Bioinformatics Analysis to Identify RNA–Protein Interactions in Oogenesis

  • Ravinder SinghEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1328)


Hundreds of RNA-binding proteins are known, but the biological functions are known for only a few of them. They regulate various aspects of RNA processing or biogenesis such as splicing, polyadenylation, and translation. Here I describe a bioinformatics approach that we developed to identify potential new mRNA target(s) of the Drosophila master sex-switch protein Sex-lethal (SXL) by combining computational analysis with genetic and biochemical investigation. This approach could be used to identify new RNA–protein interactions during oogenesis in the female germline and should be applicable to numerous other posttranscriptional regulatory events.

Key words

Sex determination RNA-binding proteins Germline Oogenesis Polyadenylation Translation 



I thank Andrew Rahn and Mark Robida for their notes consulted for materials and methods and the American Cancer Society and the Butcher Foundation for grants to RS that supported this work.


  1. 1.
    Ule J, Jensen K, Mele A et al (2005) CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37(4):376–386CrossRefPubMedGoogle Scholar
  2. 2.
    Hafner M, Landthaler M, Burger L et al (2010) PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins. J Vis Exp (41), doi: 10.3791/2034
  3. 3.
    Zhang B, Kraemer B, SenGupta D et al (1999) Yeast three-hybrid system to detect and analyze interactions between RNA and protein. Methods Enzymol 306:93–113CrossRefPubMedGoogle Scholar
  4. 4.
    Hamady M, Peden E, Knight R et al (2006) Fast-Find: a novel computational approach to analyzing combinatorial motifs. BMC Bioinformatics 7:1PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Heimiller J, Sridharan V, Huntley J et al (2014) Drosophila polypyrimidine tract-binding protein (DmPTB) regulates dorso-ventral patterning genes in embryos. PLoS One 9(7):e98585Google Scholar
  6. 6.
    Schutt C, Nothiger R (2000) Structure, function and evolution of sex-determining systems in Dipteran insects. Development 127(4):667–677PubMedGoogle Scholar
  7. 7.
    Mahowald AP, Wei G (1994) Sex determination of germ cells in Drosophila. Ciba Found Symp 182:193–202Google Scholar
  8. 8.
    Steinmann-Zwicky M (1992) How do germ cells choose their sex? Drosophila as a paradigm. Bioessays 14(8):513–518Google Scholar
  9. 9.
    Salz HK (2013) Sex, stem cells and tumors in the Drosophila ovary. Fly 7(1):3–7Google Scholar
  10. 10.
    Samuels ME, Bopp D, Colvin RA et al (1994) RNA binding by Sxl proteins in vitro and in vivo. Mol Cell Biol 14(7):4975–4990PubMedCentralPubMedGoogle Scholar
  11. 11.
    Fujii S, Amrein H (2002) Genes expressed in the Drosophila head reveal a role for fat cells in sex-specific physiology. EMBO J 21(20):5353–5363Google Scholar
  12. 12.
    Kelley RL, Solovyeva I, Lyman LM et al (1995) Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell 81(6):867–877Google Scholar
  13. 13.
    Hager J, Cline T (1997) Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination. Development 124(24):5033–5048Google Scholar
  14. 14.
    Vied C, Halachmi N, Salzberg A et al (2003) Antizyme is a target of Sex-lethal in the Drosophila germline and appears to act downstream of Hedgehog to regulate Sex-lethal and Cyclin B. Dev Biol 253(2):214–229Google Scholar
  15. 15.
    Chau J, Kulnane LS, Salz HK (2009) Sex-lethal facilitates the transition from germline stem cell to committed daughter cell in the Drosophila ovary. Genetics 182(1):121–132Google Scholar
  16. 16.
    Chau J, Kulnane LS, Salz HK (2012) Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis. Proc Natl Acad Sci U S A 109(24):9465–9470Google Scholar
  17. 17.
    Gawande B, Robida MD, Rahn A et al (2006) Drosophila Sex-lethal protein mediates polyadenylation switching in the female germline. EMBO J 25(6):1263–1272Google Scholar
  18. 18.
    Robida MD, Rahn A, Singh R (2007) Genome-wide identification of alternatively spliced mRNA targets of specific RNA-binding proteins. PLoS One 2(6):e520PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Adams MD, Celniker SE, Holt RA et al (2000) The genome sequence of Drosophila melanogaster. Science 287(5461):2185–2195Google Scholar
  20. 20.
    Singh R, Valcarcel J, Green MR (1995) Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science 268(5214):1173–1176CrossRefPubMedGoogle Scholar
  21. 21.
    Singh R, Banerjee H, Green MR (2000) Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal. RNA 6(6):901–911PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Sakashita E, Sakamoto H (1994) Characterization of RNA binding specificity of the Drosophila sex-lethal protein by in vitro ligand selection. Nucleic Acids Res 22(20):4082–4086Google Scholar
  23. 23.
    Kanaar R, Lee AL, Rudner DZ et al (1995) Interaction of the sex-lethal RNA binding domains with RNA. EMBO J 14(18):4530–4539PubMedCentralPubMedGoogle Scholar
  24. 24.
    Hertz GZ, Hartzell GWD, Stormo GD (1990) Identification of consensus patterns in unaligned DNA sequences known to be functionally related. Comput Appl Biosci 6(2):81–92PubMedGoogle Scholar
  25. 25.
    Serano TL, Cheung HK, Frank LH et al (1994) P element transformation vectors for studying Drosophila melanogaster oogenesis and early embryogenesis. Gene 138(1–2):181–186Google Scholar
  26. 26.
    Valcarcel J, Singh R, Zamore PD et al (1993) The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature 362(6416):171–175CrossRefPubMedGoogle Scholar
  27. 27.
    Mohr SE, Dillon ST, Boswell RE (2001) The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis. Genes Dev 15(21):2886–2899Google Scholar
  28. 28.
    Singh R, Valcarcel J (2005) Building specificity with nonspecific RNA-binding proteins. Nat Struct Mol Biol 12(8):645–653CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Molecular, Cellular and Developmental BiologyUniversity of Colorado at BoulderBoulderUSA

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