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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Available from: Eric Westhof, Oct 09, 2015
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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.
    G3-Genes Genomes Genetics 06/2015; 5(8). DOI:10.1534/g3.115.019521 · 3.20 Impact Factor
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    • "The GFP:H2B-tagged reporters are driven by a constitutive-expressed germline promoter (mex-5) and fused to the 3′UTR of the respective endogenous gene. The predicted GBMs according to the biophysical model (A) (26) and (B) according to the GBM predictor (25) are shown as gray square. The predicted strength of GLD-1 binding to the whole 3′-UTR based on the sum of their binding sites is shown at the end of the respective reporter. "
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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.
    Nucleic Acids Research 05/2014; 42(12). DOI:10.1093/nar/gku445 · 9.11 Impact Factor
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    • "The top 500 CLIPed sites selected based on copy number were subjected to a motif analysis using PhyloGibbs with the parameters: -D 0 -m 7 -N 0 -r -z 2 -y 250 (Siddharthan et al. 2005). RIP-Chip enrichment of transcripts and measured binding affinities to short oligonucleotide sequences were taken from the Supplemental data of Wright et al. (2011) and log2 array expression levels of total mRNA, mRNA in gonads, and polysomal mRNA in wild-type and gld-1 mutant worms were found in the Supporting Information of Scheckel et al. (2012). "
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    ABSTRACT: To understand the function of the hundreds of RNA-binding proteins (RBPs) that are encoded in animal genomes it is important to identify their target RNAs. Although it is generally accepted that the binding specificity of an RBP is well described in terms of the nucleotide sequence of its binding sites, other factors such as the structural accessibility of binding sites or their clustering, to enable binding of RBP multimers, are also believed to play a role. Here we focus on GLD-1, a translational regulator of Caenorhabditis elegans, whose binding specificity and targets have been studied with a variety of methods such as CLIP (cross-linking and immunoprecipitation), RIP-Chip (microarray measurement of RNAs associated with an immunoprecipitated protein), profiling of polysome-associated mRNAs and biophysical determination of binding affinities of GLD-1 for short nucleotide sequences. We show that a simple biophysical model explains the binding of GLD-1 to mRNA targets to a large extent, and that taking into account the accessibility of putative target sites significantly improves the prediction of GLD-1 binding, particularly due to a more accurate prediction of binding in transcript coding regions. Relating GLD-1 binding to translational repression and stabilization of its target transcripts we find that binding sites along the entire transcripts contribute to functional responses, and that CDS-located sites contribute most to translational repression. Finally, biophysical measurements of GLD-1 affinity for a small number of oligonucleotides appear to allow an accurate reconstruction of the sequence specificity of the protein. This approach can be applied to uncover the specificity and function of other RBPs.
    RNA 08/2013; 19(10). DOI:10.1261/rna.037531.112 · 4.94 Impact Factor
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