PUM1 and PUM2 exhibit different modes of regulation for SIAH1 that involve cooperativity with NANOS paralogues
- 317 Downloads
Pumilio (PUM) proteins are RNA-binding proteins that posttranscriptionally regulate gene expression in many organisms. Their PUF domain recognizes specific PUM-binding elements (PBE) in the 3′ untranslated region of target mRNAs while engaging protein cofactors such as NANOS that repress the expression of target mRNAs through the recruitment of effector complexes. Although the general process whereby PUM recognizes individual mRNAs has been studied extensively, the particulars of the mechanism underlying PUM–NANOS cooperation in mRNA regulation and the functional overlap among PUM and NANOS paralogues in mammals have not been elucidated. Here, using the novel PUM1 and PUM2 mRNA target SIAH1 as a model, we show mechanistic differences between PUM1 and PUM2 and between NANOS1, 2, and 3 paralogues in the regulation of SIAH1. Specifically, unlike PUM2, PUM1 exhibited PBE-independent repression of SIAH1 3′UTR-dependent luciferase expression. Concordantly, the PUF domains of PUM1 and PUM2 showed different EMSA complex formation patterns with SIAH1 3′UTRs. Importantly, we show direct binding of NANOS3, but not NANOS2, to SIAH1 3′UTR, which did not require PBEs or the PUF domain. To the best of our knowledge, this is the first report, showing that an NANOS protein directly binds RNA. Finally, using NANOS1 and NANOS3 constructs carrying mutations identified in infertile patients, we show that these mutations disrupt repression of the SIAH1-luciferase reporter and that the central region in NANOS1 appears to contribute to the regulation of SIAH1. Our findings highlight the mechanistic versatility of the PUM/NANOS machinery in mammalian posttranscriptional regulation.
Keywords3′UTR RNA-binding proteins Posttranscriptional gene regulation
This work was supported by the National Science Center Poland (Grant no. 2011/01/B/NZ2/04833 to BGM and ETIUDA scholarship no. 2014/12/T/NZ1/00497 to MS) and Ministry of Science and Higher Education (Grant no. N N401318439 to JJ). We thank Dr. Witold Filipowicz, Dr. Thomas Tuschl, Dr. Damian Brauze, Christine Rickards-Rostworowska, and Dr. Miroslawa Siatecka for the helpful discussions and for commenting on the manuscript.
MS conducted the experiments and prepared the figures, DMJ conducted the experiments and prepared the figures, MJS conducted the experiments, BGM analysed the results, AS conducted the experiments, SO performed the bioinformatic search for PUM mRNA targets, EI conducted experiments, KKZ conducted the experiments, MK conducted the experiments and prepared the manuscript, JB designed software to bioinformatically select PUM mRNA targets, and JJ conceived the experiments and prepared the manuscript. All authors reviewed the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interest.
- 9.Zamore PD, Williamson JR, Lehmann R (1997) The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins. RNA 3:1421–1433Google Scholar
- 19.Spik A, Oczkowski S, Olszak A, Formanowicz P, Blazewicz J, Jaruzelska J (2006) Human fertility protein PUMILIO2 interacts in vitro with testis mRNA encoding Cdc42 effector 3 (CEP3). Reprod Biol 6:103–113Google Scholar
- 20.Kusz-Zamelczyk K, Sajek M, Spik A, Glazar R, Jedrzejczak P, Latos-Bielenska A, Kotecki M, Pawelczyk L, Jaruzelska J (2013) Mutations of NANOS1, a human homologue of the Drosophila morphogen, are associated with a lack of germ cells in testes or severe oligo-astheno-teratozoospermia. J Med Genet 50:187–193. https://doi.org/10.1136/jmedgenet-2012-101230 CrossRefGoogle Scholar
- 21.Santos MG, Machado AZ, Martins CN, Domenice S, Costa EM, Nishi MY, Ferraz-de-Souza B, Jorge SA, Pereira CA, Soardi FC, de Mello MP, Maciel-Guerra AT, Guerra-Junior G, Mendonca BB (2014) Homozygous inactivating mutation in NANOS3 in two sisters with primary ovarian insufficiency. Biomed Res Int 2014:787465. https://doi.org/10.1155/2014/787465 Google Scholar
- 32.Kusz KM, Tomczyk L, Sajek M, Spik A, Latos-Bielenska A, Jedrzejczak P, Pawelczyk L, Jaruzelska J (2009) The highly conserved NANOS2 protein: testis-specific expression and significance for the human male reproduction. Mol Hum Reprod 15:165–171. https://doi.org/10.1093/molehr/gap003 CrossRefGoogle Scholar
- 39.Hubstenberger A, Courel M, Benard M, Souquere S, Ernoult-Lange M, Chouaib R, Yi Z, Morlot JB, Munier A, Fradet M, Daunesse M, Bertrand E, Pierron G, Mozziconacci J, Kress M, Weil D (2017) P-body purification reveals the condensation of repressed mRNA regulons. Mol Cell 68:144–157 e5. https://doi.org/10.1016/j.molcel.2017.09.003 CrossRefGoogle Scholar