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

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 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.

Download full-text


Available from: Stefan Luschnig,
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in cis, to produce three separate helices that meet around a central unpaired region. Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome.
    Frontiers in Genetics 04/2014; 5:79. DOI:10.3389/fgene.2014.00079
  • Source
    • "Interestingly, TgPuf1 and TgPuf2 are more homologous to their respective Puf1 and Puf2 genes in Plasmodium, suggesting that the duplication of Puf genes in these two Apicomplexan parasites occurred earlier before the divergence of these parasite taxa. In the malaria parasite P. berghei, only PbPuf2 are found to regulate the stage-transition in sporozoites, whereas deletion of PbPuf1 had no effects on this process [1,2,19,28]. In P. falciparum both Puf proteins are abundantly expressed in gametocytes and Puf2 plays an important role during gametocytogenesis [32]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Puf proteins act as translational regulators and affect many cellular processes in a wide range of eukaryotic organisms. Although Puf proteins have been well characterized in many model systems, little is known about the structural and functional characteristics of Puf proteins in the parasite Toxoplasma gondii. Using a combination of conventional molecular approaches, we generated endogenous TgPuf1 tagged with hemagglutinin (HA) epitope and investigated the TgPuf1 expression levels and localization in the tachyzoites and bradyzoites. We used RNA Electrophoretic Mobility Shfit Assay (EMSA) to determine whether the recombination TgPuf1 has conserverd RNA binding activity and specificity. TgPuf1 was expressed at a significantly higher level in bradyzoites than in tachyzoites. TgPuf1 protein was predominantly localized within the cytoplasm and showed a much more granular cytoplasmic staining pattern in bradyzoites. The recombinant Puf domain of TgPuf1 showed strong binding affinity to two RNA fragments containing Puf-binding motifs from other organisms as artificial target sequences. However, two point mutations in the core Puf-binding motif resulted in a significant reduction in binding affinity, indicating that TgPuf1 also binds to conserved Puf-binding motif. TgPuf1 appears to exhibit different expression levels in the tachyzoites and bradyzoites, suggesting that TgPuf1 may function in regulating the proliferation or/and differentiation that are important in providing parasites with the ability to respond rapidly to changes in environmental conditions. This study provides a starting point for elucidating the function of TgPuf1 during parasite development.
    Parasites & Vectors 03/2014; 7(1):141. DOI:10.1186/1756-3305-7-141 · 3.43 Impact Factor
  • Source
    • "Many Puf proteins have been identified as essential factors for several aspects of development such as germline stem cell maintenance, synaptic plasticity, embryonic axis patterning, and mating type switching [13-17]. Many approaches such as DNA microarrays, bioinformatics approaches, and RNA immunoprecipitation have been performed in Saccharomyces cerevisiae and Drosophila melanogaster to identify specific target RNAs associated with Puf proteins [15,18,19]. Many RNAs with binding motifs interact with Puf proteins at different developmental stages and in different tissues. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening. APUM5 transcriptional profiling was carried out using a bioinformatics tool. We found that APUM5 was associated with both biotic and abiotic stress responses. However, bacterial and fungal pathogen infection susceptibility was not changed in APUM5 transgenic plants compared to that in wild type plants although APUM5 expression was induced upon pathogen infection. In contrast, APUM5 was involved in the abiotic stress response. 35S-APUM5 transgenic plants showed hypersensitive phenotypes under salt and drought stresses during germination, primary root elongation at the seedling stage, and at the vegetative stage in soil. We also showed that some abiotic stress-responsive genes were negatively regulated in 35S-APUM5 transgenic plants. The APUM5-Pumilio homology domain (PHD) protein bound to the 3[prime] untranslated region (UTR) of the abiotic stress-responsive genes which contained putative Pumilio RNA binding motifs at the 3[prime] UTR. These results suggest that APUM5 may be a new post-transcriptional regulator of the abiotic stress response by direct binding of target genes 3[prime] UTRs.
    BMC Plant Biology 03/2014; 14(1):75. DOI:10.1186/1471-2229-14-75 · 3.81 Impact Factor
Show more