Article

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

ABSTRACT

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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    • "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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    • "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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