Comparative analysis of the sea urchin genome has broad implications for the primitive state of deuterostome host defense and the genetic underpinnings of immunity in vertebrates. The sea urchin has an unprecedented complexity of innate immune recognition receptors relative to other animal species yet characterized. These receptor genes include a vast repertoire of 222 Toll-like receptors, a superfamily of more than 200 NACHT domain-leucine-rich repeat proteins (similar to nucleotide-binding and oligomerization domain (NOD) and NALP proteins of vertebrates), and a large family of scavenger receptor cysteine-rich proteins. More typical numbers of genes encode other immune recognition factors. Homologs of important immune and hematopoietic regulators, many of which have previously been identified only from chordates, as well as genes that are critical in adaptive immunity of jawed vertebrates, also are present. The findings serve to underscore the dynamic utilization of receptors and the complexity of immune recognition that may be basal for deuterostomes and predicts features of the ancestral bilaterian form.
Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1184-4) contains supplementary material, which is available to authorized users.
Recent studies of translational control suggest that translation termination may not be simply the end of synthesizing a protein but rather be involved in modulating both the translation efficiency and stability of a given transcript. Using recombinant eukaryotic release factor 3 (eRF3) and cellular extracts, we have shown for Saccharomyces cerevisiae that yeast eRF3 and Pab1p can interact. This interaction, mediated by the N؉M domain of eRF3 and amino acids 473 to 577 of Pab1p, was demonstrated to be direct by the two-hybrid approach. We confirmed that a genetic interaction exists between eRF3 and Pab1p and showed that Pab1p overexpression enhances the efficiency of termination in SUP35 ( In general, termination of protein synthesis occurs when the ribosome elongation machinery encounters an in-frame termination codon on the mRNA. In eukaryotes two release factors have been identified, eukaryotic release factor 1 (eRF1), which recognizes all three stop codons, and eRF3, a GTPase that binds to eRF1 and stimulates its release activity in vitro (35,109). The eRF1 protein has a structure mimicking that of a tRNA molecule. It recognizes the stop codon in the A site of the ribosome and catalyzes the hydrolysis of the peptidyltRNA bond (88).In Saccharomyces cerevisiae eRF1 and eRF3 termination factors are encoded by the essential genes SUP45 and SUP35, respectively (10,43,53,60,105). Mutations in either of these genes give the same pleiotropic phenotypes which were selected as omnipotent nonsense suppressors (for a review see reference 49). Moreover, overexpression of both eRF1 and eRF3 is required to enhance the efficiency of termination in yeast (90). In higher eukaryotes, overproduction of eRF1 alone is sufficient to compete with a suppressor tRNA (62). Either Xenopus laevis or human eRF1 alone was also shown previously to have an antisuppressor effect against a suppressor tRNA in the reticulocyte lysate translation system (28). In vitro the eRF1 of higher eukaryotes has a release activity and does not need any other factor (28, 35), and eRF3 by itself binds GTP, but GTPase activity requires the presence of both eRF1 and ribosomes (36). It has been shown previously that eRF1 and eRF3 interact, suggesting that they form a functional complex (70,90,99,109 . Yeast eRF3 consists of an N-terminal prionforming domain (PrD), a charged M (middle) domain of unknown function, and a C-terminal domain that provides the essential translation termination activity (96,97,105). Recently, the minimum length of PrD was defined as amino acids (aa) 1 to 97 (76). It was shown previously that Hsp104 protein is required for formation and maintenance of [PSI ϩ ] aggregates of eRF3: its overproduction or inactivation cures cells of [PSI ϩ ] (14). The eRF3 family includes proteins from yeasts, humans, X. laevis, and other species that are strongly conserved in the C-terminal region, which has a significant homology with the translation elongation factor eEF1A (47,60,109). In contrast to the C-terminal part, the N-terminal region of eRF3 is ...
CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.
During vertebrate oogenesis and early embryogenesis, gene expression is governed mainly by translational control. The recruitment of Poly(A) Binding Protein (PABP) during poly(A) tail lengthening appears to be the key to translational activation during this period of development in Xenopus laevis. We showed that PABP1 and ePABP proteins are both present during oogenesis and early development. We selected ePABP as an eRF3 binding protein in a two-hybrid screening of a X. laevis cDNA library and demonstrated that this protein is associated with translational complexes. It can complement essential functions of the yeast homologue Pab1p. We discuss specific expression patterns of the finely tuned PABP1 and ePABP proteins.
An interaction between human poly(A)-binding protein (PABP) et human eRF3 has been demonstrated using a double-hybrid approach and in vitro assays. Here, we show that the binding of both proteins is conserved through evolution. We also demonstrate that the last 39 C-terminal amino acids of PABP contain the interface that interacts with eRF3. This region includes helix 5, identified by RMN, which is conserved in all known PABPs. Lastly, we demonstrate that eRF3 et PABP molecules interact in vivo.
eIF4E binding protein (4E-BP) inhibits translation of capped mRNA by binding to the initiation factor eIF4E and is known to be mostly or completely unstructured in both free and bound states. Using small angle X-ray scattering (SAXS), we report here the analysis of 4E-BP structure in solution, which reveals that while 4E-BP is intrinsically disordered in the free state, it undergoes a dramatic compaction in the bound state. Our results demonstrate that 4E-BP and eIF4E form a ‘fuzzy complex’, challenging current visions of eIF4E/4E-BP complex regulation.
Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.
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