A multispecies comparison of the metazoan 3'-processing downstream elements and the CstF-64 RNA recognition motif

BACKGROUND: The Cleavage Stimulation Factor (CstF) is a required protein complex for eukaryotic mRNA 3'-processing. CstF interacts with 3'-processing downstream elements (DSEs) through its 64-kDa subunit, CstF-64; however, the exact nature of this interaction has remained unclear. We used EST-to-genome alignments to identify and extract large sets of putative 3'-processing sites for mRNA from ten metazoan species, including Homo sapiens, Canis familiaris, Rattus norvegicus, Mus musculus, Gallus gallus, Danio rerio, Takifugu rubripes, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans. In order to further delineate the details of the mRNA-protein interaction, we obtained and multiply aligned CstF-64 protein sequences from the same species. RESULTS: We characterized the sequence content and specific positioning of putative DSEs across the range of organisms studied. Our analysis characterized the downstream element (DSE) as two distinct parts – a proximal UG-rich element and a distal U-rich element. We find that while the U-rich element is largely conserved in all of the organisms studied, the UG-rich element is not. Multiple alignment of the CstF-64 RNA recognition motif revealed that, while it is highly conserved throughout metazoans, we can identify amino acid changes that correlate with observed variation in the sequence content and positioning of the DSEs. CONCLUSION: Our analysis confirms the early reports of separate U- and UG-rich DSEs. The correlated variations in protein sequence and mRNA binding sequences provide novel insights into the interactions between the precursor mRNA and the 3'-processing machinery

C. elegans sequences that control trans-splicing and operon pre-mRNA processing

Many mRNAs in Caenorhabditis elegans are generated through a trans-splicing reaction that adds one of two classes of spliced leader RNA to an independently transcribed pre-mRNA. SL1 leaders are spliced mostly to pre-mRNAs from genes with outrons, intron-like sequences at the 5′-ends of the pre-mRNAs. In contrast, SL2 leaders are nearly exclusively trans-spliced to genes that occur downstream in polycistronic pre-mRNAs produced from operons. Operon pre-mRNA processing requires separation into individual transcripts, which is accomplished by 3′-processing of upstream genes and spliced leader trans-splicing to the downstream genes. We used a novel computational analysis, based on nonnegative matrix factorization, to identify and characterize significant differences in the cis-acting sequence elements that differentiate various types of functional site, including internal versus terminal 3′-processing sites, and SL1 versus SL2 trans-splicing sites. We describe several key elements, including the U-rich (Ur) element that couples 3′-processing with SL2 trans-splicing, and a novel outron (Ou) element that occurs upstream of SL1 trans-splicing sites. Finally, we present models of the distinct classes of trans-splicing reaction, including SL1 trans-splicing at the outron, SL2 trans-splicing in standard operons, competitive SL1-SL2 trans-splicing in operons with large intergenic separation, and SL1 trans-splicing in SL1-type operons, which have no intergenic separation

Probe-level analysis of expression microarrays characterizes isoform-specific degradation during mouse oocyte maturation.

Gene expression microarrays have provided many insights into changes in gene expression patterns between different tissue types, developmental stages, and disease states. Analyses of these data focused primarily measuring the relative abundance of transcripts of a gene, while treating most or all transcript isoforms as equivalent. Differences in the selection between transcript isoforms can, however, represent critical changes to either the protein product or the posttranscriptional regulation of the transcript. Novel analyses on existing microarray data provide fresh insights and new interpretations into transcriptome-wide changes in expression.A probe-level analysis of existing gene expression arrays revealed differences in mRNA processing, primarily affecting the 3'-untranslated region. Working with the example of microarrays drawn from a transcriptionally silent period of mouse oocyte development, probe-level analysis (implemented here as rmodel) identified genes whose transcript isoforms have differing stabilities. Comparison of micorarrays measuring cDNA generated from oligo-dT and random primers revealed further differences in the polyadenylation status of some transcripts. Additional analysis provided evidence for sequence-targeted cleavage, including putative targeting sequences, as one mechanism of degradation for several hundred transcripts in the maturing oocyte.The capability of probe-level analysis to elicit novel findings from existing expression microarray data was demonstrated. The characterization of differences in stability between transcript isoforms in maturing mouse oocytes provided some mechanistic details of degradation. Similar analysis of existing archives of expression microarray data will likely provide similar discoveries

BIOINFORMATICS APPLICATIONS NOTE Databases and ontologies PACdb: PolyA Cleavage Site and 3 ′-UTR Database

Summary: The PolyA Cleavage Site and 3 ′-UTR Database (PACdb) is a web-accessible database that catalogs putative 3 ′-processing sites and 3 ′-UTR sequences for multiple organisms. Sites have been identified primarily via expressed sequence tag-genome alignments, enabling delineation of both the specificities and heterogeneity of 3 ′-processing events

STEMS pilot trial:a pilot cluster randomised controlled trial to investigate the addition of patient direct access to physiotherapy to usual GP-led primary care for adults with musculoskeletal pain

INTRODUCTION: Around 17% of general practitioner (GP) consultations are for musculoskeletal conditions, which will rise as the population ages. Patient direct access to physiotherapy provides one solution, yet adoption in the National Health Service (NHS) has been slow. SETTING: A pilot, pragmatic, non-inferiority, cluster randomised controlled trial (RCT) in general practice and physiotherapy services in the UK. OBJECTIVES: Investigate feasibility of a main RCT. PARTICIPANTS: Adult patients registered in participating practices and consulting with a musculoskeletal problem. INTERVENTIONS: 4 general practices (clusters) randomised to provide GP-led care as usual or the addition of a patient direct access to physiotherapy pathway. OUTCOMES: Process outcomes and exploratory analyses of clinical and cost outcomes. DATA COLLECTION: Participant-level data were collected via questionnaires at identification, 2, 6 and 12 months and through medical records. BLINDING: The study statistician and research nurses were blinded to practice allocation. RESULTS: Of 2696 patients invited to complete study questionnaires, 978 participated (intervention group n=425, control arm n=553) and were analysed. Participant recruitment was completed in 6 months. Follow-up rates were 78% (6 months) and 71% (12 months). No evidence of selection bias was observed. The direct access pathway was used by 90% of patients in intervention practices needing physiotherapy. Some increase in referrals to physiotherapy occurred from one practice, although waiting times for physiotherapy did not increase (28 days before, 26 days after introduction of direct access). No safety issues were identified. Clinical and cost outcomes were similar in both groups. Exploratory estimates of between group effect (using 36-item Short Form Health Survey (SF-36) Physical Component Summary (PCS)) at 6 months was -0.28 (95% CI -1.35 to 0.79) and at 12 months 0.12 (95% CI -1.27 to 1.51). CONCLUSIONS: A full RCT is feasible and will provide trial evidence about the clinical and cost-effectiveness of patient direct access to physiotherapy. TRIAL REGISTRATION NUMBER: ISRCTN23378642

Autonomous Rock Acquisition

In order to perform efficient, robust planetary exploration with robotic systems that use manipulation during science experiments, we will be increasingly reliant on vehiclebased autonomous and semi-autonomous control modalities. Planned lander and rover missions to Mars will require manipulative capabilities for placing science equipment and acquiring geological samples. As an Earth-based operator can only communicate with the system periodically and therefore is limited to making only high level commands, we must rely on local autonomies in the system to intelligently carry out the desired actions. This paper describes an experimental system and ongoing research for developing strategies for autonomous behavior mediated by humans at a great distance. This paper is not intended to present any one aspect of our work in a highly theoretical or rigorous way; rather we give an overview of our work and communicate insights into the challenges we have faced over the past few years. 1. INTR..