Báo cáo khoa học: The predominant protein arginine methyltransferase PRMT1 is critical for zebrafish convergence and extension during gastrulation pdf

The predominant protein arginine methyltransferase
PRMT1 is critical for zebrafish convergence and extension
during gastrulation
Yun-Jung Tsai
1
, Huichin Pan
1,2
, Chuan-Mao Hung
1
, Po-Tsun Hou
1
, Yi-Chen Li
1
, Yu-Jen Lee
3
,
Yi-Ting Shen
1,4
, Trang-Tiau Wu
4,5
and Chuan Li
1,2
1 Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan
2 Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan
3 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:
(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.
FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS 905
PRMT1 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 alternatively 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
PRMT1 expression and function during embryogenesis Y J. Tsai et al.
906 FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS
stages (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 gastrulation to the early segmentation 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.
Y J. Tsai et al. PRMT1 expression and function during embryogenesis
FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS 907
4–8 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, midbrain; mhb, mid-hindbrain
boundary; ov, otic vesicle; som, somites.
PRMT1 expression and function during embryogenesis Y J. Tsai et al.
908 FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS
segmentation 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
myoD expression 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 endoderm, 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’.
Y J. Tsai et al. PRMT1 expression and function during embryogenesis
FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS 909
Fig. 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.
PRMT1 expression and function during embryogenesis Y J. Tsai et al.
910 FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS
shortening 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)
WT 100 120
MO2 5 18 51 26 176
MO2 + WT cRNA 29 39 25 7 100
MO2 + MT cRNA 8 24 50 18 161
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.
Y J. Tsai et al. PRMT1 expression and function during embryogenesis
FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS 911
could 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).
PRMT1 expression and function during embryogenesis Y J. Tsai et al.
912 FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS
nally derived. The continuous prmt1 expression indi-
cates zygotic expression. The results obtained are con-
sistent with previous reports of prmt1 expression in
mice or Xenopus early development [3,24]. We also
detected wide expression of prmt1 in various adult
zebrafish tissues. Ubiquitous expression of PRMT1
was 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 Ca
2+
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
supporting the importance of active PRMT1 methyl-
transferase in early embryogenesis. Reduced methyl-
transferase activity and protein arginine methylation
should thus be responsible for the abnormalities in
zebrafish prmt1 morphants. The defective phenotypes
in epiboly and C ⁄ E indicate wide effects of prmt1 in
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 transcriptional 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
Y J. Tsai et al. PRMT1 expression and function during embryogenesis
FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS 913
and 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 lgof
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
Ò
Works
Staufen, Germany). The homogenate was centrifuged
at 17 530 g at 4 °C 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-
3
H]-AdoMet (60 Ci ⁄ mmol; Amer-
sham Biotech, Little Chalfont, UK) were incubated at
37 °C 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 4 °C for 20 min,
and histones were extracted by shaking in 0.2 m sulfuric
acid for 1 h at 4 ° C. 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 situ hybridization 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
PRMT1 expression and function during embryogenesis Y J. Tsai et al.
914 FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS
Zeiss, Oberkochen, Germany). Probes used for krox20,
myoD, ntl, gsc, tbx6 and sox17 were obtained as generous
gifts from Dr Ching-Hua Hu (Department of Life Sciences,
National Taiwan Ocean University) and Dr Bon-chu
Chung (Institute of Molecular Biology, Academia Sinica,
Taiwan).
For immunostaining, embryos were collected and fixed as
described above. After fixation, the embryos were treated
with proteinase and acetone. Embryos were incubated with
blocking solution (5% goat serum, 0.1% Triton X-100 in
NaCl ⁄ P
i
) for 1 h and anti-PRMT1 antiserum overnight at
room temperature. Embryos were washed and incubated

with fluoroscein isothiocyanate-conjugated goat anti-(rabbit
IgG) antibody (Jackson ImmunoResearch, West Grove,
PA, USA).
Knockdown of prmt1 by AMOs and rescue cRNA
injection
Two AMOs of prmt1 (zprmt1 MO1 and MO2, Fig. 1B)
were designed and purchased from Gene Tools (Philomath,
OR, USA). Control embryos were injected with a standard
control oligonucleotide (5¢-CCTCTTACCTCAGTTACA
ATTTAT-3¢). We also injected 1.5-fold (w ⁄ w) p53 AMO
(5¢-GACCTCCTCTCCACTAAACTACGAT-3¢) to the
zprmt1 AMO, as suggested by Robu et al. [42], aiming to
avoid apoptosis induced through the off-target activation
of p53. AMO was injected into one- to two-cell stage
embryos with a microinjector Nanojector II (Drummond
Scientific Company, Broomall, PA, USA). The full prmt1
coding region was prepared from RT-PCR with primers
ASF and ZF1-R and cloned into the vector pCS2
+
. Rescue
experiments were performed with prmt1 cRNA synthesized
in vitro using the mMESSAGEmMACHINE kit in accor-
dance with the manufacturer’s protocol (Ambion Europe
Ltd, Huntingdon, UK). The 5¢ region recognized by the
AMOs was further mutated using QuikChange
Ò
II site-
directed mutagenesis kit (Stratagene, La Jolla, CA, USA)
to introduce mismatches aiming to avoid quenching in the
rescue experiments by MO1 or MO2 with the primer sets

(MO1 forward and MO1 reverse; MO2 forward and MO2
reverse; Table S1). Catalytic inactive PRMT1 with S69A,
G70A and T71A mutations at the conserved AdoMet-bind-
ing site was designed as described by Balint et al. [29]. The
point mutations were also introduced by site-directed muta-
genesis to produce the catalytic mutant by the primer set
(SGT forward and SGT reverse; Table S1). Synthesized
capped cRNA was co-injected with the AMO into the one-
cell stage embryos.
Cell culture and migration assay
Huh-7 (human hepatocarcinoma cell line) cells were cul-
tured at 37 °C in DMEM medium (Gibco, Gaithersburg,
MD, USA) supplemented with 10% fetal bovine serum
(Gibco), 1% of penicillin and 1% of l-glutamine in 5%
CO
2
. Transient siRNA-mediated PRMT1 knockdown was
performed using PRMT1 siRNA that targets nucleotides
1037–1055 (5¢-CCA TCG ACC TGG ACT TCA A-3¢)
and control siRNA (5¢-UUC UCC GAA CGU GUC
ACU U-3¢) synthesized by GenePharma (GenePharma
Co., Ltd, Shanghai, China) with Lipofectamine 2000 (Invi-
trogen, Carlsbad, CA, USA). After 44 h, cells (2.5 · 10
4
)
were seeded into wells of the Oris Cell Migration Assem-
bly Kit-FLEX (Platypus Technology, Madison, WI, USA)
and cell migration assays were carried in accordance with
the manufacturer’s instructions. After the cells were
allowed to attach for 10 h, well inserts were removed, and

the cells were allowed to migrate into the clear field for
16 h. Cells were fixed with formaldehyde, stained with
crystal violet and photographed. The pre-migration and
post-migration images were captured and analyzed using
imagej software ( The percent-
age of cell movement is defined as the migration area of
PRMT1 siRNA-treated cells over that of control siRNA-
treated cells.
Acknowledgements
The project was supported by NSC 94-2320-B-040-044,
95-2320-B-040-042, 96-2320-B-040-022-MY2 and 98-
2320-B-040-011-MY3 from National Science Council
and CSMU 94-OM-A-023, 97-OM-A-136 and 98OM-
A-060 from Chung Shan Medical University. The
authors would like to express thanks to Drs Bon-chu
Chung, Yi-Chuan Cheng and Shye-Jye Lee for valu-
able discussions and Dr Wen-Wei Chang for his valu-
able suggestions on the cell migration assay. We also
thank Yuling Lin, Pei-Hsin Chang, Hsiao-Yun Cheng,
Li-Chun Tu and Han-Ni Chuang for fish rearing,
cDNA preparation and WISH probe preparation.
References
1 Bedford MT & Clarke SG (2009) Protein arginine methyl-
ation in mammals: who, what, and why. Mol Cell 33, 1–13.
2 Lin WJ, Gary JD, Yang MC, Clarke S & Herschman
HR (1996) The mammalian immediate-early TIS21 pro-
tein and the leukemia-associated BTG1 protein interact
with a protein-arginine N-methyltransferase. J Biol
Chem 271, 15034–15044.
3 Pawlak MR, Scherer CA, Chen J, Roshon MJ &

Ruley HE (2000) Arginine N-methyltransferase 1 is
required for early postimplantation mouse development,
but cells deficient in the enzyme are viable. Mol Cell
Biol 20, 4859–4869.
4 Lee J & Bedford MT (2002) PABP1 identified as an
arginine methyltransferase substrate using high-density
protein arrays. EMBO Rep 3, 268–273.
Y J. Tsai et al. PRMT1 expression and function during embryogenesis
FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS 915
5 Wada K, Inoue K & Hagiwara M (2002) Identification
of methylated proteins by protein arginine N-methyl-
transferase 1, PRMT1, with a new expression cloning
strategy. Biochim Biophys Acta 1591, 1–10.
6 Smith JJ, Rucknagel KP, Schierhorn A, Tang J,
Nemeth A, Linder M, Herschman HR & Wahle E
(1999) Unusual sites of arginine methylation in Poly(A)-
binding protein II and in vitro methylation by protein
arginine methyltransferases PRMT1 and PRMT3.
J Biol Chem 274, 13229–13234.
7 Nichols RC, Wang XW, Tang J, Hamilton BJ,
High FA, Herschman HR & Rigby WF (2000) The
RGG domain in hnRNP A2 affects subcellular
localization. Exp Cell Res 256, 522–532.
8 Cote J, Boisvert FM, Boulanger MC, Bedford MT &
Richard S (2003) Sam68 RNA binding protein is an
in vivo substrate for protein arginine N-methyltransfer-
ase 1. Mol Biol Cell 14, 274–287.
9 Passos DO, Quaresma AJ & Kobarg J (2006) The
methylation of the C-terminal region of hnRNPQ
(NSAP1) is important for its nuclear localization.

Biochem Biophys Res Commun 346, 517–525.
10 Ostareck-Lederer A, Ostareck DH, Rucknagel KP,
Schierhorn A, Moritz B, Huttelmaier S, Flach N,
Handoko L & Wahle E (2006) Asymmetric arginine
dimethylation of heterogeneous nuclear ribonucleopro-
tein K by protein-arginine methyltransferase 1 inhibits
its interaction with c-Src. J Biol Chem 281, 11115–
11125.
11 Abramovich C, Yakobson B, Chebath J & Revel M
(1997) A protein-arginine methyltransferase binds to
the intracytoplasmic domain of the IFNAR1 chain in
the type I interferon receptor. EMBO J 16, 260–266.
12 Mowen KA, Tang J, Zhu W, Schurter BT, Shuai K,
Herschman HR & David M (2001) Arginine methyla-
tion of STAT1 modulates IFNalpha ⁄ beta-induced tran-
scription. Cell 104, 731–741.
13 Meissner T, Krause E, Lodige I & Vinkemeier U (2004)
Arginine methylation of STAT1: a reassessment. Cell
119, 587–589. Discussion 589–590.
14 Komyod W, Bauer UM, Heinrich PC, Haan S &
Behrmann I (2005) Are STATS arginine-methylated?
J Biol Chem 280, 21700–21705.
15 Weber S, Maass F, Schuemann M, Krause E, Suske G
& Bauer UM (2009) PRMT1-mediated arginine
methylation of PIAS1 regulates STAT1 signaling. Genes
Dev 23, 118–132.
16 Yamagata K, Daitoku H, Takahashi Y, Namiki K,
Hisatake K, Kako K, Mukai H, Kasuya Y & Fukamizu
A (2008) Arginine methylation of FOXO transcription
factors inhibits their phosphorylation by Akt. Mol Cell

32, 221–231.
17 Wang H, Huang ZQ, Xia L, Feng Q, Erdjument-Bro-
mage H, Strahl BD, Briggs SD, Allis CD, Wong J,
Tempst P et al. (2001) Methylation of histone H4 at
arginine 3 facilitating transcriptional activation by
nuclear hormone receptor. Science 293, 853–857.
18 Chen D, Ma H, Hong H, Koh SS, Huang SM,
Schurter BT, Aswad DW & Stallcup MR (1999)
Regulation of transcription by a protein methyltransfer-
ase. Science 284, 2174–2177.
19 Huang S, Litt M & Felsenfeld G (2005) Methylation of
histone H4 by arginine methyltransferase PRMT1 is
essential in vivo for many subsequent histone modifica-
tions. Genes Dev 19, 1885–1893.
20 An W, Kim J & Roeder RG (2004) Ordered coopera-
tive functions of PRMT1, p300, and CARM1 in tran-
scriptional activation by p53. Cell 117, 735–748.
21 Rezai-Zadeh N, Zhang X, Namour F, Fejer G,
Wen YD, Yao YL, Gyory I, Wright K & Seto E (2003)
Targeted recruitment of a histone H4-specific
methyltransferase by the transcription factor YY1.
Genes Dev 17, 1019–1029.
22 Barrero MJ & Malik S (2006) Two functional modes of
a nuclear receptor-recruited arginine methyltransferase
in transcriptional activation. Mol Cell 24, 233–243.
23 Kwak YT, Guo J, Prajapati S, Park KJ, Surabhi RM,
Miller B, Gehrig P & Gaynor RB (2003) Methylation
of SPT5 regulates its interaction with RNA polymerase
II and transcriptional elongation properties. Mol Cell
11, 1055–1066.

24 Batut J, Vandel L, Leclerc C, Daguzan C, Moreau M
& Neant I (2005) The Ca2
+
-induced methyltransferase
xPRMT1b controls neural fate in amphibian embryo.
Proc Natl Acad Sci USA 102, 15128–15133.
25 Hung CM & Li C (2004) Identification and
phylogenetic analyses of the protein arginine
methyltransferase gene family in fish and ascidians.
Gene 340, 179–187.
26 Scorilas A, Black MH, Talieri M & Diamandis EP
(2000) Genomic organization, physical mapping, and
expression analysis of the human protein arginine meth-
yltransferase 1 gene. Biochem Biophys Res Commun 278,
349–359.
27 Goulet I, Gauvin G, Boisvenue S & Cote J (2007)
Alternative splicing yields protein arginine methyltrans-
ferase 1 isoforms with distinct activity, substrate speci-
ficity, and subcellular localization. J Biol Chem 282,
33009–33021.
28 Corey DR & Abrams JM (2001) Morpholino antisense
oligonucleotides: tools for investigating vertebrate devel-
opment. Genome Biol 2, REVIEWS1015.
29 Balint BL, Szanto A, Madi A, Bauer UM, Gabor P,
Benko S, Puskas LG, Davies PJ & Nagy L (2005)
Arginine methylation provides epigenetic transcription
memory for retinoid-induced differentiation in myeloid
cells. Mol Cell Biol 25, 5648–5663.
30 Dong CW, Zhang YB, Lu AJ, Zhu R, Zhang FT,
Zhang QY & Gui JF (2007) Molecular characterisation

and inductive expression of a fish protein arginine meth-
PRMT1 expression and function during embryogenesis Y J. Tsai et al.
916 FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS
yltransferase 1 gene in response to virus infection. Fish
Shellfish Immunol 22, 380–393.
31 Michaud-Levesque J & Richard S (2009) Thrombospon-
din-1 is a transcriptional repression target of PRMT6.
J Biol Chem 284, 21338–21346.
32 Rai K, Nadauld LD, Chidester S, Manos EJ,
James SR, Karpf AR, Cairns BR & Jones DA (2006)
Zebra fish Dnmt1 and Suv39h1 regulate organ-specific
terminal differentiation during development. Mol Cell
Biol 26, 7077–7085.
33 Huot ME, Brown CM, Lamarche-Vane N & Richard S
(2009) An adaptor role for cytoplasmic Sam68 in modu-
lating Src activity during cell polarization. Mol Cell Biol
29, 1933–1943.
34 Zhu S, Liu L, Korzh V, Gong Z & Low BC (2006)
RhoA acts downstream of Wnt5 and Wnt11 to regulate
convergence and extension movements by involving
effectors Rho kinase and Diaphanous: use of zebrafish
as an in vivo model for GTPase signaling. Cell Signal
18, 359–372.
35 Huot ME, Vogel G & Richard S (2009) Identification
of a Sam68 ribonucleoprotein complex regulated by
epidermal growth factor. J Biol Chem 284, 31903–
31913.
36 Yu Z, Chen T, Hebert J, Li E & Richard S (2009) A
mouse PRMT1 null allele defines an essential role for
arginine methylation in genome maintenance and cell

proliferation. Mol Cell Biol 29, 2982–2996.
37 Kimmel CB, Ballard WW, Kimmel SR, Ullmann B &
Schilling TF (1995) Stages of embryonic development of
the zebrafish. Dev Dyn 203, 253–310.
38 Link V, Shevchenko A & Heisenberg CP (2006) Proteo-
mics of early zebrafish embryos. BMC Dev Biol 6,1.
39 Hung CJ, Chen DH, Shen YT, Li YC, Lin YW,
Hsieh M & Li C (2007) Characterization of protein
arginine methyltransferases in porcine brain. J Biochem
Mol Biol 40, 617–624.
40 Gurvich N, Berman MG, Wittner BS, Gentleman RC,
Klein PS & Green JB (2005) Association of valproate-
induced teratogenesis with histone deacetylase inhibition
in vivo. FASEB J 19, 1166–1168.
41 Westerfield M (2007) The Zebrafish Book: A Guide for
the Laboratory Use of Zebrafish (Danio rerio), 5th edn.
M Westerfield, Eugene, OR.
42 Robu ME, Larson JD, Nasevicius A, Beiraghi S, Bren-
ner C, Farber SA & Ekker SC (2007) p53 activation by
knockdown technologies. PLoS Genet 3, e78.
Supporting information
The following supplementary material is available:
Fig. S1. Expression of zebrafish prmt1 mRNA and
protein during early development.
Fig. S2. Defective phenotypes in prmt1 knock-down
zebrafish at 72, 96 or 120 hpf.
Fig. S3. Knockdown of prmt1 induces gastrulation
defects.
Table S1. Primers used in the present study.
This supplementary material can be found in the

online version of this article.
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should be addressed to the authors.
Y J. Tsai et al. PRMT1 expression and function during embryogenesis
FEBS Journal 278 (2011) 905–917 ª 2011 The Authors Journal compilation ª 2011 FEBS 917