The cardiac neural crest gene MafB ectopically directs CXCR4 expression in the trunk neural crest.

The cardiac neural crest is a subpopulation of cells arising from the caudal hindbrain. The delaminated cardiac neural crest cells migrate to the heart using the CXCR/SDF1 chemokine signaling system. These cells contribute to the formation of the cardiovascular system, including the septation of the outflow tract, which is unique to these cells. Here, we investigated the effect of ectopic expression of the cardiac neural crest gene MafB on trunk neural crest cells. First, we found that MafB has the potential to activate its own cis-regulatory element in enteric and trunk neural crest cells but not in cranial neural crest cells. Forced expression of two cardiac neural crest genes, Ets1 and Sox8, together with or without MafB, induced ectopic Sox10E2 enhancer activity in the trunk region. Finally, we uncovered that the expression of MafB, Ets1 and Sox8 can induce ectopic CXCR4 expression in the trunk neural crest cells, resulting in acquisition of responsiveness to the SDF1 signal. These results demonstrate that MafB, Ets1 and Sox8 are critical components for generation of the identity of the cardiac neural crest, especially the cell migration property.

The germ cell-specific TAP-like protein NXF-2 forms a novel granular structure and is required for tra-2 3'UTR-dependent mRNA export in Caenorhabditis elegans.

TAP is a general mRNA export receptor and is highly conserved among eukaryotes. The nematode Caenorhabditis elegans has another TAP-like protein, NXF-2, but little is known about its function. In this study, we show that NXF-2 is specifically expressed in germ cells and forms a novel granular structure that is different from that of P granules and that NXF-2 granules are anchored to the nuclear periphery in the mitotic region of the hermaphrodite gonad. In contrast, NXF-2 granules are released within the whole cytoplasm in the meiotic region, where the feminization gene tra-2 starts to function. Both inhibition of XPO-1 (an ortholog of the export receptor CRM1) and mutation of the nuclear export signal of NXF-2 caused the release of NXF-2 granules from the nuclear periphery, indicating that anchoring of NXF-2 granules depends on XPO-1 function. Moreover, inhibition of NXF-2 resulted in a substantial nuclear accumulation of the reporter mRNA carrying the tra-2 3'UTR. These results suggest that, together with XPO-1, NXF-2 exports and anchors tra-2 mRNA to the nuclear periphery to avoid precocious translation until the germ cells reach the meiotic region, thereby contributing to the regulation of tra-2 mRNA expression.

MRG-1 is required for both chromatin-based transcriptional silencing and genomic integrity of primordial germ cells in Caenorhabditis elegans.

In Caenorhabditis elegans, germline cells remain transcriptionally silenced during embryogenesis. The transcriptional silencing is achieved by two different mechanisms: One is the inhibition of RNA polymerase II in P2-P4 cells at the establishment stage, and another is chromatin-based silencing in two primordial germ cells (PGCs) at the maintenance stage; however, the molecular mechanism underlying chromatin-based silencing is less understood. We investigated the role of the chromodomain protein MRG-1, which is an essential maternal factor for germline development, in transcriptional silencing in PGCs. PGCs lacking maternal MRG-1 showed increased levels of two histone modifications (H3K4me2 and H4K16ac), which are epigenetic markers for active transcription, and precocious activation of germline promoters. Loss of MES-4, a H3K36 methyltransferase, also caused similar derepression of the germline genes in PGCs, suggesting that both MRG-1 and MES-4 function in chromatin-based silencing in PGCs. In addition, the mrg-1 null mutant showed abnormal chromosome structures and a decrease in homologous recombinase RAD-51 foci in PGCs, but the mes-4 null mutant did not show such phenotypes. Taken together, we propose that MRG-1 has two distinct functions: chromatin-based transcriptional silencing and preserving genomic integrity at the maintenance stage of PGCs.

Forkhead box B2 inhibits the malignant characteristics of the pancreatic cancer cell line Panc-1 in vitro.

Forkhead box (FOX) proteins constitute a family of transcription factors that are evolutionarily conserved in various species ranging from yeast to humans. These proteins have functions during development as well as in adulthood. To date, many reports have described the functions of FOX family genes in cancer cells, but the role of FOXB2 is not well understood. In one of the pancreas ductal adenocarcinoma cell lines, Panc-1 cells, we showed here that FOXB2 expression is barely detectable and that CpG islands in the 5' regions of the FOXB2 are highly methylated. These findings led us to hypothesize that FOXB2 acts as a tumor suppressor. To clarify our hypotheses, we investigated the effects of FOXB2 over-expression in Panc-1 cells. We obtained FOXB2 stable transfectants, and these clones exhibited reduced spheroid formation ability. Expression of ß-catenin, which is reported to be over-expressed in various cancer cells, was highly suppressed in FOXB2 stable transfectants. Moreover, side population (SP) cell fractions, which have a high tumorigenesis and metastatic potential, as well as anchorage-independent growth ability, were reduced. These results suggest that FOXB2 has the ability to inhibit the malignant characteristics of Panc-1 in vitro.

Transcriptome profiling of the cardiac neural crest reveals a critical role for MafB.

The cardiac neural crest originates in the caudal hindbrain, migrates to the heart, and contributes to septation of the cardiac outflow tract and ventricles, an ability unique to this neural crest subpopulation. Here we have used a FoxD3 neural crest enhancer to isolate a pure population of cardiac neural crest cells for transcriptome analysis. This has led to the identification of transcription factors, signaling receptors/ligands, and cell adhesion molecules upregulated in the early migrating cardiac neural crest. We then functionally tested the role of one of the upregulated transcription factors, MafB, and found that it acts as a regulator of Sox10 expression specifically in the cardiac neural crest. Our results not only reveal the genome-wide profile of early migrating cardiac neural crest cells, but also provide molecular insight into what makes the cardiac neural crest unique.

Developmental mechanisms of migratory muscle precursors in medaka pectoral fin formation.

Limb muscles are formed from migratory muscle precursor cells (MMPs) that delaminate from the ventral region of dermomyotomes and migrate into the limb bud. MMPs remain undifferentiated during migration, commencing differentiation into skeletal muscle after arrival in the limb. However, it is still unclear whether the developmental mechanisms of MMPs are conserved in teleost fishes. Here, we investigate the development of pectoral fin muscles in the teleost medaka Oryzias latipes. Expression of the MMP marker lbx1 is first observed in several somites prior to the appearance of fin buds. lbx1-positive cells subsequently move anteriorly and localize in the prospective fin bud region to differentiate into skeletal muscle cells. To address the developmental mechanisms underlying fin muscle formation, we knocked down tbx5, a gene that is required for fin bud formation. tbx5 morphants showed loss of fin buds, whereas lbx1 expression initiated normally in anterior somites. Unlike in normal embryos, expression of lbx1 was not maintained in migrating fin MMPs or within the fin buds. We suggest that fin MMPs appear to undergo two phases in their development, with an initial specification of MMPs occurring independent of fin buds and a second fin bud-dependent phase of MMP migration and proliferation. Our results showed that medaka fin muscle is composed of MMPs. It is suggested that the developmental mechanism of fin muscle formation is conserved in teleost fishes including medaka. Through this study, we also propose new insights into the developmental mechanisms of MMPs in fin bud formation.

Splicing activator RNPS1 suppresses errors in pre-mRNA splicing: A key factor for mRNA quality control.

Human RNPS1 protein was first identified as a pre-mRNA splicing activator in vitro and RNPS1 regulates alternative splicing in cellulo. RNPS1 was also known as a peripheral factor of the exon junction complex (EJC). Here we show that cellular knockdown of RNPS1 induced a reduction of the wild-type aurora kinase B (AURKB) protein due to the induced aberrant pre-mRNA splicing events, indicating that the fidelity of AURKB pre-mRNA splicing was reduced. The major aberrant AURKB mRNA was derived from the upstream pseudo 5' and 3' splice sites in intron 5, which resulted in the production of the non-functional truncated AURKB protein. AURKB, is an essential mitotic factor, whose absence is known to cause multiple nuclei, and this multinucleation phenotype was recapitulated in RNPS1-knockdown cells. Importantly this RNPS1-knockdown phenotype was rescued by ectopic expression of AURKB, implying it is a major functional target of RNPS1. We found RNPS1 protein, not as a component of the EJC, binds directly to a specific element in the AURKB exon upstream of the authentic 5' splice site, and this binding is required for normal splicing. RNPS1-knockdown induces a parallel aberrant splicing pattern in a fully distinct pre-mRNA, MDM2, suggesting that RNPS1 is a global guardian of splicing fidelity. We conclude that RNPS1 is a key factor for the quality control of mRNAs that is essential for the phenotypes including cell division.

Developmental regulation and evolution of muscle-specific microRNAs.

MicroRNAs (miRs) are a group of small RNAs that play a major role in post-transcriptional regulation of gene expression. In animals, many of the miRs are expressed in a conserved spatiotemporal manner. Muscle tissues, the major cellular systems involved in the locomotion and physiological functions of animals, have been one of the main sites for verification of miR targets and analysis of their developmental functions. During the determination and differentiation of muscle cells, numerous miRs bind to and repress target mRNAs in a highly specific but redundant manner. Interspecific comparisons of the sequences and expression of miRs have suggested that miR regulation became increasingly important during the course of vertebrate evolution. However, the detailed molecular interactions that have led to the highly complex morphological structures still await investigation. In this review, we will summarize the recent findings on the functional and developmental characteristics of miRs that have played major roles in vertebrate myogenesis, and discuss how the evolution of miRs is related to the morphological complexity of the vertebrates.

DND protein functions as a translation repressor during zebrafish embryogenesis.

Germline and somatic cell distinction is regulated through a combination of microRNA and germ cell-specific RNA-binding proteins in zebrafish. An RNA-binding protein, DND, has been reported to relieve the miR-430-mediated repression of some germ plasm mRNAs such as nanos3 and tdrd7 in primordial germ cells (PGCs). Here, we showed that miR-430-mediated repression is not counteracted by the overexpression of DND protein in somatic cells. Using a λN-box B tethering assay in the embryo, we found that tethering of DND to reporter mRNA results in translation repression without affecting mRNA stability. Translation repression by DND was not dependent on another germline-specific translation repressor, Nanos3, in zebrafish embryos. Moreover, our data suggested that DND represses translation of nanog and dnd mRNAs, whereas an RNA-binding protein DAZ-like (DAZL) promotes dnd mRNA translation. Thus, our study showed that DND protein functions as a translation repressor of specific mRNAs to control PGC development in zebrafish.

Control of the heat stress-induced alternative splicing of a subset of genes by hnRNP K.

Pre-mRNA splicing is widely repressed upon heat shock in eukaryotic cells. However, it has been shown that HSP105 pre-mRNA is alternatively spliced in response to heat stress. Using RNAi screening in HeLa cells, we found that RNA-binding proteins hnRNP K and PSF/SFPQ are necessary for the exon 12 exclusion of HSP105 during heat stress. Moreover, exon array analyses showed that a group of genes is alternatively spliced during heat stress in an hnRNP K-dependent manner, whereas hnRNP K is not necessary for the stress-induced alternative splicing of the remaining genes. Among the latter group, we found that SRp38/SRSF10 and SC35/SRSF2 are necessary for the inclusion of exon 13 of TNRC6A during heat stress. Thus, our study clearly showed that several RNA-binding proteins are involved in the splicing regulation in response to heat stress in mammalian cells.

The ELAV protein HuD stimulates cap-dependent translation in a Poly(A)- and eIF4A-dependent manner.

The RNA-binding protein HuD promotes neuronal differentiation by an unknown mechanism. Here we identify an enhancer function of HuD in translation. Translation stimulation by HuD requires both a 3' poly(A) tail and a 5' m(7)G cap structure. We also show that HuD directly interacts with eIF4A. This interaction and the poly(A)-binding activity of HuD are critical for its translational enhancer function because HuD-eIF4A- and HuD-poly(A)-binding mutants fail to stimulate translation. We show that translation of HCV IRES mRNA, which is eIF4A independent, is not stimulated by HuD. We also find that the eIF4A and poly(A)-binding activities of HuD are not only important for stimulating translation but also are essential for HuD-induced neurite outgrowth in PC12 cells. This example of cap-dependent translational regulation might explain at least in part how HuD triggers the induction of neuronal differentiation.

Roles of mRNA fate modulators Dhh1 and Pat1 in TNRC6-dependent gene silencing recapitulated in yeast.

The CCR4-NOT complex, the major deadenylase in eukaryotes, plays crucial roles in gene expression at the levels of transcription, mRNA decay, and protein degradation. GW182/TNRC6 proteins, which are core components of the microRNA-induced silencing complex in animals, stimulate deadenylation and repress translation via recruitment of the CCR4-NOT complex. Here we report a heterologous experimental system that recapitulates the recruitment of CCR4-NOT complex by TNRC6 in S. cerevisiae. Using this system, we characterize conserved functions of the CCR4-NOT complex. The complex stimulates degradation of mRNA from the 5' end by Xrn1, in a manner independent of both translation and deadenylation. This degradation pathway is probably conserved in miRNA-mediated gene silencing in zebrafish. Furthermore, the mRNA fate modulators Dhh1 and Pat1 redundantly stimulate mRNA decay, but both factors are required for poly(A) tail-independent translation repression by tethered TNRC6A. Our tethering-based reconstitution system reveals that the conserved architecture of Not1/CNOT1 provides a binding surface for TNRC6, thereby connecting microRNA-induced silencing complex to the decapping machinery as well as the translation apparatus.

Interaction and colocalization of HERMES/RBPMS with NonO, PSF, and G3BP1 in neuronal cytoplasmic RNP granules in mouse retinal line cells.

HERMES, also called RBPMS, is a conserved RNA binding protein with a single RNA recognition motif (RRM) that is abundantly expressed in retinal ganglion cells (RGCs) and in the heart in vertebrates. Here, we identified NonO and PSF as the interacting proteins of HERMES only when the neuronal differentiation of the retinal cell line RGC-5 was induced. Although NonO and PSF are nuclear paraspeckle components, these proteins formed cytoplasmic granules with HERMES in the neurites. G3BP1, a component of stress granules, was also colocalized to the granules, interacting with NonO and HERMES even in the absence of cellular stress. Consistent with a previous report that KIF5 interacts with neuronal granules, the localization of KIF5A overlapped with the cytoplasmic granules in differentiated RGC-5 cells. Thus, our study strongly suggests that the cytoplasmic granule containing HERMES, NonO, PSF, and G3BP1 is a neuronal RNA-protein granule that is transported in neurites during retinal differentiation.

Restricted distribution of mrg-1 mRNA in C. elegans primordial germ cells through germ granule-independent regulation.

The chromodomain protein MRG-1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG-1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg-1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG-1 to the PGCs. We found that MES-4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule-independent manner. We propose that C.elegans PGCs have a novel mechanism to accumulate at least some chromatin-associated proteins that are essential for germline immortality.

The Exon Junction Complex Controls the Efficient and Faithful Splicing of a Subset of Transcripts Involved in Mitotic Cell-Cycle Progression.

The exon junction complex (EJC) that is deposited onto spliced mRNAs upstream of exon-exon junctions plays important roles in multiple post-splicing gene expression events, such as mRNA export, surveillance, localization, and translation. However, a direct role for the human EJC in pre-mRNA splicing has not been fully understood. Using HeLa cells, we depleted one of the EJC core components, Y14, and the resulting transcriptome was analyzed by deep sequencing (RNA-Seq) and confirmed by RT-PCR. We found that Y14 is required for efficient and faithful splicing of a group of transcripts that is enriched in short intron-containing genes involved in mitotic cell-cycle progression. Tethering of EJC core components (Y14, eIF4AIII or MAGOH) to a model reporter pre-mRNA harboring a short intron showed that these core components are prerequisites for the splicing activation. Taken together, we conclude that the EJC core assembled on pre-mRNA is critical for efficient and faithful splicing of a specific subset of short introns in mitotic cell cycle-related genes.

Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish.

MicroRNA (miRNA) is a class of small noncoding RNA approximately 22 nt in length. Animal miRNA silences complementary mRNAs via translational inhibition, deadenylation, and mRNA degradation. However, the underlying molecular mechanisms remain unclear. A key question is whether these three outputs are independently induced by miRNA through distinct mechanisms or sequentially induced within a single molecular pathway. Here, we successfully dissected these intricate outputs of miRNA-mediated repression using zebrafish embryos as a model system. Our results indicate that translational inhibition and deadenylation are independent outputs mediated by distinct domains of TNRC6A, which is an effector protein in the miRNA pathway. Translational inhibition by TNRC6A is divided into two mechanisms: PAM2 motif-mediated interference of poly(A)-binding protein (PABP), and inhibition of 5' cap- and poly(A) tail-independent step(s) by a previously undescribed P-GL motif. Consistent with these observations, we show that, in zebrafish embryos, miRNA inhibits translation of the target mRNA in a deadenylation- and PABP-independent manner at early time points. These results indicate that miRNA exerts multiple posttranscriptional outputs via physically and functionally independent mechanisms and that direct translational inhibition is central to miRNA-mediated repression.

Inhibition of the first step in synthesis of the mycobacterial cell wall core, catalyzed by the GlcNAc-1-phosphate transferase WecA, by the novel caprazamycin derivative CPZEN-45.

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate-GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.

Functional and direct interaction between the RNA binding protein HuD and active Akt1.

The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.

Developmental expression and evolution of muscle-specific microRNAs conserved in vertebrates.

microRNAs (miRs) are small non-coding RNA molecules expressed in a tissue-specific manner in numerous organisms. Among them, miR-1, miR-206, and miR-133, which are encoded as bicistronic gene clusters in the genome, play major roles in the control of vertebrate myogenesis. To address how the gene organization and function of these miRs evolved, we identified their homologues in the cyclostomes, the chondrichthyans and the teleosts, and examined their patterns of expression during development. It was suggested that the chondrichthyans and the cyclostome lampreys possess fewer miR-1/miR-133 genes than the medaka. The medaka additionally possessed the miR-206 gene which was not found in the genomes of chondrichthyans and lampreys. In contrast, the number and genomic organization of medaka miR-1(206)/miR-133 were similar to those found in mammals. In the lamprey, shark and medaka, miR-1 and miR-133 were expressed in both skeletal and cardiac muscle cells in adults, a developmental feature traced back to chordate invertebrates such as ascidians. We further examined the expression of these miRs in different muscle tissues in medaka embryos. miR-206 was expressed in both the tail and pectoral fin muscles, whereas miR-1, which shares the similar nucleotide sequence with miR-206, was not detectable in the embryonic pectoral fins. Comparison of the relative positions with the neighboring protein-coding genes showed high conservation of synteny between the miR-1(206)/miR-133 clusters in a single species, as well as across the vertebrate taxa. Our results suggest that, after the gene duplications, these muscle-specific miRs acquired differential regulatory functions and have contributed to the establishment of diverse and complex musculature of vertebrates.

MiR-124 is involved in post-transcriptional regulation of Polypyrimidine tract binding protein 1 (PTBP1) during neural development in the medaka, Oryzias latipes.

MicroRNAs (miRNAs) comprise a group of small noncoding RNA molecules thought to have contributed to the evolution of vertebrate brain homogeneity and diversity. The miRNA miR-124 is well conserved between invertebrates and vertebrates and is expressed abundantly in the central nervous system (CNS). We identified miR-124 in the medaka, Oryzias latipes, and investigated its role in neural development. The five candidate genes for medaka precursor miR-124 are unlinked on four different chromosomes and differ in nucleotide length. Their sequences suggest that they can generate functional miRNAs through conventional miRNA biogenesis by folding into stem-loop structures. Whole-mount in situ hybridization and northern blotting revealed that mature miR-124 is specifically expressed in the CNS and the eyes starting at two days post-fertilization. We also examined the sequences and expression of medaka Polypyrimidine tract binding protein 1 (Ptbp1), a possible direct target of miR-124. The 3'UTR of medaka Ptbp1 contains predicted binding motifs (target sites) for miR-124. A GFP reporter assay for the target sites or the entire 3'UTR showed that exogenous miR-124 silences PTBP1 expression in vivo. Our study suggests that medaka miR-124 is involved in post-transcriptional regulation of target genes in neural development and that medaka miR-124 homologs may have spatiotemporal roles different from those in other vertebrates.