Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

1.

2.

3.

4.

5.

Metagene analysis. Mouse 3′-UTR sequences (mm10) were obtained from the

UCSC Table Browser by specifying the track as ALL GENCODE V22 and the table

as Basic. The 200-nt proximal regions were extracted, and duplicated sequences

were removed. The number of motifs present in each region was then counted.

Multiple alignment. Sequences were aligned by MAFFT48 or MAFFT at the MPI

Bioinformatics Toolkit49 with manual changes to align positions of GAC motifs

among species. Multiple alignment and folding of RNA were performed by

LocARNA50.

In vitro transcription. The plasmid pEFSA Dand5-3′UTR was used as a PCR

template to generate SP6 transcription matrices. Briefly, a forward primer containing the SP6 promoter sequence and a Reverse primer were used for the

amplification of the DNA fragment encoding the sequence of interest using the

Phire Green Hot Start II PCR Master Mix (ThermoFisher F126L). For future

annealing of a fluorescent probe, the complementary 5′-TGTCTGGGCAACAGGCTCAGG-3′ sequence was introduced as a 3′ tag via the Reverse primer. For

the short transcripts Dand5 3′UTR66-110 and Dand5 3′UTR226-270, the transcription

matrices were prepared without plasmid template using overlapping PCR primers

carrying the mutations of interest. After agarose gel electrophoresis, PCR amplicons were purified on NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel, Cat.

No. 740609). 500 ng of SP6 transcription matrice was used to in vitro transcribe the

RNA of interest by using the SP6 RNA polymerase kit according to the manufacturer’s conditions (Roche, Cat.No. 10 810 274 001). After 2 hours of incubation

at 37 °C, the transcripts were incubated for one additional hour in presence of

DNAse I (Roche, Cat.No. 04 716 728 001) in order to eliminate the SP6 DNA

matrix. Finally, the transcripts were purified on Quick Spin Columns (Roche, Cat.

No. 11 274 015 001).

Fluorescent EMSA. The fluorescent DNA probe 5′-CTGAGCCTGTTGCCCAGAC-3′ carrying a 5′-Dynomics 681 dye, was synthesized by Microsynth AG.

Before each experiment, 3′ tagged RNA and the fluorescent DNA probe were preannealed by denaturation (3 min at 98 °C) and renaturation for 10 min at room

temperature. One pmol of 3′-tagged RNA and 2.5 pmol of fluorescent probe were

mixed for each condition. Complexes of fluorescent RNA:DNA duplex with

recombinant GST-KH were assembled in a final volume of 20 μL containing

10 mM Tris-HCl pH8, 100 mM KCl, 2.5 mM MgCl2, 2.5% glycerol, 1 mM DTT,

and 1 µg of yeast tRNAs by incubation on ice for 30 min in the dark. For competition experiments, the recombinant GST-KH protein was used at a fixed concentration (200 nM). Increasing amounts of unlabeled competitor RNAs were

added to pre-assembled fluorescent complexes and incubated for another 30 min.

The complexes were then resolved by electrophoresis on a 5% native polyacrylamide gel containing 45 mM Tris-Borate, 1 mM EDTA, and 2.5% glycerol.

The fluorescence was detected using the Odyssey CLx Infrared Imaging System

(LI-COR Biosciences).

Reporting summary. Further information on research design is available in the Nature

Research Reporting Summary linked to this article.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Data availability

Sequencing data of this study were deposited to the Gene Expression Omnibus (GEO)

database under accession number GSE140931. Publicly available data was downloaded

from NCBI Data Base (https://www.ncbi.nlm.nih.gov), UCSC Table Browser (https://

genome.ucsc.edu). Source data are provided with this paper.

Code availability

25.

26.

27.

Raw data for biochemical experiments are provided in Source data. The custom script of

K-mer analysis is avaliable in the Github page (https://github.com/KRK13/Kmer2021).

Other data and codes are available from the corresponding authors upon reasonable

request.

28.

Received: 14 January 2020; Accepted: 9 June 2021;

30.

16

29.

Blum, M., Feistel, K., Thumberger, T. & Schweickert, A. The evolution and

conservation of left-right patterning mechanisms. Development 141,

1603–1613 (2014).

Nakamura, T. & Hamada, H. Left-right patterning: conserved and divergent

mechanisms. Development 139, 3257–3262 (2012).

Pennekamp, P. et al. The ion channel polycystin-2 is required for left-right

axis determination in mice. Curr. Biol. 12, 938–943 (2002).

Field, S. et al. Pkd1l1 establishes left-right asymmetry and physically interacts

with Pkd2. Development 138, 1131–1142 (2011).

Yoshiba, S. et al. Cilia at the node of mouse embryos sense fluid flow for leftright determination via Pkd2. Science 338, 226–231 (2012).

Mizuno, K. et al. Role of Ca(2+) transients at the node of the mouse embryo

in breaking of left-right symmetry. Sci. Adv. 6, eaba1195 (2020).

Shiratori, H. & Hamada, H. TGFbeta signaling in establishing left-right

asymmetry. Semin Cell Dev. Biol. 32, 80–84 (2014).

Marques, S. et al. The activity of the Nodal antagonist Cerl-2 in the mouse

node is required for correct L/R body axis. Genes Dev. 18, 2342–2347 (2004).

Kawasumi, A. et al. Left-right asymmetry in the level of active Nodal protein

produced in the node is translated into left-right asymmetry in the lateral plate

of mouse embryos. Dev. Biol. 353, 321–330 (2011).

Maisonneuve, C. et al. Bicaudal C, a novel regulator of Dvl signaling abutting

RNA-processing bodies, controls cilia orientation and leftward flow.

Development 136, 3019–3030 (2009).

Piazzon, N., Maisonneuve, C., Guilleret, I., Rotman, S. & Constam, D. B. Bicc1

links the regulation of cAMP signaling in polycystic kidneys to microRNAinduced gene silencing. J. Mol. Cell Biol. 4, 398–408 (2012).

Rothe, B. et al. Bicc1 Polymerization Regulates the Localization and Silencing

of Bound mRNA. Mol. Cell Biol. 35, 3339–3353 (2015).

Doidge, R., Mittal, S., Aslam, A. & Winkler, G. S. Deadenylation of

cytoplasmic mRNA by the mammalian Ccr4-Not complex. Biochem Soc.

Trans. 40, 896–901 (2012).

Collart, M. A. & Panasenko, O. O. The Ccr4—not complex. Gene 492, 42–53

(2012).

Chicoine, J. et al. Bicaudal-C recruits CCR4-NOT deadenylase to target

mRNAs and regulates oogenesis, cytoskeletal organization, and its own

expression. Dev. Cell 13, 691–704 (2007).

Suzuki, T. et al. Postnatal liver functional maturation requires Cnot complexmediated decay of mRNAs encoding cell cycle and immature liver genes.

Development 146, dev168146(2019).

Morita, M. et al. Depletion of mammalian CCR4b deadenylase triggers

elevation of the p27Kip1 mRNA level and impairs cell growth. Mol. Cell Biol.

27, 4980–4990 (2007).

Shinohara, K. et al. Two rotating cilia in the node cavity are sufficient to

break left-right symmetry in the mouse embryo. Nat. Commun. 3, 622

(2012).

Supp, D. M., Witte, D. P., Potter, S. S. & Brueckner, M. Mutation of an

axonemal dynein affects left-right asymmetry in inversus viscerum mice.

Nature 389, 963–966 (1997).

Nonaka, S., Shiratori, H., Saijoh, Y. & Hamada, H. Determination of left-right

patterning of the mouse embryo by artificial nodal flow. Nature 418, 96–99

(2002).

Maerker, M. et al. Bicc1 and Dicer regulate left-right patterning through posttranscriptional control of the Nodal-inhibitor Dand5. Nat. Commun. In Press.

Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R. & Dawson, A. P.

Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by

specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc. Natl

Acad. Sci. USA 87, 2466–2470 (1990).

Nicastro, G., Taylor, I. A. & Ramos, A. KH-RNA interactions: back in the

groove. Curr. Opin. Struct. Biol. 30, 63–70 (2015).

Nakamura, T. et al. Fluid flow and interlinked feedback loops establish leftright asymmetric decay of Cerl2 mRNA. Nat. Commun. 3, 1322 (2012).

Hollingworth, D. et al. KH domains with impaired nucleic acid binding as a

tool for functional analysis. Nucleic Acids Res 40, 6873–6886 (2012).

Lambert, N. et al. RNA Bind-n-Seq: quantitative assessment of the sequence

and structural binding specificity of RNA binding proteins. Mol. Cell 54,

887–900 (2014).

Zhang, Y. et al. Bicaudal-C spatially controls translation of vertebrate maternal

mRNAs. RNA 19, 1575–1582 (2013).

Leal-Esteban, L. C., Rothe, B., Fortier, S., Isenschmid, M. & Constam, D. B.

Role of Bicaudal C1 in renal gluconeogenesis and its novel interaction with the

CTLH complex. PLoS Genet 14, e1007487 (2018).

Li, X. et al. Adipocyte-specific disruption of mouse Cnot3 causes

lipodystrophy. FEBS Lett. 591, 358–368 (2017).

Ukita, K. et al. Wnt signaling maintains the notochord fate for progenitor cells

and supports the posterior extension of the notochord. Mech. Dev. 126,

791–803 (2009).

NATURE COMMUNICATIONS | (2021)12:4071 | https://doi.org/10.1038/s41467-021-24295-2 | www.nature.com/naturecommunications

A Self-archived copy in

Kyoto University Research Information Repository

https://repository.kulib.kyoto-u.ac.jp

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24295-2

31. Nakamura, T. et al. Generation of robust left-right asymmetry in the mouse

embryo requires a self-enhancement and lateral-inhibition system. Dev. Cell

11, 495–504 (2006).

32. Dowdle, M. E. et al. A single KH domain in Bicaudal-C links mRNA binding

and translational repression functions to maternal development. Development

146, dev172486 (2019).

33. Nakel, K., Hartung, S. A., Bonneau, F., Eckmann, C. R. & Conti, E. Four KH

domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular

structural platform. RNA 16, 2058–2067 (2010).

34. Beuth, B., Pennell, S., Arnvig, K. B., Martin, S. R. & Taylor, I. A. Structure of a

Mycobacterium tuberculosis NusA-RNA complex. EMBO J. 24, 3576–3587

(2005).

35. Park, S., Blaser, S., Marchal, M. A., Houston, D. W. & Sheets, M. D. A gradient of

maternal Bicaudal-C controls vertebrate embryogenesis via translational repression

of mRNAs encoding cell fate regulators. Development 143, 864–871 (2016).

36. Tran, U. et al. The RNA-binding protein bicaudal C regulates polycystin 2 in

the kidney by antagonizing miR-17 activity. Development 137, 1107–1116

(2010).

37. Iwakawa, H. O. & Tomari, Y. The Functions of MicroRNAs: mRNA Decay

and Translational Repression. Trends Cell Biol. 25, 651–665 (2015).

38. Behm-Ansmant, I. et al. mRNA degradation by miRNAs and GW182 requires

both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes

Dev. 20, 1885–1898 (2006).

39. Jonas, S. & Izaurralde, E. Towards a molecular understanding of microRNAmediated gene silencing. Nat. Rev. Genet 16, 421–433 (2015).

40. Takao, D. et al. Asymmetric distribution of dynamic calcium signals in the

node of mouse embryo during left-right axis formation. Dev. Biol. 376, 23–30

(2013).

41. Mochizuki, T. et al. Cloning of inv, a gene that controls left/right asymmetry

and kidney development. Nature 395, 177–181 (1998).

42. Watanabe, D. et al. The left-right determinant Inversin is a component of

node monocilia and other 9+0 cilia. Development 130, 1725–1734 (2003).

43. Wilkinson, D. G. & Nieto, M. A. Detection of messenger RNA by in situ

hybridization to tissue sections and whole mounts. Methods Enzymol. 225,

361–373 (1993).

44. Minegishi, K. et al. A Wnt5 activity asymmetry and intercellular signaling via

PCP proteins polarize node cells for left-right symmetry breaking. Dev. Cell

40, 439–452 (2017).

45. Hashimoto, M. et al. Planar polarization of node cells determines the

rotational axis of node cilia. Nat. Cell Biol. 12, 170–176 (2010).

46. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple

sequence alignment using Clustral Omega. Mol. Syst. Biol. 7, 539 (2011).

47. Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. WebLogo: a

sequence logo generator. Genome Res 14, 1188–1190 (2004).

48. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software

version 7: improvements in performance and usability. Mol. Biol. Evol. 30,

772–780 (2013).

49. Zimmermann, L. et al. A completely reimplemented MPI bioinformatics

toolkit with a new HHpred server at its core. J. Mol. Biol. 430, 2237–2243

(2018).

50. Will, S., Reiche, K., Hofacker, I. L., Stadler, P. F. & Backofen, R. Inferring

noncoding RNA families and classes by means of genome-scale structurebased clustering. PLoS Comput. Biol. 3, e65 (2007).

ARTICLE

tides for RBNS analysis, respectively. This study was supported by grants from the

Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (no.

17H01435), from Core Research for Evolutional Science and Technology (CREST) of the

Japan Science and Technology Agency (no. JPMJCR13W5) and from the Takeda Science

Foundation to H.H.; by a Grant-in-Aid (no. 18K14725) for Early-Career Scientists from

the Japan Society for the Promotion of Science (JSPS), a Kakehashi grant from BDROtsuka Pharmaceutical Collaboration Center, and a research grant (no. 2018M-018)

from the Kato Memorial Bioscience Foundation to K.M.; by grants from the NIBB

Individual Collaborative Research Program (nos. 16-312 and 17-316) and a Sinergia

grant (no. CRSI33_130662) from the Swiss National Science Foundation to

D.B.C.; and by a KAKENHI grant (no. 15H05722) from JSPS and a research grant from

The Mitsubishi Foundation to H.S.

Author contributions

K.M. performed most experiments with mouse embryos. Y.I., H.N., and K.T. generated

transgenic and mutant mice. B.R. performed biochemical analysis of Bicc1 and Cnot3.

K.R.K., E.M., and H.O. performed RBNS analysis. K.B. and H.K. performed transgenic

assays with deletion mutants. T.Y. examined the phenotype of Cnot3 CKO mutant mice.

T.A. K. and E.K. performed experiments with Pkd2 mutant embryos and Dand5 Δ200

mutant embryos, respectively. X.S. examined the localization of Cnot3 protein in mouse

embryos. H.H., D.B.C., H.S., K.M., B.R., and K.R.K. conceived the project and wrote the

paper. All authors contributed to the revision of the manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s41467-021-24295-2.

Correspondence and requests for materials should be addressed to H.S., D.B.C. or H.H.

Peer review information Nature Communications thanks Matthew Stower, Oliver

Wessely and the other, anonymous, reviewer(s) for their contribution to the peer review

of this work.

Reprints and permission information is available at http://www.nature.com/reprints

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations.

Acknowledgements

Open Access This article is licensed under a Creative Commons

Attribution 4.0 International License, which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give

appropriate credit to the original author(s) and the source, provide a link to the Creative

Commons license, and indicate if changes were made. The images or other third party

material in this article are included in the article’s Creative Commons license, unless

indicated otherwise in a credit line to the material. If material is not included in the

article’s Creative Commons license and your intended use is not permitted by statutory

regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder. To view a copy of this license, visit http://creativecommons.org/

licenses/by/4.0/.

We thank K. Okamoto (University of Tokyo) for PIV analysis software; Y. Igarashi for

the software for quantitative analysis of basal body position; H. Sasaki for NotoCreERT2/+

mice; and M. Yamagata and S. Wada for technical support and designing oligonucleo-

© The Author(s) 2021

NATURE COMMUNICATIONS | (2021)12:4071 | https://doi.org/10.1038/s41467-021-24295-2 | www.nature.com/naturecommunications

17

...