Determining the Binding Between SAGA Subunits and Spliceosomal Components

Proper gene regulation is vital to the health and development of an organism. Determining the relationship between splicing, transcription, and chromatin structure is vital for understanding gene regulation as a whole. There have been previous studies linking these elements pairwise; however, no evidence exists for a direct link between all three. Recent data shows that splicing components of the U2 small nuclear ribonucleic protein (snRNP) co-purify with Spt-Ada-Gcn5-acetyltransferase (SAGA), a highly conserved transcriptional co-activator and chromatin modifier. We hypothesize that SAGA binds with splicing components through a multi-protein binding surface with certain core components based on preliminary yeast two-hybrid data. Here, we examine the specific binding partners between SAGA and splicing components utilizing the yeast two-hybrid system in spt7Δ Saccharomyces cerevisiae as a validation for the preliminary yeast two-hybrid performed, producing recombinant proteins through sequence and ligation-independent cloning (SLIC) and Baculovirus transfections to obtain purified proteins, and co-immunoprecipitation (co-IP) to detect specific protein-protein interactions from recombinant proteins. Yeast two-hybrid results reveal that Spt7 is necessary for the transcription of reporter genes used in this assay. Therefore, this assay cannot validate previous results or detect false positives. Currently, recombinant proteins are being produced to perform co-IPs to test direct protein interactions. The results from these experiments will demonstrate the type of binding between SAGA subunits and splicing factors and provide direct evidence of a link between all three of the elements of gene regulation

Analyzing Mutations of Spt7 Protein That Disrupt Interaction with SF3B Subunits

Proper transcription, the process of converting DNA to RNA, is crucial for the health and viability of an organism. This process is regulated by many proteins, such as co-transcriptional activators; one being the protein complex known as Spt-Ada-Gcn5-acetyltransferase, or SAGA. While much is known about the roles of SAGA in cell processes, how SAGA’s subunits promote functionality is still unknown. The focus of this study is to analyze the purpose of SAGA’s SF3B subunits. These subunits are also found in the spliceosome, the compound responsible for generating mature RNA. SAGA has no known functions relating to this process, so the reason the SF3B components are in SAGA is unclear. Spt7, another SAGA subunit, interacts with both SF3B subunits. In this study, a yeast two hybrid assay was performed where different Spt7 mutants were screened. This was done by transforming yeast with Spt7 mutants, analyzing the protein interactions and sequencing the mutants to determine their mutations. A key result of this study is in the determining that the two SF3B subunits interact with different regions of Spt7. Although the overall goal is to find an Spt7 mutant that does not interact with the SF3B components but still maintains interaction with other SAGA subunits, we now have a better idea of what type of Spt7 mutant is needed. This discovery will lay the foundation for future experiments where a mutated SAGA with no SF3B components will be expressed in Drosophila melanogaster and analyzed to determine the function of SF3B subunits in SAGA

The influence of decisional conflict on treatment decision in pelvic organ prolapse-data from the SHADE-POP trial

PurposeWomen with symptomatic pelvic organ prolapse are facing the choice between several treatment options and a potentially difficult decision. The aim of this study was to examine the effect of decisional conflict, patient characteristics and other decision-related factors on treatment decision in women with pelvic organ prolapse.MethodsData from the SHADE-POP trial were used. Women with symptomatic pelvic organ prolapse who visited their gynaecologist for (new) treatment options were included. In all participants, demographical characteristics and validated questionnaires concerning decisional conflict (DCS), shared decision making (SDM-Q-9), information provision (SCIP-B), anxiety and depression (HADS) and satisfaction with care (PSQ-18) were collected 2 weeks after the visit. Analyses were performed using univariate and multivariate linear and logistic regression analyses.ResultsNinety six women with pelvic organ prolapse facing a treatment decision were included. An increase in decisional conflict as experienced by patients was related to the choice of more conservative treatment, such as pelvic floor muscle training or pessary, instead of surgery (p = 0.02). Shared decision making, better information provision and satisfaction with care were related to lower levels of decisional conflict (p = 0.001).ConclusionDecisional conflict in women with pelvic organ prolapse favours conservative treatment instead of surgery. Gaining knowledge on the effect of decisional conflict, patient characteristics and other decision-related factors on treatment decision in pelvic organ prolapse will be a step towards a better-guided treatment decision and better patient-reported outcomes for this group of patients. NL 55737.028.15, 30-10-2016

The Spliceosomal Protein SF3B5 is a Novel Component of Drosophila SAGA that Functions in Gene Expression Independent of Splicing

The interaction between splicing factors and the transcriptional machinery provides an intriguing link between the coupled processes of transcription and splicing. Here, we show that the two components of the SF3B complex, SF3B3 and SF3B5, that form part of the U2 small nuclear ribonucleoprotein particle (snRNP) are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We show that SF3B3 and SF3B5 bind to SAGA independent of RNA and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5mutant flies, we show that SF3B5 is necessary for proper development and cell viability but not for histone acetylation. Although SF3B5 does not appear to function in SAGA\u27s histone-modifying activities, SF3B5 is still required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA\u27s transcription coactivator activity that is separate from its role in splicing

The Functional Role of Splicing Factors in Transcription and Aging

Proper gene expression of protein coding genes is an integral part of the health and survival of eukaryotes. Transcription and splicing are two coordinated processes which must occur to allow for the proper expression of protein coding genes. These processes are tightly regulated and any misregulation can lead to dysfunctional proteins which can cause functional decline or disease. Splicing occurs via the spliceosome and its associated proteins, splicing factors, with more than 300 total proteins involved in active splicing. When transcribed gene products are produced, this dynamic macromolecular machine splices out introns and ligates exon-exon junctions back together to produce mRNA. Alternative splicing occurs when alternatively used exons can be included or skipped, and exon-exon junctions can be differentially used to produce multiple transcripts. Additionally trans-acting splicing factors bind to RNA elements, and this helps to determine the splicing outcome of a particular transcript, however exactly how splicing factors affect splice site choices from transcript to transcript is still unclear, and is still a highly investigated topic. Our studies will focus on how spliceosomal components and splicing factors play a role in gene regulation, through transcription, or changes in alternative splicing which can cause a functional decline with age. Genome-wide studies of aging have identified subsets of genes that show age-related changes in expression. Aging is associated with a broad induction of stress response pathways. In contrast, a wide variety of functional classes of genes are downregulated with age, often including tissue-specific genes. Whereas the upregulation of age-regulated genes is likely to be governed by stress-responsive transcription factors, questions remain as to why particular genes are susceptible to age-related transcriptional decline. Additionally recent findings showing that splicing is misregulated with age. While defects in splicing could lead to changes in protein isoform levels, they could also impact gene expression through nonsense-mediated decay of intron-retained transcripts. Moreover, the considerable variation between genome-wide aging expression studies indicates that there is a critical need to analyze the transcriptional signatures of aging in single cell types rather than whole tissues. Since age-associated decreases in gene expression could contribute to a progressive decline in cellular function, understanding the mechanisms that determine the aging transcriptome provides a potential target to extend healthy cellular lifespan. Our initial studies demonstrate an example of how splicing and transcription can be linked through a single complex. These processes are coordinated both temporally and spatially, but the regulatory mechanisms governing this coordination are still not fully understood. Here we have identified an interaction between spliceosomal components and the transcriptional machinery which provides an intriguing link between the coupled processes of transcription and splicing. We show that two components of the SF3B complex that forms part of the U2 small nuclear ribonucleoprotein particle (snRNP), SF3B3 and SF3B5, are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We also show that SF3B3 and SF3B5 bind to SAGA independent of RNA, and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5 mutant flies, SF3B5 does not appear to function in SAGA’s histone modifying activities, but is required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA’s transcription coactivator activity that is separate from its role in splicing. Next, we sought to characterize the contribution of proper splicing to aging using the Drosophila eye as a model system. Several studies have shown that the aging transcriptome is characterized by changes in alternative splicing. However, it is unclear whether these age-related changes in splicing also contribute to the progressive functional decline associated with aging. Since decreased expression of splicing factors is observed in aging photoreceptors, we sought to characterize the contribution of individual age-regulated splicing factors to age-associated changes in alternative splicing and visual function in the Drosophila eye. Here, we show that there are age-related changes in splicing of genes involved in visual function in photoreceptors, and that proper splicing of these genes requires the combinatorial activity of individual age-regulated splicing factors. Notably, all of the splicing factors tested were necessary for proper visual function and/or photoreceptor viability in the adult eye. These data suggest that the coordinated activity of multiple splicing factors is necessary for proper splicing in the eye and to prevent visual senescence. Altogether our studies highlight the importance of splicing factors in gene regulation through transcriptional co-activation and in regulating genes important for cell specific functions during aging

Improving The Sensitivity Of Lidars Using Few-Mode Pre-Amplified Receivers

An order of magnitude improvement in the signal-to-noise ratio (SNR) of a 1550 nm LiDAR receiver was achieved using a few-mode optical preamplifier as compared to a multi-mode avalanche photodiode (APD)

Identification and regulation of a new vertebrate cytochrome P450 subfamily, the CYP2Ps, and functional characterization of CYP2P3, a conserved arachidonic acid epoxygenase/19-hydroxylase

Three genes cloned from Fundulus heteroclitus (killifish) define a new P450 subfamily, CYP2P. Structurally, the CYP2Ps are related to fish CYP2Ns and mammalian CYP2Js. CYP2P transcripts are expressed predominantly in liver and intestine. CYP2P3 coexpressed with P450 oxidoreductase in a baculovirus system catalyzed benzphetamine- N-demethylation and arachidonic acid oxidation, forming 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid. CYP2P3 regio- and enantioselectivities with arachidonic acid were remarkably similar to human CYP2J2 and rat CYP2J3. Epoxyeicosatrienoic acids and their corresponding hydration products, the dihydroxyeicosatrienoic acids, were detected in killifish liver and intestine, indicating metabolism of arachidonic acid by killifish P450s in vivo. Levels of these products in killifish intestine were higher than those in mammalian intestine. 12- O-Tetradecanoyl phorbol 13-acetate suppressed expression of CYP2P2 and CYP2P3 in killifish intestine; fasting itself suppressed expression of CYP2P2/3 but not CYP2P1. In rat intestine fasting similarly depressed the levels of CYP2J proteins. The CYP2Ps and the CYP2Js appear to be derived from a common ancestral gene, likely a fatty acid monooxygenase

The genome of the sea urchin Strongylocentrotus purpuratus.

International audienceWe report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes