PRMT1 protein level, type I protein arginine methyltransferase activity, specific asymmetric protein argi-nine methylation and histone H4 R3 methylation all decreased in the AMO-injected
Trang 13 Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan
4 Department of Pediatric Surgery, Chung Shan Medical University Hospital, Taichung, Taiwan
5 School of Medicine, Chung Shan Medical University, Taichung, Taiwan
Introduction
Protein arginine methylation is a post-translational
modification involved in various cellular functions,
such as signal transduction, protein subcellular
locali-zation, transcriptional regulation, protein–protein
interactions and DNA repair [1] At least 11 protein
arginine methyltransferase (PRMT) genes have been
identified in the mammalian system that catalyze the
transfer of methyl groups from S-adenosylmethionine (AdoMet) to the side-chain x-guanido nitrogens of arginine residues in protein substrates The activity can
be further divided into types I and II, depending on the catalyses of formation of asymmetric di-x-N,N-methylarginines or symmetric di-x-N,N¢-methylargi-nine residues respectively [2,3]
Keywords
convergence and extension; gastrulation;
PRMT1; protein arginine methylation;
zebrafish
Correspondence
C Li, Department of Biomedical Sciences,
Chung Shan Medical University, Taichung,
Taiwan
Fax: +886 4 23248187
Tel: +886 4 24730022 11807
E-mail: cli@csmu.edu.tw
(Received 27 August 2010, revised 19
December 2010, accepted 5 January 2011)
doi:10.1111/j.1742-4658.2011.08006.x
Protein arginine methyltransferase (PRMT)1 is the predominant type I methyltransferase in mammals In the present study, we used zebrafish (Danio rerio) as the model system to elucidate PRMT1 expression and function during embryogenesis Zebrafish prmt1 transcripts were detected from the zygote period to the early larva stage Knockdown of prmt1 by antisense morpholino oligo (AMO) resulted in delayed growth, shortened body-length, curled tails and cardiac edema PRMT1 protein level, type I protein arginine methyltransferase activity, specific asymmetric protein argi-nine methylation and histone H4 R3 methylation all decreased in the AMO-injected morphants The morphants showed defective convergence and extension and the abnormalities were more severe at the posterior than the anterior parts Cell migration defects suggested by the phenotypes were not only observed in the morphant embryos, but also in a cellular prmt1 small-interfering RNA knockdown model Rescue of the phenotypes by co-injection of wild-type but not catalytic defective prmt1 mRNA con-firmed the specificity of the AMO and the requirement of methyltransferase activity in early development The results obtained in the present study demonstrate a direct link of early development with protein arginine methylation catalyzed by PRMT1
Abbreviations
AdoMet, S-adenosylmethionine; AMO, antisense morpholino oligo; C ⁄ E, convergence ⁄ extension; hpf, hours post-fertilization; NR, nuclear receptor; PRMT, protein arginine methyltransferase; r, rhombomere; Sam68, Src-associated substrate during mitosis with a molecular mass
of 68 kDa; siRNA, small-interfering RNA; STAT1, signal transducer and activator of transcription 1; WISH, whole-mount in situ hybridization; xPRMT1b, Xenopus protein arginine methyltransferase type I b.
Trang 2PRMT1 is the predominant and most abundant
type I methyltransferase in mammals [2,3] RNA
bind-ing proteins such as fibrillarin, Sam68 (Src-associated
substrate during mitosis with a molecular mass of
68 kDa) and many hnRNPs with arginine and glycine
rich RGG motifs [2,4,5] or a RXR sequence [6] are
typical substrates of PRMT1 Methylation of proteins
such as hnRNPA2, Sam68 and hnRNPQ that shuttle
between the cytoplasm and nucleus can affect their
subcellular localization [7–9] Arginine methylation has
been reported to affect the protein–RNA or protein–
protein interaction of some RNA binding proteins
For example, the interaction of hnRNPK with c-Src is
reduced with arginine methylation [10]
PRMT1 also plays multiple roles in various
signal-ing pathways and transcriptional regulation For
example, interaction of PRMT1 with the cytoplasmic
domain of interferon-a receptor [11], and the putative
methylation of signal transducer and activator of
tran-scription 1 (STAT1) [12–14] and protein inhibitor of
activated STAT1 [15] by PRMT1, indicate its role in
interferon signaling Furthermore, methylation of the
transcriptional factor FOXO1 by PRMT1 can inhibit
its phosphorylation by AKT and promote nuclear
localization and transactivation of FOXO1 [16] In
addition, PRMT1 is a transcriptional coactivator of
various nuclear receptors (NRs) [17] as another PRMT
family member PRMT4⁄ CARM1
(coactivator-associ-ated arginine methyltransferase) [18] Methylation of
R3 of histone H4 by PRMT1 is part of the epigenetic
histone code critical for chromatin structure and
tran-scriptional activation [19] Increased H4R3 methylation
by the recruitment of PRMT1 has been reported with
transcription factors other than NRs, including p53
[20] and YY1 [21] Furthermore, PRMT1 can directly
methylate some transcription factors, coactivators or
transcriptional elongation factor to modulate
tran-scription For example, methylation of an orphan NR
HNF4 by PRMT1 can increase its DNA binding
affin-ity [22] Methylation of the transcriptional elongation
factor SPT5 by PRMT1 also regulates its promoter
association and RNA polymerase II interaction [23]
Mouse embryos homozygous for PRMT1knockout
failed to develop beyond the onset of gastrulation
(embryonic day 6.5), indicating that PRMT1 is critical
in early embryogenesis [3] Xenopus protein arginine
methyltransferase type I b (xPRMT1b) is maternally
expressed and subsequently transcribed zygotically
throughout the developing stages Overexpression of
xPRMT1 was found to induce the expression of a
spectrum of neural markers, and antisense morpholino
oligonucleotides (AMOs) against xPRMT1b impaired
neural development, indicating that xPRMT1b plays a
role in the early steps of neural determination [24] However, the correlation of the phenotypes with pro-tein arginine methylation catalyzed by the methyltrans-ferase was not studied The PRMT genes are highly conserved from zebrafish to humans, and the identity
of the PRMT1 proteins is close to 90% [25] Because zebrafish is amenable to genetic manipulation and the transparent embryos can be directly observed under microscope, we used zebrafish (Danio rerio) as a model system to monitor the relationship between protein arginine methylation and early developmental changes
in fish embryos
Results
Ubiquitous expression of prmt1 RNA and protein
in zebrafish embryonic development Alternative splicing of prmt1 results in various mRNA and protein isoforms in mammals [3,26,27] However,
no support for alternative splicing of zebrafish prmt1 could be obtained from a database search Ensembl (ENSDARG00000010246) illustrates that zebrafish prmt1 contains 10 exons and the prmt1 mRNA appears to be analogous to the v1 form of mammalian prmt1 mRNA (connecting the first exon and the con-stitutive 102 nucleotide exon with no alternative exons
in between; Fig 1A) A primer set to amplify the puta-tive alternaputa-tively spliced region (Fig 1A) detected a single RT-PCR product of 138 bp for RNA prepared from embryos from one cell to 72 h post-fertilization (hpf) (Fig S1A) The results opposed alternative splic-ing at the 5¢ end of the zebrafish prmt1 gene The RT-PCR product further confirmed the presence of an upstream in-frame ATG within a Kozak sequence located 21 nucleotides upstream of the start site sug-gested in NCBI (NM_200650) (Fig 1B) The predicted N-terminal amino acid sequence is indicated (Fig 1C) Ubiquitous expression of prmt1 in various adult tis-sues, such as the brain, heart, spleen, swim bladder, gill, testis, ovary and muscle, was also demonstrated
by RT-PCR (Fig S1B) Western blot analyses further detected a 42 kDa PRMT1 protein signal expressed at different zebrafish embryonic stages (Fig S1C) There-fore, PRMT1 protein is expressed both maternally and zygotically, comparable to the mRNA
Spatial and temporal expression pattern of prmt1 mRNA by whole-mount in situ hybridization (WISH)
Zebrafish prmt1 mRNA was strongly and ubiquitously expressed in embryos through the one- to four-cell
Trang 3stages (Fig 2A–C), demonstrating the maternal origin
and homogeneous distribution of prmt1 mRNA during
the very early cleavages Continuing homogenous
expression at 6 and 12 hpf indicated zygotic
transcrip-tion from gastrulatranscrip-tion to the early segmentatranscrip-tion period
(Fig 2D–F) At 24 hpf, prmt1 was strongly expressed
in the head regions, including the eyes, otic vesicle,
forebrain, midbrain and hind-brain (Fig 2G)
Expres-sion in somites was also detected As development
pro-ceeded, the expression of prmt1 decreased in most
parts of the brain but continued at somites at 48 and
72 hpf (Fig 2H, I) The signals are specific to prmt1
because the sense riboprobe did not detect any
signifi-cant signals (Fig 2J) Immunofluorescent analyses of
the PRMT1 protein also revealed similar expression
patterns (Fig 2K)
Knockdown of prmt1 with specific morpholino
oligonucleotides affects zebrafish development
AMOs designed to hybridize the 5¢ region of a target
mRNA can selectively block translation and
knock-down gene activity [28] Because two in-frame ATGs are present at the 5¢ region of prmt1, we synthesized two non-overlapping AMOs to target the upstream and downstream ATG (MO1 and MO2 respectively) (Fig 1C) Injection of high-dosed MO1 (8 ng) resulted
in the lysis of some embryos and a severely truncated phenotype in most survived embryos (data not shown) Similar phenotypes with different degrees of defects were observed when MO1 was injected at 4 ng or MO2 at 8 ng (Fig 3B) The abnormalities were classi-fied as mild, moderate and severe at 48 hpf, with dif-ferent degrees of body curvature being associated with curved or shortened tails (Fig 3B–D) Other abnor-malities, such as cardiac edema, enlarged yolk and shortened yolk stalk, smaller eyes and seriously trun-cated or bended tails, were also observed in some mor-phants At 72–120 hpf, the phenotype of edema and swollen yolk became even worse (Fig S2F–H, J–L, N–P), indicating poor circulation and metabolism The ratio of morphants with abnormal phenotypes increased as the dose of the injected AMO increased (Fig 4F; 58–98% for 2–4 ng of MO1; 75–94% for
Fig 1 Genomic structure and partial nucleotide and amino acid sequences of the zebrafish prmt1 gene (A) Genomic structure of human and zebrafish prmt1 Three major human splicing variants [27] and the only identified zebrafish splicing form are shown Exons are repre-sented as boxes and introns by the connecting lines Numbers in the boxes represent the exon length in base pairs Arrows indicate the position of the start and stop codons Filled boxes are coding and open boxes are noncoding regions The start ATG in human prmt1 was in accordance with that suggested in a previous study [27] According to the zebrafish prmt1 mRNA sequence, an ATG (arrow) 21 nucleotides upstream of the previously identified ATG (NM_200650, arrowhead) is mostly likely to be the translational start site The positions of primers used in the present study are indicated HsPRMT1v3 (NP_938075.2), HsPRMT1v2 (NP_001527.3), HsPRMT1v1 (NP_938074.2), DrPRMT1 (NP_956944.1) Hs, Homo sapiens; Dr, Danio rerio (B) The DNA sequence around the ATG translational start site of prmt1 (from the 38 nucleotides of NM_200650) is shown The two in-frame ATGs are boxed AMO binding sites complementary to the antisense morpholino oligonucleotide MO1 and MO2 are underlined MO2 begins 20 bp downstream of the first ATG of zebrafish prmt1 (C) Comparison of the N-terminal sequences of human and zebrafish PRMT1.
Trang 44–8 ng of MO2) The percentage of moderate or severe
phenotypes also increased significantly with the raised
AMO dose This dose-dependent phenotypic severity
indicates the specificity of prmt1 knockdown With the
aim of observing phenotypes beyond gastrulation, we
studied the morphants by the injection of 4 ng of
MO1 or 8 ng of MO2 in subsequent experiments
Reduced level of PRMT1 protein, type I PRMT
activity and protein arginine methylation in
prmt1 morphants
A reduced PRMT1 protein level appeared to correlate
with the phenotypic severity in the MO1-injected
embryos (Fig 4A) A decrease of PRMT1 expression
was found at 24, 48 and 72 hpf in MO1 or
MO2-injected morphants (data not shown) Thus, injection
of MO1 and MO2 indeed blocked the expression of
PRMT1 protein in zebrafish embryos, both effectively
and persistently
Because PRMT1 is the predominant type I protein
arginine methyltransferase, the type I activity in the
morphants should be reduced correspondingly In vitro
methylation reaction with a typical type I PRMT
sub-strate fibrillarin showed that fibrillarin methylation
cat-alyzed by the morphant extract was reduced compared
to that by the wild-type extract (Fig 4B) The type I
activity remained low from 24–72 hpf
We further examined the level of protein arginine methylation in the embryos with an antibody ASYM24 that recognizes asymmetrically dimethylated arginines
in alternate RG sequences [8] Dozens of zebrafish embryonic proteins of different molecular masses were detected and most of the methylarginine-specific sig-nals were reduced in the prmt1 morphants (Fig 4C)
We then examined protein arginine methylation of specific PRMT1 substrates Histone H4 arginine 3 methylation catalyzed by PRMT1 was abolished in PRMT) ⁄ ) mouse embryonic stem cells [17] We thus determined H4 R3 methylation in the embryos As shown in Fig 4D, asymmetric arginine dimethylation
at this residue detected by a modification-specific anti-body was reduced in the morphants Detection with another H4-specific antibody confirmed an equal load-ing of H4 protein These results confirm that the reduction of H4 R3 methylation was not a result of decreased H4 protein but instead was caused by the reduced expression of PRMT1 in the morphants
Reduced medial–lateral convergence and a shortened anterior–posterior axis in the morphants at early segmentation stage Because defective phenotypes observed in the prmt1 morphants are probably a consequence of earlier defects, we evaluated zebrafish development at the
Fig 2 Spatial and temporal expression of prmt1 by WISH and immunofluorescent analysis Zebrafish embryos at the one-cell stage (A), two-cell stage (B), four-cell stage (C), 6 hpf (D), 12 hpf (E, F), 24 hpf (G),
48 hpf (H) and 72 hpf (I) were analyzed by WISH A dorsal view of the 12 hpf is shown
in (F) WISH with sense riboprobe is shown
in (J) Immunofluorescent analysis with anti-PRMT1 of 24-hpf embryos is shown in (K).
a, adaxial cells; e, eye; f, forebrain; h, hind-brain; m, midhind-brain; mhb, mid-hindbrain boundary; ov, otic vesicle; som, somites.
Trang 5segmentation stage with different markers to pinpoint
the defects Expression of krox20 is restricted to
rhom-bomeres (r)3 and 5 (r5) in the hindbrain region At the
10-somite stage, krox20 expression in the morphants at
r3 and r5 was laterally extended (by 1.2–2.5-fold) and
the posterior r5 is more extended than r3 (Fig 5A)
Generally, the anterior–posterior distance between r3
and r5 was reduced, and the extent of reduction was
also correlated with the degree of lateral extension We
grouped the morphants according to the degree of
abnormal krox20 expression
Abnormal somite development in the prmt1
mor-phants at 10-somite stage was clearly revealed by a
muscle and somite-specific marker myoD As shown in
Fig 5B, myoD expression in the two rows of adaxial
cells flanking notochord was irregularly bent at the
posterior end in type 1 morphants In type 2
mor-phants, the width between the two rows increased,
with lateral myoD expression being diminished at the
end of one side, and extended and compressed at the
other The width was greatly broadened and the lateral
myoDexpression was greatly expanded in type 3
mor-phants Even though the same number of segments
was present in the morphants, the distances between
the segments were extremely compressed
Generally, the markers showed shortened anterior–
posterior axes in the morphants and the abnormalities
were more severe at the posterior than the anterior
part of the embryos The percentages of the three types
of abnormal phenotypes observed for each marker gene are shown in Fig 5C
Developmental defects of prmt1 morphants at gastrulation
The shortened anterior–posterior axes in the prmt1 morphants at the segmentation stage indicate defects
in convergence and extensions (C⁄ E) at gastrulation
At gastrulation, the three germ layers and the body plan are established by directed and coordinated cell movements, including epiboly to cover the yolk cells
by spreading the blastomeres, involution to internalize the marginal cells to form the precursors of the meso-derm and endomeso-derm, and C⁄ E, in which cells accumu-late on the dorsal side and lead to axis formation Gastrulation begins at 50% of epiboly (6 hpf) and ends at 100% (10 hpf)
Defective epiboly can be observed in most mor-phants at 10 hpf Although wild-type embryos showed complete blastopore closure, the MO2-injected embryos cannot close the yolk plug and demonstrated varying degrees of open blastopores (Fig 6A) Staining with notail (ntl, expressed in the ring mesoderm and endodermal precursors around the margin as a pan-mesendodermal marker) showed shortened but wid-ened notochords in the prmt1 morphants The axial mesendoderm failed to migrate to the anterior The morphants are grouped according to the degree of
Fig 3 Defective phenotypes in prmt1
knockdown zebrafish Phenotypes of
embryos injected with zprmt1 MO at
48 hpf Uninjected wild-type embryos are
shown (A) The injected embryos are
classi-fied into mild, moderate and severe
accord-ing to the phenotypes at 48 hpf The three
types of MO injected embryos at 48 hpf are
shown in (B–D) The injected embryos
with normal body axes as the wild-type are
classified as ‘normal’ (E) Frequencies of
three phenotypes caused by injection of
prmt1 MO (2 or 4 ng of MO1 and 4, or 8 ng
of MO2) The injected embryos with normal
body axes as the wild-type are classified as
‘normal’.
Trang 6Fig 4 Reduced PRMT1 protein expression, type I protein arginine methyltransferase activity and specific protein arginine methylation in prmt1 morphants (A) Proteins were prepared from embryos either not injected, or injected with prmt1 MO1 Western blot analysis of PRMT1 protein in embryos injected with 4 ng of MO1 with phenotypes classified as mild or moderate at 48 hpf are shown Detection by anti-b-actin was used as a loading control WT, wild-type; M, morphants (B) In vitro methylation was conducted with extracts from MO2 (8 ng) injected embryos at 24, 48 and 72 hpf as the source of protein arginine methyltransferase and recombinant mouse fibrillarin as the methyl-accepting protein The samples were separated by SDS ⁄ PAGE and the methylated proteins were detected by fluorography (C) Argi-nine-methylated proteins in 48 hpf embryos were detected by western blotting with an asymmetric dimethylarginine-specific antibody ASYM24 Detection by anti-b-actin was used as a loading control (D) Western blot analysis of H4R3me2 levels in 48 hpf morphant embryos Analysis of histone H4 served to normalize levels of H4R3me2 in morphants and wild-type embryos.
Fig 5 Defective phenotypes at segmentation stage for prmt1 morphants (A) Dorsal view of embryos (10-somite stage) for krox20 staining The positions of r3 and r5 are indicated The widths of r3 and r5 and the vertical distance between r3 and r5 are indicated In type 1 mor-phants, r3 was almost normal but r5 was slightly extended laterally The width of r3 and r5 were extended to 1.5-fold in type 2 and even
to 2–2.5-fold in type 3 (B) Expression of myoD at paraxial ⁄ adaxial mesoderm at the 10-somite stage Dorsal views, anterior at top The lengths of myoD expressed paraxial ⁄ adaxial mesoderm are indicated In type 1, 2 and 3 morphants, the length was 0.8–0.9, 0.6–0.7 and 0.5 compared to normal (C) The phenotypes were classified according the degree of abnormality as type 1, 2, and 3 Percentages of wild-type and morphants embryos within each phenotypic category are shown in the bar graphs n, total embryos counted in the experiments.
Trang 7shortening and widening of the notochord (Fig 6B).
By contrast, the expression of goosecoid (gsc), a
mes-endoderm marker expressed mainly in the prechordal
plate, did not reveal clear differences between
wild-type and morphants (Fig S3A)
Expression of a ventral mesodermal marker tbx6,
a member of the Brachyury-related T-box family,
revealed a slight epibolic delay and a thickened germ
ring in morphant embryos at 6 hpf The tbx6
expres-sion at 10 hpf showed a margin with a larger
unen-closed blastopore in the morphants (Fig S3B) Even
though the expression of an endodermal marker sox17
was not eliminated from the endoderm progenitors,
the strong single dot stained by sox17 at dorsal
fore-runner cells (i.e that will become Kupffer’s vesicle)
split into two (or a few) spots in some morphants
(Fig S3C)
Rescue of the C⁄ E during gastrulation of the
zebrafish prmt1 morphants by injection of
prmt1 cRNA
To further demonstrate the direct relationship between
AMO-mediated knockdown of PRMT1 and the
phe-notypes described, rescue experiments with prmt1
cRNA were conducted No significant phenotypic
changes were observed when 50 ng of wild-type or
5¢ mutated (AMO-mismatched nucleotide sequences
without changing amino acid sequences) prmt1 RNA
were injected alone We then co-injected the AMO
with prmt1 cRNA As observed at the early
gastrula-tion stage, the cRNA (50 ng) can partially rescue the abnormal phenotypes induced by MO-2 (4 ng) The defective C⁄ E phenotypes revealed by ntl staining at
10 hpf were classified as shown in Fig 6B The per-centage of the severe phenotypes decreased greatly in co-injected embryos (Table 1) To examine whether the phenotypes of prmt1 knockdown and the rescue of the morphants were a result of the methyltransferase activ-ity of PRTM1 or the PRMT1 protein per se, we pre-pared cRNA of catalytically inactive PRMT1 Three conserved amino acids SGT at the AdoMet-binding site were mutated to AAA, as previously reported by Balint et al [29] We showed that the abnormal pheno-types of the morphants cannot be rescued by the catalytic-defective cRNA (Table 1) Increased methyl-transferase activity assayed by in vitro methylation
Fig 6 Knockdown of prmt1 induces gastrulation defects Wild-type and prmt1 MO2 (8 ng) injected embryos at 10 hpf were examined (A) The morphants showed abnormal morphology at the end of epiboly (10 hpf) as reflected by different degrees of open blastopores Dashed arrows and semicircles depict embryo lengths and angles between anterior–posterior ends Lateral views, dorsal to the right (B) ntl (staining the forerunner cell group, axial chorda mesoderm) staining of the embryos at 10 hpf Dorsal views, anterior at top The morphants are grouped according to the degree of shortening and widening of the notochord.
Table 1 Rescue of gastrulation defects by catalytic active but not catalytic inactive zebrafish prmt1 cRNA.
Normal (%)
Type1 (%) Type2 (%)
Type3 (%) Total (n)
MO2 (4 ng) were co-injected with zebrafish prmt1 cRNA (50 pg) The WT cRNA contains mismatches at the MO target site without changing the encoded amino acids The MT cRNA contains the same mismatches and mutations at the AdoMet-binding site The embryos were analyzed at 10 hpf by staining with ntl The pheno-typic categories are classified as shown in Fig 6.
Trang 8could be detected in extracts from the rescued embryos
compared to that from morphants or morphants
res-cued by catalytically inactive RNA (data not shown)
The results obtained in the present study thus confirm
that the phenotypes in the morphants were specifically
a result of the reduced PRMT1 methyltransferase
activity caused by the knockdown
Reduced PRMT1 level and defective cell
movements in human Huh7 cells
From the above analyses, prmt1 knockdown did not
affect cell speciation, although cells in the morphant
embryos appeared to migrate slower and resulted in
the observed shortened anterior–posterior axes and
lateral-expanded defects The developmental program
was not blocked but progressed with a slight delay in
the prmt1 morphants from gastrulation to
segmenta-tion The phenotypes are thus likely to be the result of
defective cell movements
Genes involved in cell movements during
embryo-genesis are usually also involved in cellular migration
We thus studied whether reduced PRMT1 can affect
cell movement in a cellular model Huh7 is a human hepatocarcinoma cell line in which cell migration can
be detected under normal growth conditions without any induction PRMT1 small-interfering RNA (siRNA) knockdown reduced the PRMT1 protein level to 60% of that of control siRNA-treated Huh7 cells (Fig 7A) Reduced cell movement can be observed in PRMT1 siRNA-treated cells compared to control cells, as shown in Fig 7B The capacity of cell movement in PRMT1 knockdown cells is reduced to
75% compared to that of control cells The decreased cell movement in PRMT1 knockdown cells
is statistically significant (Fig 7C) The results obtained indicate that PRMT1 functions in the regula-tion of cell movement
Discussion
In the present study, we demonstrate that the prmt1 gene is actively and ubiquitously expressed at both RNA and protein levels at the early developmental stages of zebrafish The mRNA and protein are pres-ent before mid-blastula transition and thus are
mater-Fig 7 Reduced cell migration in a prmt1-deficient cell model Huh7 cells were treated with control or prmt1 siRNA (A) Cell extracts from the siRNA-treated cells were immunoblotted with anti-PRMT1 Detection by anti-b-tubulin was used as a loading control Reduced PRMT1 protein expression by siRNA was normalized with the b-tubulin signal (B) Images of the pre-migration and post-migration cells stained with crystal violet are shown The white circles indicate areas covered by the stoppers before cell migration (C) Quantification of cell movement
is represented as the percentage of the area covered by migrated cells in prmt1 siRNA-treated cells compared to that in control cells Data are shown as the mean ± SD of two independent experiments performed in quadruplicate A statistically significant difference between the two siRNA-treated cells is indicated (**P < 0.01; Student’s t-test).
Trang 9was reported in different human, rodent or fish
(Japa-nese flounder Paralichthys olivaceus) adult tissues
[2,3,26,30] The results obtained in the present study
show that the ubiquitous expression of PRMT1 in
adult tissues starts at early embryogenesis
In mice, the prmt1 homozygous mutants die at
approximately embryonic day 6.5 when gastrulation
begins [3] On the other hand, prmt1 knockout
embry-onic stem cells are viable, indicating the specific
require-ment of prmt1 in early embryogenesis Knockdown
and overexpression of xPRMT1b in Xenopus were
pre-viously found to provide valuable information about
the gene in early neural development [24], although the
focus of that study was on the roles of PRMT1
involv-ing Ca2+ neural induction, and its effects on other
developmental aspects were less discussed In the
pres-ent study, we successfully knocked down the
expres-sion of prmt1 in zebrafish by injection of AMO into
one-cell embryos Observation of the defective
pheno-types of the zebrafish prmt1 morphants provides the
possibility of evaluating the effects of PRMT1 viably
beyond gastrulation We observed a shortened body
length and curved tails in the majority of prmt1
mor-phants Body axis shortening and lateral expansion in
the morphants were even obvious in the posterior part
of the embryos, as revealed by marker gene staining
Generally, prmt1 knockdown did not affect cell
specia-tion, although cells in the morphants appeared to
migrate slower, resulting in the observed
anterior–pos-terior shortening and lateral expansion Even though
prmt1 has been implicated in many cellular processes,
its involvement in cell movement or migration has not
been described In the present study, we also used a
cellular model to demonstrate that PRMT1
knock-down cells migrated more slowly in a simple cell
move-ment experiment Besides PRMT1, PRMT6
knockdown affects genes involved in cellular
move-ments and inhibits cell migration [31]
We confirmed the reduced expression of PRMT1
protein in the morphants Consistently, the level of
arginine methyltransferase activity and
arginine-methy-lated proteins was reduced upon the injection of
AMO Rescue of the prmt1 morphants with prmt1
cRNA can partially reverse the early defective
pheno-types, confirming the specificity of the AMO Most
importantly, catalytic defective mutant prmt1 cRNA
lost the ability to rescue the morphants, further
early embryogenesis Considering the substrate spec-trum and the coactivator function of PRMT1, it is likely that no single target can explain the wide range
of phenotypes There would be numerous proteins and target genes that might be affected
First, PRMT1 might affect transcriptional regulation through its coactivator activity or by direct modifica-tion of histones or various transcripmodifica-tional factors PRMT1 has been shown to be the coactivator of a few NRs [17] and can also serve as a coactivator of p53 [20] Epigenetic controls play critical roles in develop-ment The importance of methyltransferases involved
in epigenetic regulation, such as DNA methyltransfer-ase Dnmt1 and histone lysine methyltransferase Suv39h1 (specific for H3K9), have been reported in zebrafish development [32] Methylation of histone H4 R3 is responsible for active chromatin and transcrip-tional activation [17,19] We showed that overall asym-metric arginine dimethylation of H4R3 was decreased
in the prmt1morphants A low level of methylated H4 R3 bound to certain promoters at critical developmen-tal stages should be responsible for part of the abnor-mal phenotypes of the prmt1 morphants
Second, many typical PRMT1 substrates containing preference RGG or GAR sequences comprise RNA binding proteins that are abundant in the early embryos Abnormal protein arginine methylation of these substrate proteins might affect their subcellular localization, as well as interactions with RNA or pro-teins, and thus lead to the developmental defects For example, methylation of a typical RGG box-containing PRTM1 substrate Sam68 is important for its RNA binding activity and nuclear localization [8] Sam68 has also been reported to be associated with RhoA [33], the downstream key regulator of the noncanonical Wnt pathway controlling C⁄ E [34] In addition, Sam68 is required for growth factor-induced migration [35] We observed a decreased asymmetric arginine dimethyla-tion of Sam68 in both zebrafish morphants and PRMT1 siRNA knockdown cells (data not shown) Whether reduced arginine methylation of Sam68 might be related
to defective cell migration requires further investigation Furthermore, even though embryonic stem cells from mouse with a PRMT1 hypomorphic allele with residual PRMT1 activity are viable, PRMT1-deficient mouse embryonic fibroblasts showed spontaneous DNA damage, G2⁄ M accumulation, cell cycle delay
Trang 10and genome instability [36] The defects indicate that
PRMT1 is involved in the DNA damage response
pathway Knockdown of PRMT1 might thus affect
early development as a result of defective cell
prolifera-tion or apoptosis Increased apoptotic cells were
detected in the prmt1 morphants (data not shown),
which may be correlated with the phenotypes
In summary, in the present study, we demonstrate
the importance of the enzyme activity of PRMT1 with
zebrafish embryogenesis We show the relationships
between prmt1 knockdown, reduced protein arginine
methylation and H4 R3 methylation with respect to
early developmental defects at gastrulation in
zebra-fish The present study describes the first thorough
investigation of a protein arginine methyltransferase
family member in zebrafish The investigation also
establishes zebrafish as a good study platform for
pro-tein arginine methylation
Experimental procedures
Zebrafish rearing
Adult zebrafish (Danio rerio) were maintained under a
14 : 10 h light⁄ dark cycle at 28 C All embryos were
collected by natural spawning and staged according to
Kimmel et al [37]
mRNA expression analyses by RT-PCR
Total RNA was isolated from embryos at different stages
of embryogenesis and different adult tissues by TRIzol
reagent (Molecular Research Center, Inc., Cincinnati, OH,
USA) First-strand cDNA was synthesized from 5 lg of
total RNA by M-MLV Reverse Transcriptase (Promega,
Madison, WI, USA) RT-PCR was performed with the
pri-mer set ZF1-F and ZF1-R to amplify the conserved regions
in zebrafish prmt1 gene (GenBank NM_200650.1) or ASF
and ASR for putative alternative splicing at the 5¢ end of
prmt1(Fig 1A and Table S1) Amplification of the
elonga-tion factor 1a (primer set Ef1 and Ef2) was used as an
internal control
Zebrafish embryonic extract preparation, western
blot analyses and in vitro methylation
Zebrafish embryos were manually deyolked [38],
resus-pended in extraction buffer (150 mm NaCl, 100 mm
Tris⁄ HCl, pH 7.5, 5% glycerol, 1 mm dithithreitol, 1%
Tri-ton X-100, 1 mm phenylmethanesulfonyl fluoride and
com-plete protease inhibitor cocktail; Roche Diagnostics, Basel,
Switzerland) and then homogenized (400 lL per 100
embryos) by a homogenizer (IKA T10; IKA Works
Staufen, Germany) The homogenate was centrifuged
at 17 530 g at 4C for 20 min and the supernatant was stored at )20 C as the embryonic extract Aliquots of the embryonic extract (30 lg of protein) were resolved by SDS⁄ PAGE followed by western blot analyses with antibodies specific to PRMT1 (Upstate Biotechnology, Lake Placid, NY, USA) and methylarginines (ASYM24; Upstate Biotechnology) In vitro methylation was conducted
as described previously [39] Essentially, embryonic extracts (35 lg of protein), recombinant mouse fibrillarin protein and 1.5 lCi of [methyl-3H]-AdoMet (60 Ci⁄ mmol; Amer-sham Biotech, Little Chalfont, UK) were incubated at
37C for 60 min in methylation buffer (50 mm sodium phosphate, pH 7.5) with a total volume of 15 lL The sam-ples were subjected to SDS⁄ PAGE The gels were then stained, treated with EN3HANCE (Perkin Elmer, Wal-tham, MA, USA) and dried for fluorography
Isolation of zebrafish histones and assay for histone methylation
Histones were prepared essentially in accordance with the protocol previously described by Gurvich et al [40] Zebrafish embryos harvested at 48 hpf were manually deyolked and dissolved in extraction buffer Nuclei were collected by centrifugation at 17 530 g at 4C for 20 min, and histones were extracted by shaking in 0.2 m sulfuric acid for 1 h at 4C After centrifugation, histones were precipitated with ethanol at )20 C overnight, washed once with ethanol, and resuspended in distilled water Aliquots of the zebrafish embryonic extract (10 lg) were resolved by SDS⁄ PAGE followed by western blot analyses with anti-H4 (Upstate Biotechnology) and anti-H4Me R3 (Upstate Biotechnology)
WISH and immunofluorescent analysis
Zebrafish prmt1 cDNA obtained from imaGenes (Berlin, Germany) was amplified with the primers set ZF1-F and ZF1R The fragment was cloned into a modified pGEM vector with partial deletion in the multiple cloning sites and the resulting pGEM-zprmt1 was used for riboprobe preparation
In situhybridization was performed according to Wester-field [41] Essentially, after rehydration, proteinase treat-ment and prehybridization, hybridization was performed with 100–200 ng of digoxigenin-UTP labeled riboprobes The pGEM-zprmt1 plasmid was linearized by EcoRI or SalI restriction enzyme and the RNA was transcribed with SP6 or T7 RNA polymerase to prepare the antisense or sense RNA probe respectively The embryos were washed and incubated with anti-DIG antiserum and stained Embryos were then mounted in 100% glycerol for observa-tion using a dissecting microscope (Zeiss AXioskop2; Carl