POS-1 and GLD-1 repress glp-1 translation through a conserved binding-site cluster

RNA-binding proteins (RBPs) coordinate cell fate specification and differentiation in a variety of systems. RNA regulation is critical during oocyte development and early embryogenesis, in which RBPs control expression from maternal mRNAs encoding key cell fate determinants. The Caenorhabditis elegans Notch homologue glp-1 coordinates germline progenitor cell proliferation and anterior fate specification in embryos. A network of sequence-specific RBPs is required to pattern GLP-1 translation. Here, we map the cis-regulatory elements that guide glp-1 regulation by the CCCH-type tandem zinc finger protein POS-1 and the STAR-domain protein GLD-1. Our results demonstrate that both proteins recognize the glp-1 3′ untranslated region (UTR) through adjacent, overlapping binding sites and that POS-1 binding excludes GLD-1 binding. Both factors are required to repress glp-1 translation in the embryo, suggesting that they function in parallel regulatory pathways. It is intriguing that two equivalent POS-1–binding sites are present in the glp-1 3′ UTR, but only one, which overlaps with a translational derepression element, is functional in vivo. We propose that POS-1 regulates glp-1 mRNA translation by blocking access of other RBPs to a key regulatory sequence.     

Polarity-Dependent Asymmetric Distribution and MEX-5/6–Mediated Translational Activation of the Era-1 mRNA in C. elegans Embryos

The early C. elegans embryo is an attractive model system to investigate fundamental developmental processes. With the exception of mex-3 mRNA, maternally contributed mRNAs are thought to be distributed uniformly in the one-cell embryo. Here, we report and characterize the striking distribution of the mRNA encoding the novel protein ERA-1. We found that era-1 mRNA is enriched in the anterior of the one-cell embryo and present solely in anterior blastomeres thereafter. Although era-1 is not an essential gene, we uncovered that era-1 null mutant embryos are sensitive to slight impairment of embryonic polarity. We found that the asymmetric distribution of era-1 mRNA depends on anterior-posterior polarity cues and on the era-1 3’UTR. Similarly to the era-1 mRNA, the YFP-ERA-1 protein is enriched in anterior blastomeres. Interestingly, we found that the RNA-binding protein MEX-5 is required for era-1 mRNA asymmetry. Furthermore, we show that MEX-5, together with its partially redundant partner MEX-6, are needed to activate era-1 mRNA translation in anterior blastomeres. These findings lead us to propose that MEX-5/6–mediated regulation of era-1 mRNA contributes to robust embryonic development.     

MEX-5 and MEX-6 function to establish soma/germline asymmetry in early C. elegans embryos

An asymmetrical network of cortically localized PAR proteins forms shortly after fertilization of the C. elegans egg. This network is required for subsequent asymmetries in the expression patterns of several proteins that are encoded by nonlocalized, maternally expressed mRNAs. We provide evidence that two nearly identical genes, mex-5 and mex-6, link PAR asymmetry to those subsequent protein asymmetries. MEX-5 is a novel, cytoplasmic protein that is localized through PAR activities to the anterior pole of the 1-cell stage embryo. MEX-5 localization is reciprocal to that of a group of posterior-localized proteins called germline proteins. Ectopic expression of MEX-5 is sufficient to inhibit the expression of germline proteins, suggesting that MEX-5 functions to inhibit anterior expression of the germline proteins.     

C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis

Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(−) mex-3(−) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.     

Computational Identification of Post Translational Modification Regulated RNA Binding Protein Motifs

RNA and its associated RNA binding proteins (RBPs) mitigate a diverse array of cellular functions and phenotypes. The interactions between RNA and RBPs are implicated in many roles of biochemical processing by the cell such as localization, protein translation, and RNA stability. Recent discoveries of novel mechanisms that are of significant evolutionary advantage between RBPs and RNA include the interaction of the RBP with the 3’ and 5’ untranslated region (UTR) of target mRNA. These mechanisms are shown to function through interaction of a trans-factor (RBP) and a cis-regulatory element (3’ or 5’ UTR) by the binding of a RBP to a regulatory-consensus nucleic acid motif region that is conserved throughout evolution. Through signal transduction, regulatory RBPs are able to temporarily dissociate from their target sites on mRNAs and induce translation, typically through a post-translational modification (PTM). These small, regulatory motifs located in the UTR of mRNAs are subject to a loss-of-function due to single polymorphisms or other mutations that disrupt the motif and inhibit the ability to associate into the complex with RBPs. The identification of a consensus motif for a given RBP is difficult, time consuming, and requires a significant degree of experimentation to identify each motif-containing gene on a genomic scale. We have developed a computational algorithm to analyze high-throughput genomic arrays that contain differential binding induced by a PTM for a RBP of interest–RBP-PTM Target Scan (RPTS). We demonstrate the ability of this application to accurately predict a PTM-specific binding motif to an RBP that has no antibody capable of distinguishing the PTM of interest, negating the use of in-vitro exonuclease digestion techniques.     

The STAR/Maxi-KH domain protein GLD-1 mediates a developmental switch in the translational control of C. elegans PAL-1

Translational control is an essential mechanism of gene control utilized throughout development, yet the molecular mechanisms underlying translational activation and repression are poorly understood. We have investigated the translational control of the C. elegans caudal homolog, pal-1, and found that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH domain family, acts through a minimal pal-1 3' UTR element to repress pal-1 translation in the distal germline. We also provide data suggesting that GLD-1 may repress pal-1 translation after initiation. Finally, we show that GLD-1 represses the distal germline expression of the KH domain protein MEX-3, which was previously shown to repress PAL-1 expression in the proximal germline and which appears specialized to control PAL-1 expression patterns in the embryo. Hence, GLD-1 mediates a developmental switch in the control of PAL-1 repression, allowing MEX-3 to accumulate and take over the task of PAL-1 repression in the proximal germline, where GLD-1 protein levels decline.     

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.     

Role for cis-Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5′ untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA^fMet-mRNA ternary complex was inhibited unless a 5′ deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5′ UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5′ UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5′ UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.     

Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3′UTR

Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3′ untranslated region (3′UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3′UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3′UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA–ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3′UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem–loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.