A quantitative RNA code for mRNA target selection by the germline fate determinant GLD-1

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
The EMBO Journal (Impact Factor: 10.43). 02/2011; 30(3):533-45. DOI: 10.1038/emboj.2010.334
Source: PubMed


RNA-binding proteins (RBPs) are critical regulators of gene expression. To understand and predict the outcome of RBP-mediated regulation a comprehensive analysis of their interaction with RNA is necessary. The signal transduction and activation of RNA (STAR) family of RBPs includes developmental regulators and tumour suppressors such as Caenorhabditis elegans GLD-1, which is a key regulator of germ cell development. To obtain a comprehensive picture of GLD-1 interactions with the transcriptome, we identified GLD-1-associated mRNAs by RNA immunoprecipitation followed by microarray detection. Based on the computational analysis of these mRNAs we generated a predictive model, where GLD-1 association with mRNA is determined by the strength and number of 7-mer GLD-1-binding motifs (GBMs) within UTRs. We verified this quantitative model both in vitro, by competition GLD-1/GBM-binding experiments to determine relative affinity, and in vivo, by 'transplantation' experiments, where 'weak' and 'strong' GBMs imposed translational repression of increasing strength on a non-target mRNA. This study demonstrates that transcriptome-wide identification of RBP mRNA targets combined with quantitative computational analysis can generate highly predictive models of post-transcriptional regulatory networks.

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    • "Several potential new PUF-sensitive 3′ UTRs were also revealed (Fig. 7 B). A " GLD-1 group " was defined by GLD-1-sensitive 3′ UTRs that were de-repressed in early stages (distal gonad) and included previously established or predicted GLD-1 targets (Fig. 7 B; Lee and Schedl, 2001; Jungkamp et al., 2011; Wright et al., 2011; Doh et al., 2013). These results support reporter screen accuracy, with some false negatives based on previous quantifications (Hubstenberger et al., 2012). "
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    ABSTRACT: Ribonucleoproteins (RNPs) often coassemble into supramolecular bodies with regulated dynamics. The factors controlling RNP bodies and connections to RNA regulation are unclear. During Caenorhabditis elegans oogenesis, cytoplasmic RNPs can transition among diffuse, liquid, and solid states linked to mRNA regulation. Loss of CGH-1/Ddx6 RNA helicase generates solid granules that are sensitive to mRNA regulators. Here, we identified 66 modifiers of RNP solids induced by cgh-1 mutation. A majority of genes promote or suppress normal RNP body assembly, dynamics, or metabolism. Surprisingly, polyadenylation factors promote RNP coassembly in vivo, suggesting new functions of poly(A) tail regulation in RNP dynamics. Many genes carry polyglutatmine (polyQ) motifs or modulate polyQ aggregation, indicating possible connections with neurodegenerative disorders induced by CAG/polyQ expansion. Several RNP body regulators repress translation of mRNA subsets, suggesting that mRNAs are repressed by multiple mechanisms. Collectively, these findings suggest new pathways of RNP modification that control large-scale coassembly and mRNA activity during development.
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    • "We propose a third model in which the misexpression of a common target(s), which is controlled redundantly by GLD-1 and PUF-8, interferes in spermatogenesis. A large number of potential targets have been identified for GLD-1 and PUF-8, and several of these potential targets are common to both proteins (Mainpal et al. 2011; Wright et al. 2011). Therefore, it is probable that some of the potential common targets are either redundantly or synergistically controlled by GLD-1 and PUF-8. "
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    ABSTRACT: Successful meiotic progression of germ cells is crucial for gametogenesis. Defects in this process affect proper genetic transmission and sometimes lead to tumor formation in the germline. In Caenorhabditis elegans, the RNA-binding protein GLD-1 is essential for the meiotic development of oocytes. However, its role during spermatogenesis has not been understood. Here, we show that GLD-1 functions redundantly with the PUF family protein PUF-8 to ensure proper meiotic development of spermatocytes. When grown at 20°C - the standard laboratory temperature for C. elegans growth - primary spermatocytes in both gld-1 and puf-8 single mutant males and hermaphrodites complete the meiotic divisions normally. By contrast, some of the gld-1; puf-8 double mutant spermatocytes exit meiosis and form germ cell tumors in both sexes. During larval development, gld-1; puf-8 double mutant germ cells begin to express the meiotic marker HIM-3, lose P granules and form the sperm-specific membranous organelle (MO), which are characteristics of developing spermatocytes. However, some of these cells quickly lose HIM-3 and form germ cell tumors which lack MO, but contain P granules. Mutations that block meiotic progression at late pachytene or diakinetic stage fail to arrest the tumorigenesis, suggesting that the gld-1; puf-8 double mutant spermatocytes exit meiosis prior to the completion of pachytene. Together, results presented here uncover a novel function for gld-1 in the meiotic development of spermatocytes in both hermaphrodites and males. Copyright © 2015 Author et al.
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    • "Although other nucleobases like cytosine are able to form this intramolecular hydrogen bond with C19, only a uracil can protrude into the small pocket formed by the linker residues. In conclusion, our structure explains also well the 5′-UAC-3′ consensus found at the 5′end of the GBM (24,25). "
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    ABSTRACT: The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional α-helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5′end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5′end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.
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