Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/2006; 103(12):4487-92. DOI: 10.1073/pnas.0509260103
Source: PubMed

ABSTRACT Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3'-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins.

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Available from: Stefan Luschnig, Aug 14, 2015
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    • "Its sequence, however, is remarkably divergent, only 19% of protein sequence similarity amongst several dipteran species (Curtis et al., 1995). Current models argue that Nanos functions through its interaction with Pumilio, which binds RNAs containing a conserved motif in their 3 0 UTR, the Nanos Response Element (NRE), or more effectively to document the Pumilio role in this interaction, the Pumilio Response Element (PRE) (Gerber et al., 2006; Sonoda and Wharton, 1999; Wharton et al., 1998; Wharton and Struhl, 1991). Only a few mRNAs, however, have been identified as Nanos/ Pumilio targets. "
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    ABSTRACT: Nanos is an essential factor of germ line success in all animals tested. This gene encodes a Zn-finger RNA-binding protein that in complex with its partner pumilio, binds to and changes the fate of several known transcripts. We summarize here the documented functions of nanos in several key organisms, and then emphasize echinoderms as a working model for how nanos expression is regulated. Nanos presence outside of the target cells is often detrimental to the animal, and in sea urchins, nanos expression appears to be regulated at every step of transcription, and post-transcriptional activity, making this gene product exciting, every which way. © 2013 Wiley Periodicals, Inc.
    genesis 03/2014; 52(3). DOI:10.1002/dvg.22737
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    • "Tap-tagged proteins were removed from the beads by incubating 150 U of AcTEV protease (Invitrogen) for 2 h. RNA was then isolated using TRIzol reagent (Invitrogen), followed by RNeasy (Qiagen) purification as per the manufacturer's instructions (Gerber et al. 2006). Each RNA Tap tag pull-down in Figure 1 was conducted in triplicate, and averages and standard deviations from RT-qPCR experiments were used in this study. "
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    ABSTRACT: E2F transcription factors are important regulators of cell proliferation and are frequently dysregulated in human malignancies. To identify novel regulators of E2F function, we used Drosophila as a model system to screen for mutations that modify phenotypes caused by reduced levels of dE2F1. This screen identified components of the Pumilio translational repressor complex (Pumilio, Nanos, and Brain tumor) as suppressors of dE2F1-RNAi phenotypes. Subsequent experiments provided evidence that Pumilio complexes repress dE2F1 levels and that this mechanism of post-transcriptional regulation is conserved in human cells. The human Pumilio homologs Pum 1 and Pum 2 repress the translation of E2F3 by binding to the E2F3 3' untranslated region (UTR) and also enhance the activity of multiple E2F3 targeting microRNAs (miRNAs). E2F3 is an oncogene with strong proliferative potential and is regularly dysregulated or overexpressed in cancer. Interestingly, Pumilio/miRNA-mediated regulation of E2F3 is circumvented in cancer cells in several different ways. Bladder carcinomas selectively down-regulate miRNAs that cooperate with Pumilio to target E2F3, and multiple tumor cell lines shorten the 3' end of the E2F3 mRNA, removing the Pumilio regulatory elements. These studies suggest that Pumilio-miRNA repression of E2F3 translation provides an important level of E2F regulation that is frequently abrogated in cancer cells.
    Genes & Development 02/2012; 26(4):356-68. DOI:10.1101/gad.182568.111
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    • "The sequences of Dm-hb (first and second NRE, red) and Dm-bcd mRNA are shown with nucleotides that affect Drosophila Pumilio binding when mutated (white) (Curtis et al., 1997; Gerber et al., 2006; Murata and Wharton, 1995; Sonoda and Wharton, 1999; Zamore et al., 1997). The RNA consensus motif of Box B was identified in a genome-wide survey of mRNAs associated with Pum (Gerber et al., 2006). "
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