Skip to main content
SpringerLink
Log in

Exploring Translational Control of Maternal mRNAs in Zebrafish

  • Protocol
  • First Online:
Germline Development in the Zebrafish

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2218))

Abstract

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Evsikov AV, de Vries WN, Peaston AE, Radford EE, Fancher KS, Chen FH, Blake JA, Bult CJ, Latham KE, Solter D, Knowles BB (2004) Systems biology of the 2-cell mouse embryo. Cytogenet Genome Res 105(2–4):240–250. https://doi.org/10.1159/000078195

    Article  CAS  PubMed  Google Scholar 

  2. Oh B, Hwang S, McLaughlin J, Solter D, Knowles BB (2000) Timely translation during the mouse oocyte-to-embryo transition. Development 127(17):3795–3803

    CAS  PubMed  Google Scholar 

  3. Potireddy S, Vassena R, Patel BG, Latham KE (2006) Analysis of polysomal mRNA populations of mouse oocytes and zygotes: dynamic changes in maternal mRNA utilization and function. Dev Biol 298(1):155–166. https://doi.org/10.1016/j.ydbio.2006.06.024

    Article  CAS  PubMed  Google Scholar 

  4. Salles FJ, Darrow AL, O'Connell ML, Strickland S (1992) Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation. Genes Dev 6(7):1202–1212. https://doi.org/10.1101/gad.6.7.1202

    Article  CAS  PubMed  Google Scholar 

  5. Winata CL, Lapinski M, Pryszcz L, Vaz C, Bin Ismail MH, Nama S, Hajan HS, Lee SGP, Korzh V, Sampath P, Tanavde V, Mathavan S (2018) Cytoplasmic polyadenylation-mediated translational control of maternal mRNAs directs maternal-to-zygotic transition. Development 145(1). https://doi.org/10.1242/dev.159566

  6. Aanes H, Winata CL, Lin CH, Chen JP, Srinivasan KG, Lee SG, Lim AY, Hajan HS, Collas P, Bourque G, Gong Z, Korzh V, Alestrom P, Mathavan S (2011) Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition. Genome Res 21(8):1328–1338. https://doi.org/10.1101/gr.116012.110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sampath P, Lee QY, Tanavde V (2011) Identifying translationally regulated genes during stem cell differentiation. Curr Protoc Stem Cell Biol Chapter 1:Unit1B 8. https://doi.org/10.1002/9780470151808.sc01b08s18

    Article  PubMed  Google Scholar 

  8. Sampath P, Pritchard DK, Pabon L, Reinecke H, Schwartz SM, Morris DR, Murry CE (2008) A hierarchical network controls protein translation during murine embryonic stem cell self-renewal and differentiation. Cell Stem Cell 2(5):448–460. https://doi.org/10.1016/j.stem.2008.03.013

    Article  CAS  PubMed  Google Scholar 

  9. Mercer JF, Wake SA (1985) An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot hybridization. Nucleic Acids Res 13(22):7929–7943. https://doi.org/10.1093/nar/13.22.7929

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sheets MD, Fox CA, Hunt T, Vande Woude G, Wickens M (1994) The 3′-untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation. Genes Dev 8(8):926–938. https://doi.org/10.1101/gad.8.8.926

    Article  CAS  PubMed  Google Scholar 

  11. Murray EL, Schoenberg DR (2008) Assays for determining poly(A) tail length and the polarity of mRNA decay in mammalian cells. Methods Enzymol 448:483–504. https://doi.org/10.1016/S0076-6879(08)02624-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nilsen TW (2015) Measuring the length of poly(A) tails. Cold Spring Harb Protoc 2015(4):413–418. https://doi.org/10.1101/pdb.prot081034

    Article  PubMed  Google Scholar 

  13. Salles FJ, Strickland S (1999) Analysis of poly(A) tail lengths by PCR: the PAT assay. Methods Mol Biol 118:441–448. https://doi.org/10.1385/1-59259-676-2:441

    Article  CAS  PubMed  Google Scholar 

  14. Rassa JC, Wilson GM, Brewer GA, Parks GD (2000) Spacing constraints on reinitiation of paramyxovirus transcription: the gene end U tract acts as a spacer to separate gene end from gene start sites. Virology 274(2):438–449. https://doi.org/10.1006/viro.2000.0494

    Article  CAS  PubMed  Google Scholar 

  15. Janicke A, Vancuylenberg J, Boag PR, Traven A, Beilharz TH (2012) ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications. RNA 18(6):1289–1295. https://doi.org/10.1261/rna.031898.111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Couttet P, Fromont-Racine M, Steel D, Pictet R, Grange T (1997) Messenger RNA deadenylylation precedes decapping in mammalian cells. Proc Natl Acad Sci U S A 94(11):5628–5633. https://doi.org/10.1073/pnas.94.11.5628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bazzini AA, Lee MT, Giraldez AJ (2012) Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish. Science 336(6078):233–237. https://doi.org/10.1126/science.1215704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Westerfield M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene, OR

    Google Scholar 

  19. Alestrom P, D’Angelo L, Midtlyng PJ, Schorderet DF, Schulte-Merker S, Sohm F, Warner S (2019) Zebrafish: housing and husbandry recommendations. Lab Anim 2019:23677219869037. https://doi.org/10.1177/0023677219869037

    Article  CAS  Google Scholar 

  20. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310. https://doi.org/10.1002/aja.1002030302

    Article  CAS  PubMed  Google Scholar 

  21. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21. https://doi.org/10.1093/bioinformatics/bts635

    Article  CAS  Google Scholar 

  22. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14(4):417–419. https://doi.org/10.1038/nmeth.4197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11(10):R106. https://doi.org/10.1186/gb-2010-11-10-r106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Patil DP, Bakthavachalu B, Schoenberg DR (2014) Poly(A) polymerase-based poly(A) length assay. Methods Mol Biol 1125:13–23. https://doi.org/10.1007/978-1-62703-971-0_2

    Article  PubMed  PubMed Central  Google Scholar 

  25. Martin G, Keller W (1998) Tailing and 3′-end labeling of RNA with yeast poly(A) polymerase and various nucleotides. RNA 4(2):226–230

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

M.Ł. is supported by the DIAMENTOWY scholarship from the Polish Ministry of Science and Higher Education. The project no. POIR.04.04.00-00-1AF0/16-00/ is carried out within the First TEAM programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. This work was supported by funding from A*STAR and BMRC, Singapore.

Author information

Authors and Affiliations

  1. International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland

    Cecilia Lanny Winata & Maciej Łapiński

  2. Max Planck Institute for Heart and Lung Research, Bad-Nauheim, Germany

    Cecilia Lanny Winata

  3. Skin Research Institute of Singapore, Agency for Science Technology and Research, Singapore, Singapore

    Hisyam Ismail & Prabha Sampath

  4. Vision Research Foundation, Sankara Nethralaya, Chennai, India

    Sinnakaruppan Mathavan

  5. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Prabha Sampath

Authors
  1. Cecilia Lanny Winata
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maciej Łapiński
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hisyam Ismail
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sinnakaruppan Mathavan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Prabha Sampath
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cecilia Lanny Winata .

Editor information

Editors and Affiliations

  1. Institute for Developmental Biochemistry, University of Göttingen Medical Center Georg-August-Universität, Göttingen, Germany

    Roland Dosch

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Winata, C.L., Łapiński, M., Ismail, H., Mathavan, S., Sampath, P. (2021). Exploring Translational Control of Maternal mRNAs in Zebrafish . In: Dosch, R. (eds) Germline Development in the Zebrafish. Methods in Molecular Biology, vol 2218. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0970-5_29

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0970-5_29

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0969-9

  • Online ISBN: 978-1-0716-0970-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation