Substrate Specificity and Plasticity of FERM-Containing Protein Tyrosine Phosphatases

SummaryEpidermal growth factor receptor (EGFR) pathway substrate 15 (Eps15) is a newly identified substrate for protein tyrosine phosphatase N3 (PTPN3), which belongs to the FERM-containing PTP subfamily comprising five members including PTPN3, N4, N13, N14, and N21. We solved the crystal structures of the PTPN3-Eps15 phosphopeptide complex and found that His812 of PTPN3 and Pro850 of Eps15 are responsible for the specific interaction between them. We defined the critical role of the additional residue Tyr676 of PTPN3, which is replaced by Ile939 in PTPN14, in recognition of tyrosine phosphorylated Eps15. The WPD loop necessary for catalysis is present in all members but not PTPN21. We identified that Glu instead of Asp in the WPE loop contributes to the catalytic incapability of PTPN21 due to an extended distance beyond protonation targeting a phosphotyrosine substrate. Together with in vivo validations, our results provide novel insights into the substrate specificity and plasticity of FERM-containing PTPs

miRNA arm selection and isomiR distribution in gastric cancer

BACKGROUND: MicroRNAs (miRNAs) are small non-protein-coding RNAs. miRNA genes need several biogenesis steps to form function miRNAs. However, the precise mechanism and biology involved in the mature miRNA molecules are not clearly investigated. In this study, we conducted in-depth analyses to examine the arm selection and isomiRs using NGS platform. METHODS: We sequenced small RNAs from one pair of normal and gastric tumor tissues with Solexa platform. By analyzing the NGS data, we quantified the expression profiles of miRNAs and isomiRs in gastric tissues. Then, we measured the expression ratios of 5p arm to 3p arm of the same pre-miRNAs. And, we used Kolmogorov-Smirnov (KS) test to examine isomiR pattern difference between tissues. RESULTS: Our result showed the 5p arm and 3p arm miRNA derived from the same pre-miRNAs have different tissue expression preference, one preferred normal tissue and the other preferred tumor tissue, which strongly implied that there could be other mechanism controlling mature miRNA selection in addition to the known hydrogen-bonding selection rule. Furthermore, by using the KS test, we demonstrated that some isomiR types preferentially occur in normal gastric tissue but other types prefer tumor gastric tissue. CONCLUSIONS: Arm selections and isomiR patterns are significantly varied in human cancers by using deep sequencing NGS data. Our results provided a novel research topic in miRNA regulation study. With advanced bioinformatics and molecular biology studies, more robust conclusions and insight into miRNA regulation can be achieved in the near future

Discovery and characterization of medaka miRNA genes by next generation sequencing platform

Background MicroRNAs (miRNAs) are endogenous non-protein-coding RNA genes which exist in a wide variety of organisms, including animals, plants, virus and even unicellular organisms. Medaka (Oryzias latipes) is a useful model organism among vertebrate animals. However, no medaka miRNAs have been investigated systematically. It is beneficial to conduct a genome-wide miRNA discovery study using the next generation sequencing (NGS) technology, which has emerged as a powerful sequencing tool for high-throughput analysis. Results In this study, we adopted ABI SOLiD platform to generate small RNA sequence reads from medaka tissues, followed by mapping these sequence reads back to medaka genome. The mapped genomic loci were considered as candidate miRNAs and further processed by a support vector machine (SVM) classifier. As result, we identified 599 novel medaka pre-miRNAs, many of which were found to encode more than one isomiRs. Besides, additional minor miRNAs (also called miRNA star) can be also detected with the improvement of sequencing depth. These quantifiable isomiRs and minor miRNAs enable us to further characterize medaka miRNA genes in many aspects. First of all, many medaka candidate pre-miRNAs position close to each other, forming many miRNA clusters, some of which are also conserved across other vertebrate animals. Secondly, during miRNA maturation, there is an arm selection preference of mature miRNAs within precursors. We observed the differences on arm selection preference between our candidate pre-miRNAs and their orthologous ones. We classified these differences into three categories based on the distribution of NGS reads. Finally, we also investigated the relationship between conservation status and expression level of miRNA genes. We concluded that the evolutionally conserved miRNAs were usually the most abundant ones. Conclusions Medaka is a widely used model animal and usually involved in many biomedical studies, including the ones on development biology. Identifying and characterizing medaka miRNA genes would benefit the studies using medaka as a model organism

Designating eukaryotic orthology via processed transcription units

Orthology is a widely used concept in comparative and evolutionary genomics. In addition to prokaryotic orthology, delineating eukaryotic orthology has provided insight into the evolution of higher organisms. Indeed, many eukaryotic ortholog databases have been established for this purpose. However, unlike prokaryotes, alternative splicing (AS) has hampered eukaryotic orthology assignments. Therefore, existing databases likely contain ambiguous eukaryotic ortholog relationships and possibly misclassify alternatively spliced protein isoforms as in-paralogs, which are duplicated genes that arise following speciation. Here, we propose a new approach for designating eukaryotic orthology using processed transcription units, and we present an orthology database prototype using the human and mouse genomes. Currently existing programs cover less than 69% of the human reference sequences when assigning human/mouse orthologs. In contrast, our method encompasses up to 80% of the human reference sequences. Moreover, the ortholog database presented herein is more than 92% consistent with the existing databases. In addition to managing AS, this approach is capable of identifying orthologs of embedded genes and fusion genes using syntenic evidence. In summary, this new approach is sensitive, specific and can generate a more comprehensive and accurate compilation of eukaryotic orthologs

Acetylome of acinetobacter baumannii SK17 reveals a highly-conserved modification of histone-like protein HU

Lysine acetylation is a prevalent post-translational modification in both eukaryotes and prokaryotes. Whereas this modification is known to play pivotal roles in eukaryotes, the function and extent of this modification in prokaryotic cells remain largely unexplored. Here we report the acetylome of a pair of antibiotic-sensitive and -resistant nosocomial pathogen Acinetobacter baumannii SK17-S and SK17-R. A total of 145 lysine acetylation sites on 125 proteins was identified, and there are 23 acetylated proteins found in both strains, including histone-like protein HU which was found to be acetylated at Lys13. HU is a dimeric DNA-binding protein critical for maintaining chromosomal architecture and other DNA-dependent functions. To analyze the effects of site-specific acetylation, homogenously Lys13-acetylated HU protein, HU(K13ac) was prepared by genetic code expansion. Whilst not exerting an obvious effect on the oligomeric state, Lys13 acetylation alters both the thermal stability and DNA binding kinetics of HU. Accordingly, this modification likely destabilizes the chromosome structure and regulates bacterial gene transcription. This work indicates that acetyllysine plays an important role in bacterial epigenetics

Transcriptional regulation of miR-196b by ETS2 in gastric cancer cells

E26 transformation-specific sequence (ETS)-2 is a transcriptional modulator located on chromosome 21, alterations in its expression have been implicated with a reduced incidence of solid tumors in Down syndrome patients. MicroRNAs (miRNAs) are thought to participate in diverse biological functions; however, the regulation of miRNAs is not well characterized. Recently, we reported that miR-196b is highly expressed in gastric cancers. Herein, we demonstrate that miR-196b expression was significantly repressed by ETS2 during gastric cancer oncogenesis. We demonstrate that knockdown of endogenous ETS2 expression increases miR-196b expression. A genomic region between −751 and −824 bp upstream of the miR-196b transcriptional start site was found to be critical for the repression activity. This putative regulatory promoter region contains three potential ETS2-binding motifs. Mutations within the ETS2 binding sites blocked the repression activity of ETS2. Furthermore, knockdown of ETS2 or overexpression of miR-196b significantly induced migration and invasion in gastric cancer cells. In addition, alterations in ETS2 and miR-196b expression in gastric cancer cell lines affected the expression of epithelial–mesenchymal transition-related genes. The levels of vimentin, matrix metalloproteinase (MMP)-2 and MMP9 were drastically induced, but levels of E-cadherin were decreased in shETS2- or miR-196b-transfected cells. Our data indicate that ETS2 plays a key role in controlling the expression of miR-196b, and miR-196b may mediate the tumor suppressor effects of ETS2. We demonstrated that miR-196b was transcriptionally regulated by ETS2 and there was an inverse expression profile between miR-196b and ETS2 in clinical samples. This finding could be beneficial for the development of effective cancer diagnostic and alternative therapeutic strategies

Hsp90 Interacts Specifically with Viral RNA and Differentially Regulates Replication Initiation of Bamboo mosaic virus and Associated Satellite RNA

Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3′ untranslated region (3′ UTR) of BaMV genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3′ UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3′ UTR of BaMV RNA during the initiation of BaMV RNA replication

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe