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

University of Bayreuth, Bayreuth, Bavaria, Germany
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2006; 103(12):4487-92. DOI: 10.1073/pnas.0509260103
Source: PubMed


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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    • "A single Pumilio-like recognition motif (UGUAAACA) is present in the P. berghei uis4 mRNA at nucleotide positions 284–292 of the ORF (UGUAAACA). The related UGUAHAUA (H = A/C/U) motif is recognized by human Pum1[44], Drosophila Pumilio[45], yeast Puf3[46]and most importantly by P. falciparum Puf2[7]. In the human malaria parasite pfs25 and pfs28 are directly bound by Puf2 and kept translationally silent prior to transmission; in the absence of Puf2 the translation levels of these proteins are upregulated. "
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    • "The tandem affinity purification (TAP-tag) method involves the fusion of a double-tag either at the amino or carboxy terminus of the protein followed by transfection of the studied cell type (Figure 1B). In vivo-formed RNA-protein complexes are purified by two-step affinity chromatography with tag-specific binding proteins (Puig et al., 2001; Gerber et al., 2006). However, the presence of the tag could interfere with native interactions, yielding false, or at least incomplete patterns of binding. "
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    Preview · Article · Dec 2015
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    • "Researchers now regularly study these interactions by immunoprecipitating RNA-protein complexes (RNPs) and identifying the associated RNA cargo using genomic readouts including microarrays and more recently next-generation sequencing. These techniques are frequently referred to as RIP-Chip, PAR-CLIP and RIP-Seq (Tenenbaum et al., 2000; Brown et al., 2001; Intine et al., 2003; De Silanes et al., 2004; Gerber et al., 2004, 2006; Di Marco et al., 2005; Hogan et al., 2008; Mazan-Mamczarz et al., 2008; Calaluce et al., 2010; Abdelmohsen et al., 2012; Guttman and Rinn, 2012; LeGendre et al., 2013; Singh et al., 2014). These methods have proven to be powerful tools for studying the role of RBPs in post-transcriptional gene expression and for subtly dissecting this complex process to uncover the players involved. "
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