RNA-binding protein (RBP) regulation is widespread across biology from development to learning and memory. Often RBPs contain multiple modular domains, which contribute to distinct RNA- binding activity or interactions necessary for regulation. However, assays to determine specific regulatory activity of individual domains are limited. The tethered function assay (TFA) provides a direct method to assess the functional activity of RBPs and their domains. The assay consists of two key components: (i) a reporter plasmid that encodes an mRNA, such as luciferase or GFP, engineered with high-affinity binding sites for an exogenous RNA-binding factor, and (ii) an effector construct that expresses a chimera of the RBP fused to the RNA-binding factor. Co- transfection of these constructs allows for measurement of reporter activity as a quantitative readout for RBP regulatory function. We developed an inducible TFA (iTFA) system through generation of a stable inducible mammalian cell line to express a reporter mRNA encoding nanoluciferase (nLuc) with six high-affinity MS2 binding sites within its 3' untranslated region. These cells can be transfected with a single plasmid that expresses an RBP fused to the MS2 coat protein. This approach enhances efficiency and reproducibility through reduction of transfection burden to a single plasmid and simplified normalization. We used this platform to dissect the individual and combined regulatory effects of YBX3 domains, a modular RBP with differential regulatory activity. The iTFA system provides a streamlined, tunable platform for functional analysis of RBPs that facilitates rapid interrogation of RBP or domain-specific activities in mammalian cells.

Analysis of proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting (western blotting) is a vital part of the molecular biologist's toolkit. This technique separates a complex protein mixture by molecular weight and then assays the presence of target proteins using specific antibodies. Immunoblotting has a variety of applications. Examples include use as a targeted approach to study protein-protein interactors or as a control to confirm expression or depletion of protein targets. However, the successful execution of immunoblotting requires complicated, multistep experiments. Protocols must be optimized for each organism, target protein, and application. Therefore, knowledge gaps exist for the use of immunoblots in many models, including the model frog Xenopus laevis. Due to their large size, abundant material for biochemical experiments, and facile handling, X. laevis oocytes and embryos have been vital for studying principles of translational control. However, this species lacks specific protocols for robust and routine immunoblotting. Here, we offer an in-depth protocol for western blotting optimized for samples from multiple Xenopus developmental stages. We then analyze translational regulators across development.

BACKGROUND:Frem1 has been linked to human face shape variation, dysmorphology, and malformation, but little is known about its regulation and biological role in facial development.RESULTS:During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Finally, we found that FREM1 is sufficient to induce cNCC proliferation in a concentration-dependent manner and that Shh pathway antagonism reduces Frem1 expression during pathogenesis of midfacial hypoplasia.CONCLUSIONS:By demonstrating that the Shh signaling pathway regulates Frem1 expression in cNCCs, these findings provide novel insight into the mechanisms underlying variation in midfacial morphogenesis.

Bicaudal-C (Bicc1) is an evolutionarily conserved RNA binding protein that functions in a regulatory capacity in a variety of contexts. It was originally identified as a genetic locus in Drosophila that when disrupted resulted in radical changes in early development. In the most extreme phenotypes embryos carrying mutations developed with mirror image duplications of posterior structures and it was this striking phenotype that was responsible for the name Bicaudal. These seminal studies established Bicc1 as an important regulator of Drosophila development. What was not anticipated from the early work, but was revealed subsequently in many different organisms was the broad fundamental impact that Bicc1 proteins have on developmental biology; from regulating cell fates in vertebrate embryos to defects associated with several human disease states. In the following review we present a perspective of Bicc1 focusing primarily on the molecular aspects of its RNA metabolism functions in vertebrate embryos.

BioID is a proximity labeling strategy whose goal is to identify in vivo protein-protein interactions. The central components of this strategy are modified biotin ligase enzymes that promiscuously add biotin groups to proteins in close proximity. The transferred biotin group provides a powerful tag for purification and thus identification of interacting proteins. While a variety of modified biotin ligases were created for BioID, the original enzymes were inefficient, required long incubation times, and high intracellular biotin concentrations for protein labeling. These limitations hinder the application of BioID in contexts such as developing embryos where processes such as cell division and cell fate decisions occur rapidly. Recently, a new biotin ligase called TurboID was developed that addressed many of the deficiencies of previous enzymes. In this paper we compare TurboID to the BioID2 biotin ligase in developing Xenopus embryos. We find that the TurboID enzyme has several advantages over the BioID2 enzyme. TurboID labels proteins efficiently without the addition of additional biotin and occurs at a range of temperatures compatible with the culturing of Xenopus embryos. Biotinylation events occurred rapidly and were limited by TurboID expression and not its activity. Thus, TurboID is an efficient tool for BioID applications in Xenopus embryos and its use should facilitate the identification of interacting proteins in specific networks and complexes during Xenopus development.

ABSTRACT The Bicaudal-C (Bicc1) multi-KH domain RNA binding protein controls cell fates by binding and repressing the translation of specific target mRNAs. To provide new insights into Bicc1-RNA binding we used quantitative assays to analyze interactions between Xenopus Bicc1 protein and the 32-nt Bicc1 RNA target site from Xl Cripto-1 mRNA. High-affinity binding by Xl Bicc1(K d =34nM) required both a 5’ leader sequence and a 3’loop sequence. Bicc1 proteins from humans ( Hs Bicc1), zebrafish ( Dr Bicc1) and fruit flies ( Dm Bicc1) also bound the Xl Cripto-1 RNA with high-affinity via an interaction requiring the 5’ leader sequence, providing evidence for a conserved mechanism of RNA recognition by Bicc1. However, in contrast to Xl Bicc1, each of the other species’ Bicc1s showed a significantly relaxed requirement for the 3’ loop sequence. Analyses of mutant forms of Xl Bicc1, revealed that the KH1 domain promoted Bicc1’s requirement for the loop sequence via a GXXG-independent mechanism. In addition, a fusion protein comprised of the Xl KH1 and the Dm Bicc1-KH2-KH3 subdomains was sufficient to establish high-affinity, loop-sensitive Bicc1-binding to Xl Cripto-1 RNA. These data support a model wherein the KH2 domain interacts with the leader sequence via a GXXG-mediated KH-RNA interface, defining the conserved core of the metazoan Bicc1-RNA interaction, while the KH1 domain is required for the formation of a more flexible, secondary protein-RNA interface that allows recognition of the 3’ loop sequence. This secondary interface differs between species and possibly between target mRNAs, establishing the potential for a range of translational repression activities for Bicc1 target mRNAs.

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus , zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3′-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5 , Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3′-UTR mRNA reporter was responsive to pkd2 , suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.

Bicaudal‐C (Bicc1) is an RNA binding protein that functions as translational repressor in all animal species. Its repression activities are required for a multitude of biological processes such as guiding the earliest steps of embryonic development and organ homeostasis. For example, defects in Bicc1 expression or its activities can give rise to polycystic kidney disease in humans, a disorder that is characterized by an abnormal collection of cysts in the kidney. In addition, Bicc1 functions in vertebrate embryos to control the translation of specific maternal mRNAs that encode key developmental regulatory proteins. During the early stages of vertebrate development defects in Bicc1 trigger cell fate changes that lead to embryonic malformations similar to some human birth defects. A general feature is that in all of its biological contexts Bicc1 binds to specific mRNAs and it is through this binding that it selects specific mRNAs for translational repression. My project is focused on defining the molecular determinants of the Bicc1 protein required for that specific mRNA binding. Recent results from the Sheets lab identified a 70 amino acid region of the Bicc1 protein, the KH2 domain that is required for mRNA binding. However, the particular molecular features within the KH2 domain important for RNA binding activity are unknown. To address this issue I am generating mutations of the KH2 domain that change evolutionarily conserved amino acid residues in all Bicc1 proteins. These mutant proteins will be analyzed with gel shift RNA binding assays in vitro as well as in vivo RNA binding experiments performed in Xenopus embryos. My goal is to define the amino acid residues in the KH2 domain that are responsible for providing specificity for Bicc1 binding to specific mRNAs, guide its repression activities and mediate its biological functions.Support or Funding InformationThis work is supported by a Hilldale Undergraduate Research Fellowship to SD and funding from NICHD (R01HD091921) to MDS. Megan Dowdle is supported by a SciMed GRS Advanced Opportunity Fellowship and Biotechnology Training Program of the National Institutes of Health (NIGMS T32GM008349).

The earliest steps of animal development depend upon posttranscriptional events that drive the embryonic cell cycle and guide cell fate decisions. The analysis of post-transcriptional regulatory events has relied upon the use of chimeric reporter mRNAs that encode firefly luciferase fused to potential regulatory sequences. A new and more sensitive luciferase developed recently called NanoLuc has the potential to improve reporter studies and provide new insights into the regulation of embryonic processes. Here I describe how to create and analyze reporter mRNAs encoding NanoLuc luciferase using extracts from microinjected Xenopus embryos.

Bicaudal-C (Bicc1) is a conserved RNA-binding protein that represses the translation of selected mRNAs to control development. In Xenopus embryos, Bicc1 binds and represses specific maternal mRNAs to control anterior-posterior cell fates. However, it is not known how Bicc1 binds its RNA targets or how binding affects Bicc1-dependent embryogenesis. Focusing on the KH domains, we analyzed Bicc1 mutants for their ability to bind RNA substrates in vivo and in vitro Analyses of these Bicc1 mutants demonstrated that a single KH domain, KH2, was crucial for RNA binding in vivo and in vitro, while the KH1 and KH3 domains contributed minimally. The Bicc1 mutants were also assayed for their ability to repress translation, and results mirrored the RNA-binding data, with KH2 being the only domain essential for repression. Finally, maternal knockdown and rescue experiments indicated that the KH domains were essential for the regulation of embryogenesis by Bicc1. These data advance our understanding of how Bicc1 selects target mRNAs and provide the first direct evidence that the RNA binding functions of Bicc1 are essential for both Bicc1-dependent translational repression and maternal vertebrate development.

Sonic Hedgehog (Shh) signaling plays key regulatory roles in embryonic development and postnatal homeostasis and repair. Modulation of the Shh pathway is known to cause malformations and malignancies associated with dysregulated tissue growth. However, our understanding of the molecular mechanisms by which Shh regulates cellular proliferation is incomplete. Here, using mouse embryonic fibroblasts, we demonstrate that the Forkhead box gene Foxd1 is transcriptionally regulated by canonical Shh signaling and required for downstream proliferative activity. We show that Foxd1 deletion abrogates the proliferative response to SHH ligand while FOXD1 overexpression alone is sufficient to induce cellular proliferation. The proliferative response to both SHH ligand and FOXD1 overexpression was blocked by pharmacologic inhibition of cyclin-dependent kinase signaling. Time-course experiments revealed that Shh pathway activation of Foxd1 is followed by downregulation of Cdkn1c, which encodes a cyclin-dependent kinase inhibitor. Consistent with a direct transcriptional regulation mechanism, we found that FOXD1 reduces reporter activity of a Fox enhancer sequence in the second intron of Cdkn1c. Supporting the applicability of these findings to specific biological contexts, we show that Shh regulation of Foxd1 and Cdkn1c is recapitulated in cranial neural crest cells and provide evidence that this mechanism is operational during upper lip morphogenesis. These results reveal a novel Shh-Foxd1-Cdkn1c regulatory circuit that drives the mitogenic action of Shh signaling and may have broad implications in development and disease.

Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Vertebrate Bicaudal-C (Bicc1) has important biological roles in the formation and homeostasis of multiple organs, but direct experiments to address the role of maternal Bicc1 in early vertebrate embryogenesis have not been reported. Here, we use antisense phosphorothioate-modified oligonucleotides and the host-transfer technique to eliminate specifically maternal stores of both bicc1 mRNA and Bicc1 protein from Xenopus laevis eggs. Fertilization of these Bicc1-depleted eggs produced embryos with an excess of dorsal-anterior structures and overexpressed organizer-specific genes, indicating that maternal Bicc1 is crucial for normal embryonic patterning of the vertebrate embryo. Bicc1 is an RNA-binding protein with robust translational repression function. Here, we show that the maternal mRNA encoding the cell-fate regulatory protein Wnt11b is a direct target of Bicc1-mediated repression. It is well established that the Wnt signaling pathway is crucial to vertebrate embryogenesis. Thus, the work presented here links the molecular function of Bicc1 in mRNA target-specific translation repression to its biological role in the maternally controlled stages of vertebrate embryogenesis.

In all animals, a critical period in early development is when embryonic cells switch from relying solely upon maternally deposited RNAs and proteins to relying upon molecules encoded by the zygotic genome. Xenopus embryos have served as a model for examining this switch, as well as the maternally controlled stages that prepare for it. In Xenopus, the robust activation of zygotic transcription occurs at the 12th cleavage division and is referred to as the midblastula transition (MBT). Prior to MBT, gene expression is regulated by post-transcriptional events including mRNA and protein localization, protein post-translational modification, and mRNA translation. After the MBT, appropriate transcriptional regulation of the zygotic genome becomes critical and predominates. However, it is important to realize that the first key cell fate decisions that have profound impacts on development occur prior to the MBT and these are governed by regulating the expression of maternally deposited regulatory mRNAs and proteins. In this chapter, I will discuss post-transcriptional mechanisms that function during the maternal stages of Xenopus development with an emphasis on mechanisms known to directly modulate cell fate decisions. Emerging approaches and technologies that will help better understand this phase of development will also be discussed.

In all animals, a critical period in early development is when embryonic cells switch from relying solely upon maternally deposited RNAs and proteins to relying upon molecules encoded by the zygotic genome. Xenopus embryos have served as a model for examining this switch, as well as the maternally controlled stages that prepare for it. In Xenopus, the robust activation of zygotic transcription occurs at the 12th cleavage division and is referred to as the midblastula transition (MBT). Prior to MBT, gene expression is regulated by post-transcriptional events including mRNA and protein localization, protein post-translational modification, and mRNA translation. After the MBT, appropriate transcriptional regulation of the zygotic genome becomes critical and predominates. However, it is important to realize that the first key cell fate decisions that have profound impacts on development occur prior to the MBT and these are governed by regulating the expression of maternally deposited regulatory mRNAs and proteins. In this chapter, I will discuss post-transcriptional mechanisms that function during the maternal stages of Xenopus development with an emphasis on mechanisms known to directly modulate cell fate decisions. Emerging approaches and technologies that will help better understand this phase of development will also be discussed.

Background: Bicaudal-C RNA binding proteins are important translational repressors in many different biological contexts. Results: A minimal site for RNA binding and translation repression by Bicaudal-C was identified. Conclusion: The RNA binding site for Bicaudal-C forms a stem-loop structure important for binding and translational repression. Significance: This will facilitate the identification of Bicaudal-C binding sites in other mRNAs.Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

The Xenopus Cripto-1 protein is confined to the cells of the animal hemisphere during early embryogenesis where it regulates the formation of anterior structures. Cripto-1 protein accumulates only in animal cells because cripto-1 mRNA in cells of the vegetal hemisphere is translationally repressed. Here, we show that the RNA binding protein, Bicaudal-C (Bic-C), functioned directly in this vegetal cell-specific repression. While Bic-C protein is normally confined to vegetal cells, ectopic expression of Bic-C in animal cells repressed a cripto-1 mRNA reporter and associated with endogenous cripto-1 mRNA. Repression by Bic-C required its N-terminal domain, comprised of multiple KH motifs, for specific binding to relevant control elements within the cripto-1 mRNA and a functionally separable C-terminal translation repression domain. Bic-C-mediated repression required the 5' CAP and translation initiation factors, but not a poly(A) tail or the conserved SAM domain within Bic-C. Bic-C-directed immunoprecipitation followed by deep sequencing of associated mRNAs identified multiple Bic-C-regulated mRNA targets, including cripto-1 mRNA, providing new insights and tools for understanding the role of Bic-C in vertebrate development.