Early origin and adaptive evolution of the GW182 protein family, the key component of RNA silencing in animals

<div><p>The GW182 proteins are a key component of the miRNA-dependent post-transcriptional silencing pathway in animals. They function as scaffold proteins to mediate the interaction of Argonaute (AGO)-containing complexes with cytoplasmic poly(A)-binding proteins (PABP) and PAN2-PAN3 and CCR4-NOT deadenylases. The AGO-GW182 complexes mediate silencing of the target mRNA through induction of translational repression and/or mRNA degradation. Although the GW182 proteins are a subject of extensive experimental research in the recent years, very little is known about their origin and evolution. Here, based on complex functional annotation and phylogenetic analyses, we reveal 448 members of the GW182 protein family from the earliest animals to humans. Our results indicate that a single-copy GW182/TNRC6C progenitor gene arose with the emergence of multicellularity and it multiplied in the last common ancestor of vertebrates in 2 rounds of whole genome duplication (WGD) resulting in 3 genes. Before the divergence of vertebrates, both the AGO- and CCR4-NOT-binding regions of GW182s showed significant acceleration in the accumulation of amino acid changes, suggesting functional adaptation toward higher specificity to the molecules of the silencing complex. We conclude that the silencing ability of the GW182 proteins improves with higher position in the taxonomic classification and increasing complexity of the organism. The first reconstruction of the molecular journey of GW182 proteins from the ancestral metazoan protein to the current mammalian configuration provides new insight into development of the miRNA-dependent post-transcriptional silencing pathway in animals.</p></div

Nicotine affects protein complex rearrangement in <i>Caenorhabditis elegans</i> cells

<p>Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) <i>in vivo</i> by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)–MS/MS and spectral counting. As a result, we identified dozens of <i>C. elegans</i> proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer’s disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.</p

Additional file 3: of Alignment-free sequence comparison: benefits, applications, and tools

Supplementary methods. (DOCX 37 kb

Additional file 2: Table S1. of Alignment-free sequence comparison: benefits, applications, and tools

Ranking list of alignment-free methods and the Smith-Waterman algorithm based on the area under the curve measures across four structural levels of the SCOP2 database. (DOCX 39 kb

Annotation and profiling of barley <i>GLYCOGEN SYNTHASE3/Shaggy</i>-like genes indicated shift in organ-preferential expression

<div><p>GLYCOGEN SYNTHASE KINASE3/Shaggy-like kinases (GSKs) represent a highly conserved group of proteins found in all eukaryotes. In plants they are encoded by multigene families and integrate signaling of brassinosteroids, auxin and abscisic acid in wide range of physiological and developmental processes with a strong impact on plant responses to environmental and biotic factors. Based on comprehensively studied structures of 10 <i>Arabidopsis thaliana GSK</i> genes and encoded proteins we report identification and phylogenetic reconstruction of 7 transcriptionally active <i>GSK</i> genes in barley. We re-evaluated annotation of the <i>GSK</i> genes in the current barley genome (Hv_IBSC_PGSB_v2) and provided data that a single gene annotated in the previous barley genome ensemble should be retained in the current one. The novel structure of another <i>GSK</i>, predicted in Hv_IBSC_PGSB_v2 to encode both GSK and amine oxidase domains, was proposed and experimentally confirmed based on the syntenic region in <i>Brachypodium distachyon</i>. The genes were assigned to 4 groups based on their encoded amino acid sequences and protein kinase domains. The analysis confirmed high level of conservation of functional protein domains and motifs among plant GSKs and the identified barley orthologs. Each of the seven identified <i>HvGSK</i> genes was expressed indicating semi-constitutive regulation in all tested organs and developmental stages. Regulation patterns of <i>GSKs</i> from the indicated groups showed a shift in organ-preferential expression in <i>A</i>. <i>thaliana</i> and barley illustrating diversification of biological roles of individual <i>HvGSKs</i> in different plant species.</p></div

Relative expression profile of the 7 <i>HvGSK</i> genes in selected organs and developmental stages of barley plants.

<p>The results represent the mean values and SD of a ratio of the studied gene transcript to the transcript of ADP-ribosylation factor used as an internal reference.</p

Schematic structure of HORVU5Hr1G119790.1 transcript predicted to encode amine oxidase and Glycogen Synthase Kinase (GSK) and HORVU5Hr1G119790.18 transcript annotated as <i>HvGSK4</i>.<i>1</i>.

<p>Enlarged exon10-exon11 fragment with non-transcribed junction between amine oxidase and GSK encoding regions. Indicated are nucleotide coordinates from the current barley genome Hv_IBSC_PGSB_v2.Enlarged exon9-exon12 region of HORVU5Hr1G119790.1 and HORVU5Hr1G119790.18 (<i>HvGSK4</i>.<i>1</i>) transcripts. Indicated are annealing sites of primers and results of amplification using barley gDNA or cDNA as template. Nucleotide alignment of amplicon A and the corresponding fragment of HORVU5Hr1G119790 gene are presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199364#pone.0199364.s005" target="_blank">S4 Table</a>.</p

Phylogenic tree of <i>GSK</i> genes of <i>Arabidopsis thaliana</i> and <i>Hordeum vulgare</i> used for classification of barley GSK family members.

<p>The phylogeny was reconstructed based on amino acid sequence of kinase domains using maximum likelihood algorithm. GSK protein from <i>Physcomitrella patens</i> (PpGSK) was used as an outgroup.</p