Intronic Alternative Splicing Regulators Identified by Comparative Genomics in Nematodes

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene

Examining mindfulness-based stress reduction: Perceptions from minority older adults residing in a low-income housing facility

<p>Abstract</p> <p>Background</p> <p>Mindfulness-based stress reduction (MBSR) programs are becoming increasingly common, but have not been studied in low income minority older populations. We sought to understand which parts of MBSR were most important to practicing MBSR members of this population, and to understand whether they apply their training to daily challenges.</p> <p>Methods</p> <p>We conducted three focus groups with 13 current members of an MBSR program. Participants were African American women over the age of 60 in a low-income housing residence. We tape recorded each session and subsequently used inductive content analysis to identify primary themes.</p> <p>Results and discussion</p> <p>Analysis of the focus group responses revealed three primary themes stress management, applying mindfulness, and the social support of the group meditation. The stressors they cited using MBSR with included growing older with physical pain, medical tests, financial strain, and having grandchildren with significant mental, physical, financial or legal hardships. We found that participants particularly used their MBSR training for coping with medical procedures, and managing both depression and anger.</p> <p>Conclusion</p> <p>A reflective stationary intervention delivered in-residence could be an ideal mechanism to decrease stress in low-income older adult's lives and improve their health.</p

The relationship between doses of mindfulness-based programs and depression, anxiety, stress, and mindfulness: a dose-response meta-regression of randomized controlled trials

Abstract Objectives: Research with mindfulness-based programs (MBPs) has found participating in an MBP to predict beneficial outcomes, however, there is currently mixed research regarding the most helpful dose. This review aimed to determine whether different doses related to MBPs significantly predict outcomes. Methods: Systematic literature searches of electronic databases and trial registration sites for all randomized controlled trials of MBPs identified 203 studies (N=15,971). Depression was the primary outcome at post-program and follow-up, with secondary outcomes being mindfulness, anxiety and stress. Doses examined related to session numbers, duration and length, facilitator contact and practice. Dose-response relationships were analyzed using meta-regression in R with separate analyses for inactive and active controls. Results: Initial meta-analyses found significant between-group differences favoring MBPs for all outcomes. Meta-regression results suggested significant dose-response relationships for the mindfulness outcome for doses relating to face-to-face contact (d=0.211; C.I.[0.064,0.358]), program intensity (d=0.895; C.I.[0.315,1.474]) and actual program use (d=0.013; C.I.[0.001,0.024]). The majority of results for psychological outcomes, including depression, were not significant. Conclusions: This meta-regression examines dose-response relationships for different types and doses relating to MBPs. Considered together, MBPs appeared helpful compared to controls, supporting previous research. Based on meta-regression results, there was no evidence that larger doses are more helpful than smaller doses for predicting psychological outcomes; a finding consistent with some previous research particularly with non-clinical populations. Additionally, greater contact, intensity and actual use of MBPs predicting increased mindfulness corresponds with previous research and theory. Potential limitations and recommendations for future research are explored

Obesity, metabolic factors and risk of different histological types of lung cancer: A Mendelian randomization study.

BACKGROUND: Assessing the relationship between lung cancer and metabolic conditions is challenging because of the confounding effect of tobacco. Mendelian randomization (MR), or the use of genetic instrumental variables to assess causality, may help to identify the metabolic drivers of lung cancer. METHODS AND FINDINGS: We identified genetic instruments for potential metabolic risk factors and evaluated these in relation to risk using 29,266 lung cancer cases (including 11,273 adenocarcinomas, 7,426 squamous cell and 2,664 small cell cases) and 56,450 controls. The MR risk analysis suggested a causal effect of body mass index (BMI) on lung cancer risk for two of the three major histological subtypes, with evidence of a risk increase for squamous cell carcinoma (odds ratio (OR) [95% confidence interval (CI)] = 1.20 [1.01-1.43] and for small cell lung cancer (OR [95%CI] = 1.52 [1.15-2.00]) for each standard deviation (SD) increase in BMI [4.6 kg/m2]), but not for adenocarcinoma (OR [95%CI] = 0.93 [0.79-1.08]) (Pheterogeneity = 4.3x10-3). Additional analysis using a genetic instrument for BMI showed that each SD increase in BMI increased cigarette consumption by 1.27 cigarettes per day (P = 2.1x10-3), providing novel evidence that a genetic susceptibility to obesity influences smoking patterns. There was also evidence that low-density lipoprotein cholesterol was inversely associated with lung cancer overall risk (OR [95%CI] = 0.90 [0.84-0.97] per SD of 38 mg/dl), while fasting insulin was positively associated (OR [95%CI] = 1.63 [1.25-2.13] per SD of 44.4 pmol/l). Sensitivity analyses including a weighted-median approach and MR-Egger test did not detect other pleiotropic effects biasing the main results. CONCLUSIONS: Our results are consistent with a causal role of fasting insulin and low-density lipoprotein cholesterol in lung cancer etiology, as well as for BMI in squamous cell and small cell carcinoma. The latter relation may be mediated by a previously unrecognized effect of obesity on smoking behavior

Images from the Intronerator Genome Browser Showing Alternatively Spliced Genes

<p>Gene isoforms predicted by the Wormbase Consortium are shown in blue, and WABA homology alignments for C. briggsae to this region of the C. elegans genome are shown in purple. Dark purple indicates a region of WABA high homology, light purple corresponds to low homology, and white indicates no homology between species. Regions of alternatively spliced genes: (A) W01F3.1, (B) ZC477.9, (C) ZK637.8, (D) H24G06.1, and (E) C11D2.6 are shown.</p

Conserved Intronic Elements of <i>unc-52</i> Contain Putative Regulators of Alternative Splicing

<div><p>(A) C. elegans and C. briggsae sequence alignment is shown for the alternatively spliced portion of <i>unc-52</i>. Not all spliced isoforms are predicted by Wormbase software (blue); see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020086#pcbi-0020086-g004" target="_blank">Figure 4</a>A for observed alternative splicing patterns<i>.</i> PhastCons sequence alignment is shown with WABA-designated conservation in bold. Upper line of sequence is <i>C. elegans; C. briggsae</i> is below. High-scoring conserved motifs identified in our pentamer/hexamer analysis of conserved intronic elements flanking alternatively spliced exons, GCATG, TCTATC, CTATCC, CTATC, and TGCAC are underlined.</p><p>(B) Diagram of alternative splicing reporter constructs for testing putative <i>cis</i>-regulatory splicing motifs. Part of exon 15 through part of exon 19 of <i>unc-52</i> was cloned into a GFP/lacZ fusion vector with an <i>unc-54</i> promoter and nuclear localization sequence suitable for expression in <i>C. elegans.</i> Site-directed mutagenesis of the wild-type substrate was performed in order to test putative <i>cis</i>-splicing regulatory elements<i>.</i> A table of the splicing reporter constructs and their alterations is shown. Asterisks denote highly conserved intronic nucleotides deleted by site-directed mutagenesis. To maintain the intron length, yet remove motifs in question, a reporter was also made in which native sequence was replaced with the reverse complement sequence (shown in bold) and a HindIII site (italics) for diagnostic purposes.</p></div

An Intronic Element Regulates <i>let-2</i> Alternative Splicing

<p>The top of this figure shows the alignment of C. elegans and C. briggsae sequences for the alternatively spliced region of <i>let-2</i>. Blue tracks indicate the splicing for the embryonic (top) and adult (bottom) isoforms. The purple track indicates homology between the C. briggsae and C. elegans genomes as determined by WABA. Dark purple tracks indicate regions of strong homology (>70%). The sequence of the first conserved element of intron 10 is shown. The box indicates the part deleted and replaced with the sequence GAA in the del1.2 splicing reporter construct. The lower left part of the figure shows the results of ³²P RT-PCR reactions with primers specific for the splicing reporter. Products for exon 9- or exon 10-containing messages are indicated. In embryos, only usage of exon 9 is detected for either reporter. At the L4 stage, 34% of the wild-type reporter messages contain exon 10 while del1.2 mutant messages contain only a trace amount of this exon.</p

HRP-2, the Caenorhabditis elegans Homolog of Mammalian Heterogeneous Nuclear Ribonucleoproteins Q and R, Is an Alternative Splicing Factor That Binds to UCUAUC Splicing Regulatory Elements*

Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence