Modeling the binding specificity of the RNA-binding protein GLD-1 suggests a function of coding region–located sites in translational repression

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 (crosslinking 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.

Control of a neuronal morphology program by an RNA-binding zinc finger protein, Unkempt

“…4A,B; Abdelmohsen et al 2011;Darnell et al 2011;Cho et al 2012;Brummer et al 2013). To determine whether Unkempt associates with polyribosomes, we fractionated the lysates of SH-SY5Y cells and mouse embryonic brains on linear sucrose density gradients and examined the sedimentation pattern of Unkempt ( Fig.…”

Post-transcriptional control of executioner caspases by RNA-binding proteins

“…2A). This site (GMB 1) contained a sequence that matches the GLD-1-binding motif (GBM) that we inferred previously using motif-based enrichment of the reads from the HITS-CLIP experiment (Brümmer et al 2013). Inspection of an additional published data set of GLD-1-binding sites generated via photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) (Jungkamp et al 2011) identified evidence for a second candidate GLD-1-binding site (GBM 2) 300 base pairs (bp) downstream from GBM 1 in the ced-3 3 ′ UTR ( Fig.…”

“…S4C). In order to test whether GLD-1 binds directly to the ced-3 mRNA, we reanalyzed a set of transcriptome-wide GLD-1-binding sites that we generated previously by high-throughput sequencing of RNA isolated by crosslinking and immunoprecipitation (HITS-CLIP) of a transgenic line expressing a rescuing GLD-1::STREP/HA transgene (Brümmer et al 2013). We identified an enrichment of clipped reads at one site in the ced-3 3 ′ UTR ( Fig.…”

“…Two GBMs in the ced-3 3 ′ UTR mediate ced-3 mRNA repression by GLD-1. (A) GLD-1 binds to the ced-3 3 ′ UTR: HITS-CLIP of GLD-1::STREP/HA (Brümmer et al 2013) and PAR-CLIP of GLD-1::GFP::Flag (Jungkamp et al 2011) identified two candidate GBMs in the ced-3 3 ′ UTR. (B) Following GLD-1 immunoprecipitation, ced-3 mRNA showed a level of enrichment similar to that of the p53 tumor suppressor homolog cep-1, a known GLD-1 target (Schumacher et al 2005).…”

“…The HITS-CLIP data set was generated as described previously (Brümmer et al 2013). The data from the experiment are publicly available on the CLIPz server (http://www.clipz.unibas.ch).…”