Josane F. Sousa

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

BackgroundMelanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of αvβ3-integrin and low levels of RHOC.MethodsTwo subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified.ResultsWe identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library.ConclusionThis study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

BackgroundThe ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers.ResultsOf the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes.ConclusionsThe versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

The DNA puff BhB10-1 gene is differentially expressed in various tissues of Bradysia hygida late larvae and constitutively transcribed in transgenic Drosophila

We extended the characterization of the DNA puff BhB10-1 gene of Bradysia hygida by showing that, although its mRNA is detected only at the end of the fourth larval instar, BhB10-1 expression is not restricted to the salivary gland, the tissue in which this gene is amplified. Different amounts of BhB10-1 mRNA were detected in other larval tissues such as gut, Malpighian tubules, fat body, brain and cuticle, suggesting that this gene is expressed differentially in the various tissues analyzed. Analysis of transgenic Drosophila carrying the BhB10-1 transcription unit and flanking sequences revealed that the tested fragment promotes transcription in a constitutive manner. We suggest that either cis-regulatory elements are missing in the transgene or factors that temporally regulate the BhB10-1 gene in B. hygida are not conserved in Drosophila.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

BackgroundThe sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome.ResultsBioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury.ConclusionUsing the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

Myosin-Va contributes to manifestation of malignant-related properties in melanoma cells.

TO THE EDITOR Melanoma is a highly metastatic and therapeutically resistant cancer, whose incidence has more than tripled in the last decades (Smalley et al., 2010). Physiologically, melanocytes produce and store melanin pigments in the melanosomes, which are transported to the cell periphery and transferred to keratinocytes, a process that requires the tripartite complex Rab27a/melanophilin/myosin-Va (Hume and Seabra, 2011). Myosin-Va is an actin-based molecular motor that also serves a multitude of other functions, such as plasma membrane receptor recycling, exocytosis, association with nuclear speckles and the centrosome (see Woolner and Bement, 2010); interaction with PTEN, thereby modulating PI3K pathway (van Diepen et al., 2009), interaction with Bcl-xL, proposed to promote invasion of islet-tumor cells (Du et al., 2007); as biomarker of invasiveness for nonfunctioning pituitary adenomas (Galland et al., 2010). Moreover, myosin-Va was shown to be up-regulated by Snail to promote cancer cell invasion (Lan et al., 2010), and was postulated to control apoptosis by sequestering the pro-apoptotic protein Bmf, which is unleashed upon loss of cell attachment (Puthalakath et al., 2001). Up-regulation of MYO5A gene in melanoma and other cancer types was revealed in different microarray studies compiled here (Table S1; Figure S1). However, these data did not clarify whether MYO5A up-regulation was associated with melanocyte transformation or simply reflected tissue specificity since comparison was against normal skin and melanocytes are minor cells in the skin. Here, we extended this evidence by showing that MYO5A is up-regulated in a variety of melanoma cell lines in comparison with primary melanocytes (Figure 1a), as well as in metastatic cells in comparison to paired vertical growth phase cells (Figure 1b and S2), implicating myosin-Va in malignant transformation and/or melanoma progression. Interestingly, in this WM panel, myosin-Va expression correlated with that of the oncogenic transcription factor MITF (Sousa and Espreafico, 2008). Figure 1 Myosin-Va is highly expressed in melanoma cells and its knockdown impairs cell adhesion and spreading on fibronectin-coated surface To investigate the role of myosin-Va in melanoma cells, we knocked down this protein using three different shRNAs (shMYO5A#1-3) carried by lentiviral vectors (Figure S3 and Qin et al., 2003) and an siRNA (siMYO5A). Once efficient knockdown was attained (Figures 1c-e), functional studies were performed. Upon adhesion to fibronectin-coated glass coverslips, MYO5A-depleted cells showed numerous small blebs on their surface and reduced lamellipodia/filopodia formation (Figure 1f), besides deficient adhesion (Figure 1g) and spreading (Figure 1h). Next, we examined the role of myosin-Va in adhesion-independent growth. The ability to form colony in soft agar, as analyzed after 25-30 days of incubation, was at least 50% lower for MYO5A-depleted cells than controls, for the three different shRNAs used (Figure 2a). Proliferation rates under adherent conditions were determined by crystal violet staining for WM1617 (Figure 2b) or ATP measurements for UACC-257 (Figure 2f), and no differences were observed, in the time courses analyzed, between MYO5A-knockdown and control cells. Subsequently, we analyzed transwell migration and invasion and found rates 50 to 70% lower for shMYO5A#2/3-transfected WM1617 cells than controls (Figure 2c). Similar decrease in transwell invasion was observed for siMYO5A-transfected UACC-257 cells (Figure 2e). Next, we performed spheroid assays (as in Smalley and Herlyn, 2008) with shMYO5A#1-transduced cells. Compact spheroids with intact appearance were added to a tri-dimensional collagen gel and imaged after 24 and 48 hours of culture. Myosin-Va-depleted cells exhibited migration distances from spheroid margin to invasion front 50 to 60% shorter than controls (Figure 2d). Also, knockdown cells that migrated out of the spheroids looked smaller than controls after 48 hours, suggesting that myosin-Va-depleted cells differ in the sensitivity to microenvironment factors during migration in collagen matrix. Figure 2 Ablation of myosin-Va inhibits colony formation, migration and invasiveness of metastatic melanoma cells without affecting cell proliferation The multifunctional character of myosin-Va makes us believe that this molecular motor, in addition to its role in cell adhesion/motility by promoting focal adhesion dynamics and filopodia/lamillipodia growth (supported by work in progress from our group, Nader et al.), may also perform a role in extracellular matrix proteolysis, mediating surface exposure and positioning of matrix metalloproteinases. Indeed, the alignment of metalloproteinases along the cytoskeleton seems to be a prerequisite for cell invasion in melanoma. Also, co-localization of metalloproteinases with myosin-Va (Sbai et. al., 2011) in astrocytes, and a role for RAB27A (Bobrie et al., 2012) in the release of metalloproteinase-9 to promote metastasis of mammary carcinoma cells have been shown. Moreover, evidence that RAB27A (Akavia et al., 2010) functions as a driver of cancer supports the hypothesis that, likewise, myosin-Va promotes malignancy by functioning in vesicular trafficking. Indeed, endocytosis and recycling of plasma membrane receptors require Rab GTPases and molecular motors with reflexes in adhesion dynamics, cell signaling and metabolism in many instances shown to drive oncogenic transformation and invasion (Mosesson et al., 2008). Furthermore, the relevance of our findings is supported by recent report demonstrating that the formation of filopodia is a critical step in the metastasis cascade (Shibue, et. al., 2013). Additionally, we cannot rule out the possibility that some of the effects observed could be due to an increase in the rates of apoptosis in the MYO5A knockdown cells. Although we have not observed alteration of viability after myosin-Va depletion in short term culturing under regular conditions, increase of apoptosis rates under adhesion blockage and poor recovery of frozen stocks were noted. In fact, recent independent findings reinforce participation of myosin-Va in the control of apoptosis. Bmf sequestration to the actin cytoskeleton, presumably in complex with myosin-Va/DLC2, promotes resistance to MEK-inhibitors (Van Brocklin et al., 2009). Accordingly, overexpression of myosin-Va tail fragments harboring the binding site for DLC2 leads to apoptosis in melanoma cells likely by disrupting Bmf and probably also Bim anchorage (Izidoro-Toledo and Borges et. al., 2013). Finally, miR-145, which is a transcriptional target of p53 and known to act as a tumor suppressor, was recently shown to target myosin-Va (Dynoodt et. al., 2012). Therefore, myosin-Va may integrate mechanisms that interconnect invasion/migration machinery and resistance to apoptosis. Interdependencies between these processes are reviewed in Alexander and Friedl (2012). In summary, the data presented here show that myosin-Va promotes adhesion dynamics, anchorage-independent survival, migration and invasion in vitro. Therefore, up-regulation of myosin-Va during melanoma progression may be part of a general mechanism that promotes malignant properties.