Genome-Wide Association between Branch Point Properties and Alternative Splicing

The branch point (BP) is one of the three obligatory signals required for pre-mRNA splicing. In mammals, the degeneracy of the motif combined with the lack of a large set of experimentally verified BPs complicates the task of modeling it in silico, and therefore of predicting the location of natural BPs. Consequently, BPs have been disregarded in a considerable fraction of the genome-wide studies on the regulation of splicing in mammals. We present a new computational approach for mammalian BP prediction. Using sequence conservation and positional bias we obtained a set of motifs with good agreement with U2 snRNA binding stability. Using a Support Vector Machine algorithm, we created a model complemented with polypyrimidine tract features, which considerably improves the prediction accuracy over previously published methods. Applying our algorithm to human introns, we show that BP position is highly dependent on the presence of AG dinucleotides in the 3′ end of introns, with distance to the 3′ splice site and BP strength strongly correlating with alternative splicing. Furthermore, experimental BP mapping for five exons preceded by long AG-dinucleotide exclusion zones revealed that, for a given intron, more than one BP can be chosen throughout the course of splicing. Finally, the comparison between exons of different evolutionary ages and pseudo exons suggests a key role of the BP in the pathway of exon creation in human. Our computational and experimental analyses suggest that BP recognition is more flexible than previously assumed, and it appears highly dependent on the presence of downstream polypyrimidine tracts. The reported association between BP features and the splicing outcome suggests that this, so far disregarded but yet crucial, element buries information that can complement current acceptor site models

The metastasis-associated protein S100A4 exists in several charged variants suggesting the presence of posttranslational modifications

<p>Abstract</p> <p>Background</p> <p>S100A4 is a metastasis-associated protein which has been linked to multiple cellular events, and has been identified extracellularly, in the cytoplasm and in the nucleus of tumor cells; however, the biological implications of subcellular location are unknown. Associations between a variety of posttranslational protein modifications and altered biological functions of proteins are becoming increasingly evident. Identification and characterization of posttranslationally modified S100A4 variants could thus contribute to elucidating the mechanisms for the many cellular functions that have been reported for this protein, and might eventually lead to the identification of novel drugable targets.</p> <p>Methods</p> <p>S100A4 was immuoprecipitated from a panel of <it>in vitro </it>and <it>in vivo </it>sources using a monoclonal antibody and the samples were separated by 2D-PAGE. Gels were analyzed by western blot and silver staining, and subsequently, several of the observed spots were identified as S100A4 by the use of MALDI-TOF and MALDI-TOF/TOF.</p> <p>Results</p> <p>A characteristic pattern of spots was observed when S100A4 was separated by 2D-PAGE suggesting the presence of at least three charge variants. These charge variants were verified as S100A4 both by western immunoblotting and mass spectrometry, and almost identical patterns were observed in samples from different tissues and subcellular compartments. Interestingly, recombinant S100A4 displayed a similar pattern on 2D-PAGE, but with different quantitative distribution between the observed spots.</p> <p>Conclusion</p> <p>Endogenously expressed S100A4 were shown to exist in several charge variants, which indicates the presence of posttranslational modifications altering the net charge of the protein. The different variants were present in all subcellular compartments and tissues/cell lines examined, suggesting that the described charge variants is a universal phenomenon, and cannot explain the localization of S100A4 in different subcellular compartments. However, the identity of the specific posttranslational modification and its potential contribution to the many reported biological events induced by S100A4, are subject to further studies.</p

Young Aphids Avoid Erroneous Dropping when Evading Mammalian Herbivores by Combining Input from Two Sensory Modalities

Mammalian herbivores may incidentally ingest plant-dwelling insects while foraging. Adult pea aphids (Acyrthosiphon pisum) avoid this danger by dropping off their host plant after sensing the herbivore's warm and humid breath and the vibrations it causes while feeding. Aphid nymphs may also drop (to escape insect enemies), but because of their slow movement, have a lower chance of finding a new plant. We compared dropping rates of first-instar nymphs with those of adults, after exposing pea aphids to different combinations of simulated mammalian breath and vibrations. We hypothesized that nymphs would compensate for the greater risk they face on the ground by interpreting more conservatively the mammalian herbivore cues they perceive. Most adults dropped in response to breath alone, but nymphs rarely did so. Breath stimulus accompanied by one concurrent vibrational stimulus, caused a minor rise in adult dropping rates. Adding a second vibration during breath had no additional effect on adults. The nymphs, however, relied on a combination of the two types of stimuli, with a threefold increase in dropping rates when the breath was accompanied by one vibration, and a further doubling of dropping rates when the second vibration was added. The age-specificity of the aphids' herbivore detection mechanism is probably an adaptation to the different cost of dropping for the different age groups. Relying on a combination of stimuli from two sensory modalities enables the vulnerable nymphs to avoid costly mistakes. Our findings emphasize the importance of the direct trophic effect of mammalian herbivory for plant-dwelling insects

The CUGBP2 Splicing Factor Regulates an Ensemble of Branchpoints from Perimeter Binding Sites with Implications for Autoregulation

Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor. However, the sequence motifs utilized by this factor for specific target exon selection and its role in splicing silencing are not understood. Here, we use chemical modification footprinting to map the contact sites of CUGBP2 to GU-rich motifs closely positioned at the boundaries of the branch sites of the NI exon, and we demonstrate a mechanistic role for this specific arrangement of motifs for the regulation of branchpoint formation. General support for a branch site-perimeter–binding model is indicated by the identification of a group of novel target exons with a similar configuration of motifs that are silenced by CUGBP2. These results reveal an autoregulatory role for CUGBP2 as indicated by its direct interaction with functionally significant RNA motifs surrounding the branch sites upstream of exon 6 of the CUGBP2 transcript itself. The perimeter-binding model explains how CUGBP2 can effectively embrace the branch site region to achieve the specificity needed for the selection of exon targets and the fine-tuning of alternative splicing patterns

Epigenetic deregulation of multiple S100 gene family members by differential hypomethylation and hypermethylation events in medulloblastoma

Deregulated expression of genes encoding members of the S100 family of calcium-binding proteins has been associated with the malignant progression of multiple tumour types. Using a pharmacological expression reactivation approach, we screened 16 S100 genes for evidence of epigenetic regulation in medulloblastoma, the most common malignant brain tumour of childhood. Four family members (S100A2, S100A4, S100A6 and S100A10) demonstrated evidence of upregulated expression in multiple medulloblastoma cell lines, following treatment with the DNA methyltransferase inhibitor, 5′-aza-2′-deoxycytidine. Subsequent analysis revealed methylation of critical CpG sites located within these four genes in an extended cell line panel. Assessment of these genes in the non-neoplastic cerebellum (from which medulloblastomas develop) revealed strong somatic methylation affecting S100A2 and S100A4, whereas S100A6 and S100A10 were unmethylated. Assessed against these normal tissue-specific methylation states, S100A6 and S100A10 demonstrated tumour-specific hypermethylation in medulloblastoma primary tumours (5 out of 40 and 4 out of 35, respectively, both 12%) and cell lines (both 7 out of 9, 78%), which was associated with their transcriptional silencing. Moreover, S100A6 hypermethylation was significantly associated with the aggressive large cell/anaplastic morphophenotype (P=0.026). In contrast, pro-metastatic S100A4 displayed evidence of hypomethylation relative to the normal cerebellum in a significant proportion primary tumours (7 out of 41, 17%) and cell lines (3 out of 9, 33%), which was associated with its elevated expression. In summary, these data characterise complex patterns of somatic methylation affecting S100 genes in the normal cerebellum and demonstrate their disruption causing epigenetic deregulation of multiple S100 family members in medulloblastoma development. Epigenetic events affecting S100 genes have potential clinical utility and merit further investigation as molecular biomarkers for this disease

Origins of the Tumor Microenvironment: Quantitative Assessment of Adipose-Derived and Bone Marrow–Derived Stroma

To meet the requirements for rapid tumor growth, a complex array of non-neoplastic cells are recruited to the tumor microenvironment. These cells facilitate tumor development by providing matrices, cytokines, growth factors, as well as vascular networks for nutrient and waste exchange, however their precise origins remain unclear. Through multicolored tissue transplant procedures; we have quantitatively determined the contribution of bone marrow-derived and adipose-derived cells to stromal populations within syngeneic ovarian and breast murine tumors. Our results indicate that subpopulations of tumor-associated fibroblasts (TAFs) are recruited from two distinct sources. The majority of fibroblast specific protein (FSP) positive and fibroblast activation protein (FAP) positive TAFs originate from mesenchymal stem/stromal cells (MSC) located in bone marrow sources, whereas most vascular and fibrovascular stroma (pericytes, α-SMA+ myofibroblasts, and endothelial cells) originates from neighboring adipose tissue. These results highlight the capacity for tumors to utilize multiple sources of structural cells in a systematic and discriminative manner

Proteomes and Signalling Pathways of Antler Stem Cells

As the only known example of complete organ regeneration in mammals, deer antler in the growing or velvet phase is of major interest in developmental biology. This regeneration event initiates from self-renewing antler stem cells that exhibit pluripotency. At present, it remains unclear how the activation and quiescence of antler stem cells are regulated. Therefore, in the present study proteins that were differentially expressed between the antler stem cells and somatic cells (facial periosteum) were identified by a gel-based proteomic technique, and analysed using Ingenuity Pathway Analysis software. Several molecular pathways (PI3K/Akt, ERK/MAPK, p38 MAPK, etc.) were found to be activated during proliferation. Also expressed were the transcription factors POU5F1, SOX2, NANOG and MYC, which are key markers of embryonic stem cells. Expression of these proteins was confirmed in both cultured cells and fresh tissues by Western blot analysis. Therefore, the molecular pathways and transcription factors identified in the current study are common to embryonic and adult stem cells. However, expression of embryonic stem cell transcription factors would suggest that antler stem cells are, potentially, an intermediary stem cell type between embryonic and the more specialized tissue-specific stem cells like those residing in muscle, fat or from a hematopoietic origin. The retention of this embryonic, pluripotent lineage may be of fundamental importance for the subsequent regenerative capacity of antlers