled from the
Guofa Pearl Farm, Guangxi Zhuang Autonomous Region, China. Total RNA was extracted
from the different tissues using an RNA isolation kit (Biotecx Laboratories,
Houston, USA). The integrity of RNA was determined by fractionation on 1.2%
formaldehyde-denatured agarose gel and staining with ethidium bromide. The
quantity of RNA was determined by measuring A260 with
an Ultrospec 3000 ultraviolet/visible spectrophotometer (Amersham, Piscataway,
USA).
cDNA synthesis and degenerate
polymerase chain reaction (PCR)
Total RNA (1 mg) from gonad was used as a template for the reverse transcription
(RT) reaction with Superscript II and oligo(dT) primers (Invitrogen, Grand
Island, USA). A pair of degenerate oligonucleotide primers were designed based
on the amino acid sequences of WDLRLMM (DPF, 5‘-TGGGAYCTGCGTCTWATGATG-3‘)
and KDYAF(I/V)H (DPR, 5‘-TGRAYGAASGCATAGTCTTT-3‘) conserved in
RNA-binding proteins. The PCR was carried out according to the manufacturer’s
instructions on a Tgradient Thermocycler (Biometra, Gottingen, Germany) for 35
cycles of denaturation (94 ºC for 30 s), annealing (48 ºC for 30 s), and
extension (72 ºC for 1 min).
Rapid amplification of cDNA
ends (RACE)
Single-stranded cDNA for all RACE reactions was prepared from the total
RNA of gonad, using PowerScript (Clontech, Palo Alto, USA). 5‘-RACE and
3‘-RACE were carried out according to the manufacturer抯 instructions, using the
SMART RACE cDNA amplification kit (Clontech) and Advantage 2 cDNA polymerase
mix (Clontech). The gene-specific primers 5RP (TCGGCCCAATCTACGATT-ACATCAC)
and 3RP (GTTTGTTGGAAACATCCCTA-AGAGC), used for 5‘-RACE and 3‘-RACE,
respectively, were prepared based on the nucleotide sequence of the cDNA
fragment amplified by RT-PCR. The 3‘-RACE and 5‘-RACE were
carried out under the conditions recommended by the kit mentioned above.
DNA sequencing and sequence
analysis
All amplified products, containing the 5‘ and 3‘ ends,
were subcloned into pGEM-T easy vector (Promega, Madison, USA) for sequencing.
To confirm cloning and sequencing accuracy, the entire cDNA from gonad was
re-amplified with high fidelity polymerase (TaKaRa, Dalian, China), using a
pair of gene-specific primers FLPF (5‘-GGGCATTTGATTCTGCCATCT-3‘)
and FLPR (5‘-CACAGAAACTACTTTTGGTGCC-3‘). The purified PCR
products were subcloned into pGEM-T easy vector followed by re-sequencing. The
nucleotide sequence was compared against GenBank, using the blast algorithm at the National Center
for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/),
to identify its coding proteins. Multiple alignments and phylogenetic
analysis were created using the ClustalX program [23].
Construction of expression
vectors
The pcDNA3.1/Pf-HRPH expression plasmid was constructed by amplifying
the full-length sequence of Pf-HRPH open reading frame (ORF) using high
fidelity PCR with primers EPF (5‘-CGCGGATCCATGGCACAGAATGGAATAATGGAGC-3‘)
and EPR (5‘-CCGCTCGAGACCCCAATTTTGCCCAAATGAATC-3‘), then
inserting the PCR product into pcDNA3.1/myc-His A vector digested with BamHI
and XhoI. The recombination plasmid was confirmed by DNA sequencing.
Cell culture, transient
transfection, and Western blot analysis
HeLa cells were seeded into 60 mm plates (2´106 cells/plate) 24 h prior to transfection. Cells
were transfected with 6 mg pcDNA3.1/Pf-HRPH expression constructs. Transfection was carried
out using Sofast transfection reagent (Sunma, Xiamen, China) according to the
manufacturer抯
instructions. Forty-eight hours after the transfection, the nuclear and
cytoplasmic proteins were extracted according to the manufacturer抯 instructions, using a nuclear and cytoplasmic
extraction kit (Pierce, Rockford, USA). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was carried out in discontinuous
vertical slab gels containing 10% (V/V) acrylamide in the
resolving gel. Proteins were transferred to polyvinylidene difluoride membranes
using a semidry transfer unit, and non-specific binding was inhibited by
blocking the membranes for 1 h at room temperature with 5% (W/V)
non-fat dried milk in 0.1% (V/V) Tween/Tris-buffered saline.
Membranes were then incubated overnight at 4 ºC in rabbit monoclonal antibody
against His (Sigma, St. Louis, USA). Signals were developed by incubating
membranes with an alkaline phosphatase-conjugated goat anti-rabbit
immunoglobulin G for 2 h followed by detection with nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, as described previously [24].
Real-time PCR assay
First-strand cDNA was synthesized as described above. The primer
pairs of the putative pearl oyster RNA-binding protein (RBP) (RTRBPF, 5‘-GGAAACATCCCTAAGAGC-3‘
and RTRBPR, 5‘-ACGATTACA TCACAACCC-3‘) and glyceraldehyde
3-phosphate dehydrogenase (GAPDH) (GAPF, 5‘-GATGGTGCCGAGTATGTG-3‘
and GAPR, 5‘-GATTATCTTGGCGAGTGG-3‘) were designed using Primer
Premier 5.0 software (Premier, Toronto, Canada). The cDNA mixtures were diluted
10-fold in sterile distilled water, and 1 ml was subjected to
real-time PCR using SYBR green I dye (TaKaRa, Dalian, China). Reactions were
carried out in 25 ml volume of a solution containing 1´PCR buffer, 1.5 mM dNTP mixture, 1´SYBR green I, 15 mM MgCl2,
0.25 U Ex Taq R-PCR version (TaKaRa), and 10 mM each primer
(sense and antisense). PCR consisted of 40 cycles at 95 ºC for 15 s,
56 ºC for 30 s, and 56 ºC for 30 s, and measurements were
made at the end of the 56 ºC annealing step. Standard curves were
generated using from 10–4 to 1 mg of cDNA. For
each reaction, the crossing point (CP) was optimized using Mx3000P
software (version 2.0; Stratagene, La Jolla, USA). The PCR efficiency (E)
was calculated by the standard curve method: E=10(–1/slope); this value was optimized for each primer pair by constructing
standard curves from serial dilutions of each positive cDNA control to ensure
that E ranged from 95% to 105%. It is emphasized that the
copy numbers/0.005 mg total RNA, as cited in the above text for test
samples, were derived by reference to these standard curves and do not precisely indicate the number of mRNA molecules, as the efficiency of RT was not directly determined. All of the
real-time PCR reactions were carried out in quadruplicate. The relative
expression ratio of Pf-HRPH was calculated based on the delta-delta method
for comparing relative expression results and is defined as: ratio=2–[DCP(Pf-HPRH)-DCP(GAPDH)]=2-DDCP. Assays were carried out on an
Mx3000P real-time PCR system (Stratagene) and analyzed using Mx3000P software.
Statistical analysis
The data from the experiments were analyzed using anova in spss version 11.5 software (SPSS, Chicago, USA).
Results
Isolation and sequence
analysis of cDNA encoding pearl oyster RBP
A cDNA product of 569 bp was obtained by RT-PCR with two degenerate
oligonucleotides (DPF and DPR) using total gonad RNA as the template. Based on
the sequence, two gene-specific primers were synthesized for 5‘-RACE
(5RP) and 3‘-RACE (3RP), respectively, and used to amplify the 5‘
and 3‘ nucleotide sequence of putative pearl oyster RBP cDNA by
PCR. To confirm the entire sequence, two specific primers (FLPF and FLPR)
corresponding to the 5‘– and 3‘-untranslated region (UTR)
sequences of putative RBP mRNA were designed and RT-PCR was carried out.
The PCR products were cloned and sequenced. They matched well the sequence
expected from the results of 5‘– and 3‘-RACE.
As shown in Fig. 1, the complete cDNA sequence including the
poly(A) tail was 2737 bp. It contains a 5‘-UTR sequence (99 bp), an
ORF consisting of 1872 bp, a TAA stop and a 3‘-UTR sequence (763
bp). Two putative polyadenylation signals (AATAAA) are recognized at positions
2701–2706 and 2711–2716, shown in bold (Fig. 1). This cDNA sequence has been
registered in GenBank (accession No. EF207319). The ORF of this cDNA encodes a
protein consisting of 624 amino acid residues. The molecular weight and
isoelectric point of the deduced protein were predicted to be 69 kDa and 8.7,
respectively.
The amino acid sequence predicted from the nucleotide sequence
reveals that a relatively acidic N-terminal region of approximately 160 amino
acids was followed by three tandemly arranged RRMs containing the canonical
conserved octameric RNP-1 and hexameric RNP-2 consensus motifs [Fig. 2(A)],
suggesting that the putative protein might function as an RNA-binding protein.
The C-terminal sequence is characterized by its high proportion of glycine
(22.3%) and arginine (10.9%). Approximately 50 amino acids after the third RRM,
a region of approximately 120 amino acids was found that contained seven RGG
repeats as well as eight RG dipeptides, forming another type of RNA-binding
domain. Pf-HRPH resembles mammalian hnRNP A1, A2/B1 with four blocks of RG-rich
sequence, while hnRNP R and Q with 12 blocks of RG-rich sequences [Fig. 2(B)].
Interestingly, two tyrosine-rich acidic polypeptide sequences (Asp478-Asp-Tyr-Tyr-Gly-Tyr483 and Asp487-Tyr-Tyr-Gly-Gly-Tyr492) were interspersed between
the second and third RGG repeats. In addition, the putative Pf-HRPH contains
three Pro-rich motifs (Pro468-Arg-Met-Pro-Pro-Pro-Pro474, Gly491-Tyr-Ala-Pro-Pro-Ile-Pro497 and Gly502-Arg-Met-Pro-Pro-Pro-Pro508) within the RGG boxes region. A putative bipartite nuclear
localization sequence (Lys567-Arg-Lys-X10-Lys-Lys-Arg582) was also found in the
C-terminal.
The RRM is a common motif for RNA binding in eukaryotes. An
alignment of the RNA-binding proteins containing three RRMs was used to
construct the phylogeny tree (Fig. 3). Phylogenetic analysis using
ClustalX software indicated that the putative RBP of P. fucata was
homologous to hnRNPs. So the pearl oyster RBP was designated as Pf-HRPH.
Sequence alignment of the mouse hnRNP Q and Pf-HRPH proteins showed 51% amino
acid identity over the entire length of the protein chains, and the highest
protein sequence homology was observed within the RNA-binding domain (62%
sequence identity and 75% sequence similarity).
Expression of Pf-HRPH
in various tissues
The tissue-specific expression of oyster Pf-HRPH mRNA in
different tissues was examined using quantitative real-time PCR. The real-time
PCR was conducted with specific primers (RTRBPF and RTRBPR) and cDNAs
reverse-transcripted from the total RNA of various tissues as the template. The
pearl oyster GAPDH was used as a control of equal quantities of total
RNA used in real-time PCR. As shown in Fig. 4, the Pf-HRPH mRNA
was detected in gill, gonad, and viscera with a relatively high expression
level, whereas in muscle and foot it was detected at very low expression
levels.
Subcellular localization of
Pf-HRPH
Based on sequence analysis, there is a bipartite nuclear targeting
sequence at the C-terminus of Pf-HRPH. To determine the location of the
Pf-HRPH, the whole ORF was inserted into the multiple cloning site of the
expression vector pcDNA3.1 to construct the expression plasmid
pcDNA3.1/Pf-HRPH. In a transient transfection assay into HeLa cells, both the
nuclear and cytoplasmic protein extracts were analyzed to detect Pf-HRPH/His
recombinant protein using the anti-His antibody. As shown in Fig. 5, a
protein of approximately 70 kDa was detected in the total protein of
transfected cells, but not in untransfected cells, indicating that the protein
is the Pf-HRPH/His-Myc recombinant product. In addition, the 70 kDa product was
found only in the nucleus of transfected HeLa cells, but the cytosol was
negative, suggesting that Pf-HRPH is a nuclear protein.
Discussion
Nearly two decades after its discovery, RRM remains an exciting and
active area of study. In recent years it has become evident that RRM-type
protein is actually a member of a highly diverged protein family with
homologous sequences found from viruses to mammalian species. In this report we
present the cloning of a pearl oyster RRM-containing protein that shows high
similarity to hnRNPs. As far as we know, the putative Pf-HRPH is the first
heterogeneous nuclear ribonucleoprotein homolog cloned in mollusks. Like other
hnRNPs, the putative pearl oyster RBP shows a modular structure. In the
C-terminal region, as a whole, arginine and glycine residues
were enriched, some portions of which formed RGG boxes. RGG boxes are
characteristic motifs of RNA-binding proteins [3,4], and are also involved in
protein-protein interactions [3]. In nucleolin (with four RNP
motifs and an RGG box), specific binding to pre-ribosoma1 RNA requires the four
RNP motifs, and the RGG box region increases overall RNA affinity (10-fold)
[25]. As the Pf-HRPH has a similar molecular architecture, composed of three
RNA-binding domains in a conserved spatial arrangement and a low complexity
glycine-rich C-terminus containing RG or RGG repeat sequences, the RGG boxes
possibly function as helping domains, as postulated for hnRNP A1, A2/B1, hnRNP
R and hnRNP Q [3,26]. Interspersed between several of the RGG repeats, there were regions enriched in tyrosine and acidic residues that contain
one copy of YYGY and YYGGY, respectively. These tyrosine-rich regions contain several consensus sites for tyrosine phosphorylation in which acidic residues are found on the NH2-terminal
side of the
tyrosine [27,28]. The presence of a proline-rich
sequence within the RGG box region is particularly noteworthy, as similar
sequences have also been found in other RNA-binding proteins, such as the hnRNP
R [22]. Such proline-rich domains always provide potential binding sites for
other proteins through hydrophobic interactions [29–31], suggesting that
Pf-HRPH could bind to RNA and to other proteins.
RRM-containing proteins display a wide subcellular distribution that
reflects a vast spectrum of functions [32–34]. RG/RGG repeat domains
are frequently associated with methylation of the arginine residues, which can
result in subcellular localization as well as modulation of RNA or protein
interactions [35–37]. A putative bipartite nuclear localization signal is in the
C-terminus of Pf-HRPH (residues 567–582) at the C-terminal side of the RG/RGG-rich
region. When transfected in HeLa cells, the C-terminus Myc-His-tagged Pf-HRPH
was detected only in the cell nucleus, revealing that the region of 175 amino acids
in the C-terminus is implicated in nuclear localization. So it is speculated
that Pf-HRPH mainly processes mRNAs in the nucleus.
The hnRNPs are important molecules for the regulation of processes
involving mRNAs, including mRNA processing, masking, translational activation,
turnover, and localization [38], indirectly regulating many physiological
processes, especially in oogenesis and embryogenesis [10,20–22]. The
real-time PCR result shows that Pf-HRPH mRNAs were abundantly expressed
in gonad, suggesting that Pf-HRPH plays important roles in oogenesis. Several
RNA-binding proteins have been reported to be essential for morphogenesis and
cytodifferentiation of digestive organs [37–42]. In mollusks, the gills
and viscera are key organs of the digestive system. High expression of Pf-HRPH
mRNAs in gill and viscera implies that Pf-HRPH might be necessary for digestive
organ development in pearl oyster.
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