The Planet, 2007, Winter

https://cedar.wwu.edu/planet/1047/thumbnail.jp

The Planet, 2006, Fall

https://cedar.wwu.edu/planet/1046/thumbnail.jp

Molecular Cell Short Article trans-Splicing to Spliceosomal U2 snRNA Suggests Disruption of Branch Site-U2 Pairing during Pre-mRNA Splicing

SUMMARY Pairing between U2 snRNA and the branch site of spliceosomal introns is essential for spliceosome assembly and is thought to be required for the first catalytic step of splicing. We have identified an RNA comprising the 5 0 end of U2 snRNA and the 3 0 exon of the ACT1-CUP1 reporter gene, resulting from a trans-splicing reaction in which a 5 0 splice site-like sequence in the universally conserved branch site-binding region of U2 is used in trans as a 5 0 splice site for both steps of splicing in vivo. Formation of this product occurs in functional spliceosomes assembled on reporter genes whose 5 0 splice sites are predicted to bind poorly at the spliceosome catalytic center. Multiple spatially disparate splice sites in U2 can be used, calling into question both the fate of its pairing to the branch site and the details of its role in splicing catalysis

The Planet, 2007, Spring

https://cedar.wwu.edu/planet/1048/thumbnail.jp

Endogenous U2.U5.U6 snRNA complexes in S. pombe are intron lariat spliceosomes

Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2.U5.U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing

Modeling views in the layered view model for XML using UML

In data engineering, view formalisms are used to provide flexibility to users and user applications by allowing them to extract and elaborate data from the stored data sources. Conversely, since the introduction of Extensible Markup Language (XML), it is fast emerging as the dominant standard for storing, describing, and interchanging data among various web and heterogeneous data sources. In combination with XML Schema, XML provides rich facilities for defining and constraining user-defined data semantics and properties, a feature that is unique to XML. In this context, it is interesting to investigate traditional database features, such as view models and view design techniques for XML. However, traditional view formalisms are strongly coupled to the data language and its syntax, thus it proves to be a difficult task to support views in the case of semi-structured data models. Therefore, in this paper we propose a Layered View Model (LVM) for XML with conceptual and schemata extensions. Here our work is three-fold; first we propose an approach to separate the implementation and conceptual aspects of the views that provides a clear separation of concerns, thus, allowing analysis and design of views to be separated from their implementation. Secondly, we define representations to express and construct these views at the conceptual level. Thirdly, we define a view transformation methodology for XML views in the LVM, which carries out automated transformation to a view schema and a view query expression in an appropriate query language. Also, to validate and apply the LVM concepts, methods and transformations developed, we propose a view-driven application development framework with the flexibility to develop web and database applications for XML, at varying levels of abstraction

Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post- translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B’. Results also show that unstructured post- ranslationally modified C-terminal tails are responsible for the dynamics of Sm-B/B’ and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.This work was funded by: BBSRC (OVM), BBSRC and EPSRC (HH and NM), EU Prospects (HH), European Science Foundation (NM), the Royal Society (CVR), and fellowship from JSPS and HFSP (YM and DAPK respectively)

Designing websites with eXtensible web (xWeb) methodology

Today, eXtensible Markup Language (XML) is fast emerging as the dominant standard for storing, describing, representing and interchanging data among various enterprises systems and databases in the context of complex web enterprises information systems (EIS). Conversely, for web EIS (such as e-commerce and portals) to be successful, it is important to apply a high level, model driven solutions and meta-data vocabularies to design and implementation techniques that are capable of handling heterogonous schemas and documents. For this, we need a methodology that provides a higher level of abstraction of the domain in question with rigorously defined standards that are to be more widely understood by all stakeholders of the system. To-date, UML has proven itself as the language of choice for modeling EIS using OO techniques. With the introduction of XML Schema, which provides rich facilities for constraining and defining enterprise XML content, the combination of UML and XML technologies provide a good platform (and the flexibility) for modeling, designing and representing complex enterprise contents for building successful EIS. In this paper, we show how a layered view model coupled with a proven user interface analysis framework (WUiAM) is utilized in providing architectural construct and abstract website model (called eXtensible Web, xWeb), to model, design and implement simple, user-centred, collaborative websites at varying levels of abstraction. The uniqueness xWeb is that the model data (web user interface definitions, website data descriptions and constraints) and the web content are captured and represented at the conceptual level using views (one model) and can be deployed (multiple platform specific models) using one or more implementation models

An SF1 affinity model to identify branch point sequences in human introns

Splicing factor 1 (SF1) binds to the branch point sequence (BPS) of mammalian introns and is believed to be important for the splicing of some, but not all, introns. To help identify BPSs, particularly those that depend on SF1, we generated a BPS profile model in which SF1 binding affinity data, validated by branch point mapping, were iteratively incorporated into computational models. We searched a data set of 117 499 human introns for best matches to the SF1 Affinity Model above a threshold, and counted the number of matches at each intronic position. After subtracting a background value, we found that 87.9% of remaining high-scoring matches identified were located in a region upstream of 3′-splice sites where BPSs are typically found. Since U2AF65 recognizes the polypyrimidine tract (PPT) and forms a cooperative RNA complex with SF1, we combined the SF1 model with a PPT model computed from high affinity binding sequences for U2AF65. The combined model, together with binding site location constraints, accurately identified introns bound by SF1 that are candidates for SF1-dependent splicing