ACKNOWLEDGMENTS
We thank I. Vieira for coordinating the INCT Biodiversidade e Uso da
Terra na Amazônia and the Large Scale Biosphere-Atmosphere Program
(LBA) for logistical and infrastructure support during field
measurements.Funding:This work was supported by grants
from Brazil (CNPq 574008/2008-0, 458022/2013-6, and
400640/2012-0; CNPq-CAPES-PELD 441659/2016-0; Embrapa
SEG:02.08.06.005.00; The Nature Conservancy–Brasil; and CAPES
scholarships), the UK (Darwin Initiative 17-023; NE/F01614X/1; NE/
G000816/1; NE/F015356/2; NE/l018123/1; and NE/K016431/1),
Formas 2013-1571, and Australian Research Council grant DP120100797.
Individual support included FAPESP funding (2017/25383-0 and 2019/
05440-5, respectively) to C.G.L. and T.F.M.; H2020-MSCA-RISE
(691053-ODYSSEA) funding to E.B. and J.B.; Fulbright Brasil funding to
R.M.H.; NSF-Belmont Forum award (1852113) to L.C.; and CNPq funding
(144452/2010-3, 307597/2016-4, 436007/2018-5, 303548/2017-7,
308970/2019-5, and 313183/2014-7, respectively) to V.C.d.O., L.J., R.P.L.,
P.S.P., N.H., and J.Z. Institutional support was provided by the Herbário
IAN in Belém, LBA in Santarém, and FAPEMAT. This is paper no. 76
in the Sustainable Amazon Network publication series.Ethics
statement:All biodiversity sampling was undertaken in compliance with
Brazilian environmental regulations under the following licenses: (i) Sisbio
license 24164 for collecting plants, issued by the Chico Mendes Institute
for Biodiversity Conservation (ICMBio); (ii) Sisbio license 10061-1 for
collecting dung beetles, issued by the Institute of Environment and
Renewable Natural Resources (IBAMA); (iii) Sisbio licenses 10199-2 and
24355-2 for collecting fish, both issued by ICMBio (iv) Sisbio license
10873-1 for collecting insects, issued by IBAMA; and (v) Sisbio license
19102-4 for collecting Odonata, Ephemeroptera, Plecoptera and
Trichoptera, issued by ICMBio. No license was required for bird sampling
because the methods were observational and did not involve collecting
or handling of specimens. Socioeconomic data were collected following
the UK Research Integrity Office Principles for Research involving human
participants, human material, and personal data and were collected
with informed consent. Further approval for opportunity cost data
collection was obtained from the Brazilian Agricultural Research
Corporation (Embrapa) under CAAE 29054920.4.0000.5173 and
Stanford University under IRB Protocol 19044.Author
contributions:T.A.G., J.F., J.B., L.P., C.G.L., G.D.L., and S.F.B.F.
designed the research with additional taxon-specific input from
E.B., A.C.L., R.M.H., L.E.O.C.A., J.G.d.B., N.H., L.J., R.P.L., J.L.,
N.G.M., J.L.N., J.M.B.O.-J., V.H.F.O., V.C.d.O., P.S.P., R.R.C.S., and
J.Z.; C.G.L., E.B., A.C.L., R.M.H., J.G.d.B., N.H., L.J., R.P.L., J.L.,
N.G.M., J.L.N., J.M.B.O.-J., V.H.F.O., V.C.d.O., L.P., R.R.C.S., and J.Z.collected the field data or analyzed biological samples. S.F.B.F. and
T.A.G. processed the remote sensing data. G.D.L. and C.G.L.
undertook the analyses with input from J.B., J.R.T., R.M., R.D.G.,
T.F.M., and S.F.B.F.; T.F.M., T.A.G., L.P., and R.D.G. processed the
socioeconomic data; C.G.L., G.D.L., and J.B. led the writing, with all
authors contributing to reviewing and editing.Competing
interests:Authors declare no competing interests.Data and
materials availability:The data and computer code used in these
analyses are available at https://doi.org/10.6084/m9.figshare.- The Zonation conservation planning software is available
at: https://github.com/cbig/zonation-core.
SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/370/6512/117/suppl/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S13
Table S1
References ( 29 – 50 )
2 January 2020; accepted 10 August 2020
10.1126/science.aba7580SEX DETERMINATION
The mouseSrylocus harbors a cryptic exon that is
essential for male sex determination
Shingo Miyawaki1,2, Shunsuke Kuroki1,2, Ryo Maeda1,2, Naoki Okashita1,2, Peter Koopman^3 , Makoto Tachibana1,2*
The mammalian sex-determining geneSryinduces male development. Since its discovery 30 years ago,
Sryhas been believed to be a single-exon gene. Here, we identified a cryptic second exon of mouseSryand
a corresponding two-exon typeSry(Sry-T) transcript. XY mice lackingSry-Twere sex-reversed, and ectopic
expression ofSry-Tin XX mice induced male development.Sry-Tmessenger RNA is expressed similarly
to that of canonical single-exon typeSry(Sry-S), but SRY-T protein is expressed predominantly because of
the absence of a degron in the C terminus of SRY-S.Sryexon2 appears to have evolved recently in mice
through acquisition of a retrotransposon-derived coding sequence to replace the degron. Our findings
suggest that in nature, SRY-T, not SRY-S, is the bona fide testis-determining factor.
S
exual differentiation is essential for the
survival and evolution of a species. Ex-
pression of the Y chromosomal geneSry
is required for male development in mam-
mals ( 1 ). Since its discovery in 1990 ( 2 , 3 ),
Sryhas been considered a single-exon gene,
containing only one open reading frame (ORF)
and encoding one protein. The mouseSrylocus
has a complex genomic structure, with 2.8 kb of
specific sequence containing an ORF, flanked
by ~50 kb of palindromic sequences ( 4 ). Using a
comprehensive transcriptomics approach, we
identified a sequence within the flanking pal-
indrome that is essential for male development.
We previously developed a method to en-
rich somatic cell populations from mouse
embryonic gonads ( 5 , 6 ) (fig. S1A). Using this
method, NR5A1-expressing mouse pre-Sertoli
cells were purified for RNA-sequencing (RNA-
seq) analysis. We identified a transcribed
sequence located within the palindromic se-
quences 3′to the knownSryORF (Fig. 1A and
fig. S1B). This sequence was transcribed sim-
ilarly to the knownSrytranscript in terms of
temporal and spatial specificity (fig. S1, C and
D) but lacked a cap site, as evidenced by cap
analysis gene expression sequencing (CAGE-
seq) (Fig. 1A and fig. S1B). Long-read RNA-seq
identified this sequence as the 3′portion of a
newSrytranscript that starts at the known
Srytranscription start site and is spliced once
at typical 5′GT/3′AG splicing site sequences
with an internal polypyrimidine tract (Fig. 1B
and fig. S1, E and F). Thus, this sequence rep-
resents a second exon ofSry.
Sequencing data indicated the presence
of two mRNA transcripts, the known single-
exon type (Sry-S) and a two-exon type (Sry-T)
(Fig. 1B and fig. S2). Most (~70%) ofSry
exon2 is occupied by one long interspersed
nuclear element (LINE) L3 and three long
terminal repeats (LTRs) (Fig. 1C and fig. S3).
Sry-Texpression levels were approximately
half those ofSry-Sin pre-Sertoli cells (fig. S1G)and, as evidenced by single-cell transcriptome
analysis, individual pre-Sertoli cells expressed
both transcripts ( 7 ) (fig. S1H).
The predicted SRY-T protein shares amino
acids 1 to 377 with SRY-S, including the high-
mobility group DNA-binding domain and poly-
glutamine (poly-Q) sequences that are important
for transcriptional activation ( 8 , 9 ). Beyond this,
the C-terminal 15 amino acids of SRY-T are en-
coded by the second exon, whereas absence of
splicing results in a different 18 amino acids
at the C terminus of SRY-S (Fig. 1D). We used
CRISPR/Cas9 to establish the knock-in mouse
lineSry-T:3xFlag, in whichSryexon2 is targeted
to produce SRY-T with a triple Flag-epitope tag
at the C terminus (fig. S4). We prepared protein
lysates from embryonic day 11.5 (E11.5)Sry-
T:3xFlag gonads and enriched SRY using anti-
bodies against the poly-Q sequence ( 10 ) (fig.
S4A). Anti-FLAG immunoblotting identified a
protein with the expected molecular weight of
SRY-T-3xFLAG (~55 kDa) in the anti-SRY im-
mune complexes (Fig. 1E). These results indicate
thatSry-Tis translated into a protein in vivo.
Next, we generated mice lacking theSry-T
transcript by deletingSryexon2 (Fig. 2, A and
B, and fig. S5, A and B). Elimination ofSry-T
alone in XY mice resulted in complete male-
to-female sex reversal (Fig. 2C and fig. S5, C
to G), with a global gene expression profile of
E11.5 XYSry-T-deficient gonadal somatic cells
similar to that of their XX counterparts (fig.
S5H).Sry-Sexpression was maintained at
normal levels in these mice (fig. S5I), suggest-
ingthatinwild-type(WT)XYmice,Sry-Tand
not the previously knownSry-Sis necessary
and sufficient for male sex determination.
To gain insights into this male-to-female sex
reversal, we investigated total SRY protein ex-
pression in NR5A1+gonadal somatic cells inSCIENCEsciencemag.org 2 OCTOBER 2020•VOL 370 ISSUE 6512 121
(^1) Laboratory of Epigenome Dynamics, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan. (^2) Division of Epigenome Dynamics, Institute of
Advanced Medical Sciences, Tokushima University, 3-18-15 Kuramoto-Cho, Tokushima, 770-8503, Japan.^3 Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland
4072, Australia.
*Corresponding author. Email: [email protected]
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