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Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Restless Legs Syndrome (RLS) is characterized by an urge to move the legs, usually accompanied by uncomfortable sensations. RLS symptoms generally happen at night and can be relieved by movements. Genetic studies have linked polymorphisms in BTBD9 to a higher risk of RLS. Knockout of BTBD9 homolog in mice (Btbd9) and fly results in RLS-like phenotypes. A dysfunctional dopaminergic system is associated with RLS. However, the function of BTBD9 in the dopaminergic system and RLS is not clear. Here, we made use of the simple Caenorhabditis elegans nervous system. Loss of hpo-9, the worm homolog of BTBD9, resulted in hyperactive egg-laying behavior. Analysis of genetic interactions between hpo-9 and genes for dopamine receptors (dop-1, dop-3) indicated that hpo-9 and dop-1 worked similarly. Reporter assays of dop-1 and dop-3 revealed that hpo-9 knockout led to a significant increase of DOP-3 expression. This appears to be evolutionarily conserved in mice with an increased D(2) receptor (D(2)R) mRNA in the striatum of the Btbd9 knockout mice. Furthermore, the striatal D(2)R protein was significantly decreased and the dynamin I was increased. Overall, activities of DA neurons in the substantia nigra were not altered, but the peripheral D(1)R pathway was potentiated in the Btbd9 knockout mice. Finally, we generated and characterized dopamine neuron-specific Btbd9 knockout mice and detected active-phase sleepiness, suggesting that dopamine neuron-specific loss of Btbd9 is sufficient to disturb the sleep. Our results suggest that increased activities in the D(1)R pathway, decreased activities in the D(2)R pathway, or both may contribute to RLS.

The spinal cord contains a highly collateralized network of descending dopamine (DA) fibers that stem from the dorso-posterior hypothalamic A11 region in the brain, however, the modulatory actions of DA have generally only been assessed in lumbar segments L2-L5. In contrast to these exclusively sensorimotor segments, spinal cords segments T1-L2 and, in mouse, L6-S2, additionally contain the intermediolateral (IML) nucleus, the origin of autonomic nervous system (ANS). Here, we tested if the different spinal circuits in sensorimotor and IML-containing segments react differently to the modulation of the monosynaptic reflex (MSR) by DA. Bath-application of DA (1 μM) led to a decrease of MSR amplitude in L3-L5 segments; however, in IML-containing segments (T10-L2, and S1/2) the MSR response was facilitated. We did not observe any difference in the response between thoracic (sympathetic) and lumbosacral (parasympathetic) segments. Application of the D2-receptor agonists bromocriptine or quinpirole mimicked the effects of DA, while blocking D2 receptor pathways with raclopride or application with the D1-receptor agonist SKF 38393 led to an increase of the MSR in L3-L5 segments and a decrease of the MSR in IML-containing segments. In contrast, in the presence of the gap-junction blockers, carbenoloxone and quinine, DA modulatory actions in IML-containing segments were similar to those of sensorimotor L3-L5 segments. We suggest that DA modulates MSR amplitudes in the spinal cord in a segment-specific manner, and that the differential outcome observed in ANS segments may be a result of gap junctions in the IML.

Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae. Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q6 (ubiquinone or CoQ6) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism.

Despite its relatively streamlined genome, there are important examples of regulated RNA splicing in Saccharomyces cerevisiae, such as splicing of meiotic transcripts. Like other eukaryotes, S. cerevisiae undergoes a dramatic reprogramming of gene expression during meiosis, including regulated splicing of a number of crucial meiosis-specific RNAs. Splicing of a subset of these is dependent upon the splicing activator Mer1. Here we show a crucial role for the chromatin remodeler Swi/Snf in regulation of splicing of meiotic genes and find that the complex affects meiotic splicing in two ways. First, we show that Swi/Snf regulates nutrient-dependent downregulation of ribosomal protein encoding RNAs, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs (the ribosomal protein genes) to Mer1-regulated transcripts. We also demonstrate that Mer1 expression is dependent on Snf2, its acetylation state and histone H3 lysine 9 acetylation at the MER1 locus. Hence, Snf2 exerts systems level control of meiotic gene expression through two temporally distinct mechanisms, demonstrating that it is a key regulator of meiotic splicing in S. cerevisiae. We also reveal an evolutionarily conserved mechanism whereby the cell redirects its energy from maintaining its translational capacity to the process of meiosis.

BackgroundAs higher education institutions strive to effectively support an increasingly diverse student body, they will be called upon to provide their faculty with tools to teach more inclusively, especially in science, technology, engineering, and mathematics (STEM) classrooms where recruitment and retention of students from underrepresented and disadvantaged groups present long-standing challenges. Pedagogical training approaches to creating inclusive classrooms involve interventions that raise awareness of student and instructor social identities and explore barriers to learning, such as implicit bias, microaggressions, stereotype threat, and fixed mindset. Such efforts should focus on embracing diversity as an asset leveraged to benefit all students in their learning. In this paper, we describe the impact of multiday, off-campus immersion workshops designed to impart faculty with these tools. Based on analysis of workshop participant data, we report the resulting changes in faculty knowledge of factors affecting classroom climate and student success in STEM, attitudes about students, and motivation to adopt new teaching practices aimed at fostering equitable and culturally responsive learning environments.ResultsKey findings indicate that attendees (1) increased their knowledge of social identities and the barriers to learning in STEM classrooms, particularly those faced by students from underrepresented groups in STEM or socioeconomically challenged backgrounds; (2) changed their attitudes about students’ abilities as science majors, shifting away from a fixed-mindset perspective in which characteristics, such as intelligence, are perceived as innate and unalterable; and (3) modified their teaching approaches to promote inclusivity and cultural responsiveness.ConclusionFaculty members, who are linchpins in the evolution of college classrooms into settings that provide students with equitable opportunities to succeed academically in STEM, can benefit from participating in immersion workshops structured to support their awareness of issues affecting classroom culture related to race/ethnicity, LGBTQ status, religious affiliation, ability, socioeconomic status, and other social identities that contribute to disparities in STEM achievement and persistence.

The transcription factor Gcr1 controls expression of over 75% of the genes in actively growing yeast. Yet, despite its widespread effects, regulation of Gcr1 itself remains poorly understood. Here we show that posttranscriptional Gcr1 regulation is nutrient-dependent. Moreover, GCR1 RNA contains a long, highly conserved intron, which allows the cell to generate multiple RNA and protein isoforms whose levels change upon glucose depletion. Intriguingly, an isoform generated by intron retention is exported from the nucleus, and its translation is initiated from a conserved, intronic translation start site. Expression of gene products from both the spliced and unspliced RNAs is essential, as cells expressing only one isoform cannot adjust their metabolic program in response to environmental changes. Finally, we show that the Gcr1 proteins form dimers, providing an elegant mechanism by which this one gene, through its regulation, can perform the repertoire of transcriptional activities necessary for fine-tuned environmental response.

Pre-mRNA splicing is vital for the proper function and regulation of eukaryotic gene expression. Saccharomyces cerevisiae has been used as a model organism for studies of RNA splicing because of the striking conservation of the spliceosome components and its catalytic activity. Nonetheless, there are relatively few annotated alternative splice forms, particularly when compared to higher eukaryotes. Here, we describe a method to combine large scale RNA sequencing data to accurately discover novel splice isoforms in Saccharomyces cerevisiae. Using our method, we find extensive evidence for novel splicing of annotated intron-containing genes as well as genes without previously annotated introns and splicing of transcripts that are antisense to annotated genes. By incorporating several mutant strains at varied temperatures, we find conditions which lead to differences in alternative splice form usage. Despite this, every class and category of alternative splicing we find in our datasets is found, often at lower frequency, in wildtype cells under normal growth conditions. Together, these findings show that there is widespread splicing in Saccharomyces cerevisiae, thus expanding our view of the regulatory potential of RNA splicing in yeast.Author SummaryPre-mRNA splicing is a fundamental step in eukaryotic gene expression. Saccharomyces cerevisiae, also known as brewer’s yeast, is a model organism for the study of pre-mRNA splicing in eukaryotes. Through the process of pre-mRNA splicing, a single gene is capable of encoding multiple mature mRNA products, but it is often difficult to identify the splice events that lead to these mRNA products. Here, we describe a method to accurately discover novel splice events in Saccharomyces cerevisiae and find evidence for extensive splicing in Saccharomyces. By utilizing a variety of strains and growth conditions, we are able to characterize many splice forms and correlate cellular conditions with prevalence of novel splice events.

Productive splicing of human precursor messenger RNAs (pre-mRNAs) requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5'SS usage, while the deposition of the EJC core directly masks reconstituted 3'SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full-length mRNAs.

SUMMARY In the eukaryotic cell, spliceosomes assemble onto pre-mRNA cotranscriptionally. Spliceosome assembly takes place in the context of the chromatin environment, suggesting that the state of the chromatin may affect splicing. The molecular details and mechanisms through which chromatin affects splicing, however, are still unclear. Here, we show a role for the histone methyltransferase Set2 and its histone modification, H3K36 methylation, in pre-mRNA splicing through high-throughput sequencing. Moreover, the effect of H3K36 methylation on pre-mRNA splicing is mediated through the chromodomain protein Eaf3. We find that Eaf3 is recruited to intron-containing genes and that Eaf3 interacts with the splicing factor Prp45. Eaf3 acts with Prp45 and Prp19 after formation of the precatalytic B complex around the time of splicing activation, thus revealing the step in splicing that is regulated by H3K36 methylation. These studies support a model whereby H3K36 facilitates recruitment of an “adapter protein” to support efficient, constitutive splicing., Graphical Abstract, In Brief Leung et al. demonstrate that H3K36 trimethylation facilitates efficient pre-mRNA splicing through the association of chromodomain protein Eaf3. Eaf3 binds to methylated H3K36 at intron-containing genes to stabilize association of the splicing factor Prp45 and regulate proper cotranscriptional spliceosome assembly.

The most common lesion in DNA occurring due to clinical treatment with Temozolomide or cellular exposures to other methylating agents is 7-methylguanine (N7-Me-dG). It can undergo a secondary reaction to form N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG). MeFapy-dG undergoes epimerization in DNA to produce either α or β deoxyribose anomers. Additionally, conformational rotation around the formyl bond, C5–N(5) bond, and glycosidic bond may occur. To characterize and quantitate the mixture of these isomers in DNA, a (13)C-MeFapy-dG lesion, in which the CH(3) group of the MeFapy-dG was isotopically labeled, was incorporated into the trimer 5′-TXT-3′ and the dodecamer 5′-CATXATGACGCT-3′ (X = (13)C-MeFapy-dG). NMR spectroscopy of both the trimer and dodecamer revealed that the MeFapy-dG lesion exists in single strand DNA as ten configurationally and conformationally discrete species, eight of which may be unequivocally assigned. In the duplex dodecamer, the MeFapy-dG lesion exists as six configurationally and conformationally discrete species. Analyses of NMR data in the single strand trimer confirm that for each deoxyribose anomer, atropisomerism occurs around the C5–N(5) bond to produce R(a) and S(a) atropisomers. Each atropisomer exhibits geometrical isomerism about the formyl bond yielding E and Z conformations. (1)H NMR experiments allow the relative abundances of the species to be determined. For the single strand trimer, the α and β anomers exist in a 3:7 ratio, favoring the β anomer. For the β anomer, with respect to the C5–N(5) bond, the R(a) and S(a) atropisomers are equally populated. However, the Z geometrical isomer of the formyl moiety is preferred. For the α anomer, the E–S(a) isomer is present at 12%, whereas all other isomers are present at 5–7%. DNA processing enzymes may differentially recognize different isomers of the MeFapy-dG lesion. Moreover, DNA sequence-specific differences in the populations of configurational and conformational species may modulate biological responses to the MeFapy-dG lesion.

The United States first sent humans into space during six flights of Project Mercury from May 1961 to May 1963. These flights were brief, with durations ranging from about 15 min to just over 34 h. A primary purpose of the project was to determine if humans could perform meaningful tasks while in space. This was supported by a series of biomedical measurements on each astronaut before, during (when feasible), and after flight to document the effects of exposure to the spaceflight environment. While almost all of the data presented here have been published in technical reports, this is the first integrated summary of the main results. One unexpected finding emerges: the major physiological changes associated with these short-term spaceflights are correlated more strongly with time spent by the astronaut in a spacesuit than with time spent in space per se. Thus, exposure to the direct stressors of short-duration (up to 34 h) spaceflight was not the dominant factor influencing human health and performance. This is relevant to current spaceflight programs and especially to upcoming commercial flights in which time spent in space (as on a suborbital flight) will be minor compared to the time spent in associated preparation, ascent, and return.

N6-(2-Deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) arises from N7-methylation of deoxyguanosine followed by imidazole ring opening. The lesion has been reported to persist in animal tissues. Previous in vitro replication bypass investigations of the MeFapy-dG adduct revealed predominant insertion of C opposite the lesion, dependent on the identity of the DNA polymerase (Pol) and the local sequence context. Here we report crystal structures of ternary Pol·DNA·dNTP complexes between MeFapy-dG-adducted DNA template:primer duplexes and the Y-family polymerases human Pol η and P2 Pol IV (Dpo4) from Sulfolobus solfataricus. The structures of the hPol η and Dpo4 complexes at the insertion and extension stages, respectively, are representative of error-free replication, with MeFapy-dG in the anti conformation and forming Watson–Crick pairs with dCTP or dC.

Splicing occurs co-transcriptionally, but relative rates of splicing and transcription that might reveal mechanisms of their coordinated control have remained mysterious. Now, Carrillo Oesterreich et al. show that the fastest introns are gone nearly as soon as the 3′ splice site is transcribed and that introns have distinct splicing kinetics with respect to polymerase progression along the gene.

Introduction: Short Message Service (SMS) appointment reminders may provide a wide-reaching, low cost approach to reducing operational inefficiencies and improving access to care. Previous studies indicate this modality may improve attendance rates, yet there is a need for large-scale, pragmatic studies that include unintended consequences and operational costs.Methods: This pragmatic investigation was a before-after analysis that compared visit attendance outcomes among patients who opted into SMS appointment reminders with outcomes among those who declined over an 18-month evaluation period from March 25, 2013, to September 30, 2014. Eligibility in our integrated safety net health care system included age greater than 17, English or Spanish as a primary language, and a cell phone number in our scheduling system.Results: 47,390 patients were invited by SMS to participate, of which 20,724 (43.7 percent) responded with 18,138 opting in (81.5 percent of respondents). Participants received SMS reminders for 77,783 scheduled visits; comparison group patients (N=72,757) were scheduled for 573,079 visits during the evaluation period. Intervention and comparison groups had, respectively, attendance rates of 72.8 percent versus 66.1 percent (p

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio–temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5′ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP (small nuclear RNA [snRNA] plus associated proteins), as H2A.Z shows extensive genetic interactions with U2 snRNP-associated proteins, and RNA sequencing (RNA-seq) reveals that introns with nonconsensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z's role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled, and unspliced RNAs are released. Together, these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

The spliceosome catalyzes the removal of introns from pre-messenger RNA (mRNA) and subsequent pairing of exons with remarkable fidelity. Some exons are known to be skipped or included in the mature mRNA in a cell type- or context-dependent manner (cassette exons), thereby contributing to the diversification of the human proteome. Interestingly, splicing is initiated (and sometimes completed) co-transcriptionally. Here, we develop a kinetic mathematical modeling framework to investigate alternative co-transcriptional splicing (CTS) and, specifically, the control of cassette exons’ inclusion. We show that when splicing is co-transcriptional, default splice patterns of exon inclusion are more likely than when splicing is post-transcriptional, and that certain exons are more likely to be regulatable (i.e. cassette exons) than others, based on the exon–intron structure context. For such regulatable exons, transcriptional elongation rates may affect splicing outcomes. Within the CTS paradigm, we examine previously described hypotheses of co-operativity between splice sites of short introns (i.e. ‘intron definition’) or across short exons (i.e. ‘exon definition’), and find that models encoding these faithfully recapitulate observations in the fly and human genomes, respectively.

Intron sequences are removed from newly synthesized RNA and usually rapidly degraded. However, it now seems that introns have a surprising role — helping yeast cells survive when nutrients are scarce. Discovery of a physiological role for introns in yeast cells.

Recent studies have revealed a close relationship between transcription, histone modification, and RNA processing. In fact, genome-wide analyses that correlate histone marks with RNA processing signals raise the possibility that specific RNA processing factors may modulate transcription and help to "write" chromatin marks. Here we show that the nuclear cap binding complex (CBC) directs recruitment of transcription elongation factors and establishes proper histone marks during active transcription. A directed genetic screen revealed that deletion of either subunit of the CBC confers a synthetic growth defect when combined with deletion of genes encoding either Ctk2 or Bur2, a component of the Saccharomyces cerevisiae ortholog of P-TEFb. The CBC physically associates with these complexes to recruit them during transcription and mediates phosphorylation at Ser-2 of the C-terminal domain (CTD) of RNA polymerase II. To understand how these interactions influence downstream events, histone H3K36me3 was examined, and we demonstrate that CBCΔ affects proper Set2-dependent H3K36me3. Consistent with this, the CBC and Set2 have similar effects on the ability to rapidly induce and sustain activated gene expression, and these effects are distinct from other histone methyltransferases. This work provides evidence for an emerging model that RNA processing factors can modulate the recruitment of transcription factors and influence histone modification during elongation.

Alternative pre-mRNA splicing allows dramatic expansion of the eukaryotic proteome and facilitates cellular response to changes in environmental conditions. The Saccharomyces cerevisiae gene SUS1, which encodes a protein involved in mRNA export and histone H2B deubiquitination, contains two introns; non-canonical sequences in the first intron contribute to its retention, a common form of alternative splicing in plants and fungi. Here we show that the pattern of SUS1 splicing changes in response to environmental change such as temperature elevation, and the retained intron product is subject to nonsense-mediated decay. The activities of different splicing factors determine the pattern of SUS1 splicing, including intron retention and exon skipping. Unexpectedly, removal of the 3' intron is affected by splicing of the upstream intron, suggesting that cross-exon interactions influence intron removal. Production of different SUS1 isoforms is important for cellular function, as we find that the temperature sensitivity and histone H2B deubiquitination defects observed in sus1Δ cells are only partially suppressed by SUS1 cDNA, but SUS1 that is able to undergo splicing complements these phenotypes. These data illustrate a role for S. cerevisiae alternative splicing in histone modification and cellular function and reveal important mechanisms for splicing of yeast genes containing multiple introns.

INTRODUCTION Caloric restriction (CR) delays cancer growth in animals, though translation to humans is difficult. We hypothesized intermittent fasting (i.e., intermittent extreme CR), may be better tolerated and prolong survival of prostate cancer (CaP) bearing mice. METHODS We conducted a pilot study by injecting 105 male individually-housed SCID mice with LAPC-4 cells. When tumors reached 200 mm3, 15 mice/group were randomized to one of seven diets and sacrificed when tumors reached 1,500 mm3: Group 1: ad libitum 7 days/week; Group 2: fasted 1 day/week and ad libitum 6 days/week; Group 3: fasted 1 day/week and fed 6 days/week via paired feeding to maintain isocaloric conditions to Group 1; Group 4: 14% CR 7 days/week; Group 5: fasted 2 days/week and ad libitum 5 days/week; Group 6: fasted 2 day/week and fed 5 days/week via paired feeding to maintain isocaloric conditions to Group 1; Group 7: 28% CR 7 days/week. Sera from mice at sacrifice were analyzed for IGF-axis hormones. RESULTS There were no significant differences in survival among any groups. However, relative to Group 1, there were non-significant trends for improved survival for Groups 3 (HR 0.65, P = 0.26), 5 (0.60, P = 0.18), 6 (HR 0.59, P = 0.16), and 7 (P = 0.59, P = 0.17). Relative to Group 1, body weights and IGF-1 levels were significantly lower in Groups 6 and 7. CONCLUSIONS This exploratory study found non-significant trends toward improved survival with some intermittent fasting regimens, in the absence of weight loss. Larger appropriately powered studies to detect modest, but clinically important differences are necessary to confirm these findings. Prostate 70: 1037–1043, 2010. © 2010 Wiley-Liss, Inc.

The regulation of gene expression involves multiple levels of control, from those that are inheritable to those that are highly responsive to environmental changes. In this issue of Genes & Development, Dong and colleagues (pp. 1159–1173) demonstrate that the dynamically controlled immune response transcription factor NF-κB may, in fact, have a role in regulating heterochromatin and gene expression at large distances from its actual target sequences and genes.

Context: The Center for Medicare and Medicaid Innovation (CMMI) awarded Denver Health's (DH) integrated, safety net health care system $19.8 million to implement a “population health” approach into the delivery of primary care. This major practice transformation builds on the Patient Centered Medical Home (PCMH) and Wagner’s Chronic Care Model (CCM) to achieve the “Triple Aim”: improved health for populations, care to individuals, and lower per capita costs.Case description: This paper presents a case study of how DH integrated published predictive models and front-line clinical judgment to implement a clinically actionable, risk stratification of patients. This population segmentation approach was used to deploy enhanced care team staff resources and to tailor care-management services to patient need, especially for patients at high risk of avoidable hospitalization. Developing, implementing, and gaining clinical acceptance of the Health Information Technology (HIT) solution for patient risk stratification was a major grant objective.Findings: In addition to describing the Information Technology (IT) solution itself, we focus on the leadership and organizational processes that facilitated its multidisciplinary development and ongoing iterative refinement, including the following: team composition, target population definition, algorithm rule development, performance assessment, and clinical-workflow optimization. We provide examples of how dynamic business intelligence tools facilitated clinical accessibility for program design decisions by enabling real-time data views from a population perspective down to patient-specific variables.Conclusions: We conclude that population segmentation approaches that integrate clinical perspectives with predictive modeling results can better identify high opportunity patients amenable to medical home-based, enhanced care team interventions.

The synthesis of G-N(2)-(CH(2))(3)-N(2)-G trimethylene DNA interstrand cross-links (ICLs) in a 5'-CG-3' and 5'-GC-3' sequence from oligodeoxynucleotides containing N(2)-(3-aminopropyl)-2'-deoxyguanosine and 2-fluoro-O(6)-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N(2)-(3-aminopropyl)-2'-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N(2)-(3-aminopropyl)-2'-deoxyguanosine- and 2-fluoro-O(6)-(trimethylsilylethyl)inosine-containing oligodeoxynucleotides.

When messenger RNA splicing occurs co-transcriptionally, the potential for kinetic control based on transcription dynamics is widely recognized. Indeed, perturbation studies have reported that when transcription kinetics are perturbed genetically or pharmacologically splice patterns may change. However, whether kinetic control is contributing to the control of splicing within the normal range of physiological conditions remains unknown. We examined if the kinetic determinants for co-transcriptional splicing (CTS) might be reflected in the structure and expression patterns of the genome and epigenome. To identify and then quantitatively relate multiple, simultaneous CTS determinants, we constructed a scalable mathematical model of the kinetic interplay of RNA synthesis and CTS and parameterized it with diverse next generation sequencing (NGS) data. We thus found a variety of CTS determinants encoded in vertebrate genomes and epigenomes, and that these combine variously for different groups of genes such as housekeeping versus regulated genes. Together, our findings indicate that the kinetic basis of splicing is functionally and physiologically relevant, and may meaningfully inform the analysis of genomic and epigenomic data to provide insights that are missed when relying on statistical approaches alone.

Study Design. This report is composed of two studies, one cross-sectional and one prospective cohort study, that analyze the radiologic features of disc degeneration in the sand rat (Psammomys obesus). Objectives. To statistically assess progressive disc degeneration in this useful animal model in terms of a cross-sectional study and a prospective monthly evaluation of individual animals. Summary of Background Data. P. obesus is an attractive small rodent model for spontaneous age-related disc degeneration. Because disc degeneration is spontaneous, the model avoids use of chemonucleolysis or surgical injury to cause disc degeneration. Little is understood, however, about specific details of the progressive disc deterioration. Methods. This study statistically assessed 158 animals in a cross-sectional study and 22 animals in a longitudinal study, which followed individual animals to 12 months of age. Radiologic features involving irregular disc margins, disc wedging, disc narrowing, endplate calcification, subchondral sclerosis, ligament calcification, and osteophyte formation were studied. Results. Significant age-related cross-sectional changes were present for all features (P ≤ 0.005). Males showed a statistically greater incidence of wedging at 6 and 12 months, wedging at 2 and 6 months, and endplate calcification at 2 months than did females. By 6 months of age, however, endplate calcification had a higher incidence in females than in males. Prospective analysis showed that wedging, narrowing, endplate calcification, and irregular disc margins were more common at 12 months of age than at 2 months (P = 0.0001). By the age of 12 months, all lumbar sites of both males and females showed endplate calcification and the majority of animals showed narrowing and wedging. Conclusions. Radiographic signs of degeneration were evident by age 2 months; wedging, narrowing, irregular disc margins, and endplate calcification were the most common degenerative changes in older animals. These data show that the sand rat provides a reliable, useful model of spontaneous disc degeneration.

A series of novel ether-linked dimers of demethylepipodophyllotoxin are topoisomerase II poisons that exhibit higher levels of double-stranded versus single-stranded DNA cleavage than their corresponding monomers. The dimers also have higher levels of tumor cell cytotoxicity than the monomers, lending support to the two-drug model for interaction of demethylepipodophyllotoxins with human topoisomerase IIα.

Regulation of gene expression occurs at multiple levels, including the removal of non-coding sequences from protein-coding pre-messenger RNA transcripts, commonly known as RNA splicing. Correct gene expression requires high fidelity in pre-mRNA splicing, as up to 15% of mutations leading to disease result in aberrant pre-mRNA splicing. Though previously thought of as biologically distinct processes, recent studies have shown there is a complex interplay between transcription and pre-mRNA splicing, however, the regulation underlying this coordination remains unclear. Using the budding yeast Saccharomyces cerevisiae, we have identified interactions between components of the SAGA (Spt/Ada/Gcn5/Ada) transcriptional coactivator complex and the ATP-dependent spliceosomal DEAD-box protein Prp5. Prp5 plays a critical role in mediating ATP-dependent RNA remodeling in splicing, though its precise role in transcription or chromatin remodeling is unclear. Deletion of genes encoding components of the SAGA complex requ...

The synthesis of G-N2-(CH2)3-N2-G trimethylene DNA interstrand cross-links (ICLs) in a 5′-CG-3′ and 5′-GC-3′ sequence from oligodeoxynucleotides containing N2-(3-aminopropyl)-2′-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N2-(3-aminopropyl)-2′-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N2-(3-aminopropyl)-2′-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine containing oligodeoxynucleotides.

The synthesis of G-N2-(CH2)3-N2-G trimethylene DNA interstrand cross-links (ICLs) in a 5'-CG-3' and 5'-GC-3' sequence from oligodeoxynucleotides containing N2-(3-aminopropyl)-2'-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N2-(3-aminopropyl)-2'-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N2-(3-aminopropyl)-2'-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine containing oligodeoxynucleotides.

Messenger RNA splicing is a critical step in the process of eukaryotic gene expression. In splicing, the introns are removed from newly synthesized RNA by the complex molecular machinery of the spliceosome. Assembly of the spliceosome is driven by a series of interactions between the pre-mRNA, five small nuclear ribonucleoprotein particles, and a host of trans-acting, non-snRNP proteins (Will and Luhrmann 1997; Burge et al. 1999; Stevens and Abelson 1999). Extensive biochemical and genetic analyses have been carried out to identify the steps in assembly as well as the specific RNA–RNA and RNA–protein contacts that are involved. The first step of splicing is the accurate recognition of the pre-mRNA at the correct splice sites. These events are thought to be initiated when, in the absence of ATP, the U1-snRNP particle associates with the 5′ splice site via base-pairing with the 5′ end of the U1 snRNA (Mount 1982; Zhuang and Weiner 1986; Seraphin et al. 1988; Siliciano and Guthrie 1988) and association of U1-snRNP proteins with sequences around the 5′ splice site (Liu et al. 1998; Zhang and Rosbash 1999). In what has been thought to be the first energy-requiring step, U2 snRNA base-pairs with the pre-mRNA branch-point sequence (Cheng and Abelson 1987; Parker et al. 1987; Kramer 1988; Wu and Manley 1989; Zhuang and Weiner 1989; Pruzan et al. 1990; Michaud and Reed 1991; Liao et al. 1992). Once U1 and U2 are bound to the pre-mRNA, U4/U6·U5 associates with the 5′ splice site region of the pre-mRNA (Ruby and Abelson 1988; Liao et al. 1992), and the U5 snRNP is positioned such that the invariant loop 1 is aligned with exon sequences near the 5′ and 3′ splice sites (Wyatt et al. 1992; Sontheimer and Steitz 1993; Newman et al. 1995). In turn, U6 associates with the intron nucleotides at the 5′ splice site, replacing U1 interactions. Although neither U1 nor U4 appears to be involved in catalysis, it is not clear when they leave the spliceosome. The description of the interactions that take place during spliceosome assembly is based largely on analysis of splicing complexes using nondenaturing gels. This model has been extremely useful in defining the general sequence of interactions involved in the splicing reaction, but accumulating data from metazoan systems indicate that there may be more flexibility in the order of events surrounding splice-site recognition by the splicing machinery than the model predicts. Splicing of the SV40 large T-antigen intron is insensitive to removal of the 5′ end of U1 (Black and Steitz 1986), although it is inhibited by anti-U1 snRNP antibody (Fradin et al. 1984; van Santen and Spritz 1986). The requirement for U1 in 5′ splice site choice can also be bypassed by high concentrations of SR proteins or specific cis-acting sequence elements near the 5′ splice site and branch point (Crispino et al. 1994, 1996; Tarn and Steitz 1995; Crispino and Sharp 1995). These results challenge the prevailing view of spliceosome assembly that describes U1 binding as the prerequisite step for 5′ splice site recognition. Furthermore, in a series of experiments using an 11-nucleotide RNA molecule, Konarska and coworkers demonstrated that, in mammalian extracts, the U4/U6·U5 snRNP could associate with the 5′ splice site RNA oligonucleotide independently of U1 snRNP. This oligonucleotide could participate in both steps of splicing even more efficiently in the absence of U1 snRNP than when intact U1 was in the extract (Konforti and Konarska 1995). Additionally, using an in vitro cis/trans-splicing competition assay with both HeLa and nematode splicing extracts, Nilsen and coworkers demonstrated that early 5′ splice site recognition by U4/U6·U5 tri-snRNP did not require prior U2 binding (Maroney et al. 2000). These results challenge the assumption that U1 and U2 must preassemble on the pre-mRNA as a prerequisite for triple snRNP binding. Even the timing of the ATP-dependent steps in splicing appears to be more complicated than the working model of spliceosome assembly predicts. Although the first ATP-dependent step is predicted to be U2 association with the branch-point sequences, U2 and U1 both appear to associate with the pre-mRNA in an ATP-independent fashion, although U2 appears to be weakly associated in this complex (Das et al. 2000; Perriman and Ares 2000). Furthermore, U2 can bind at the consensus branch point of a minimal substrate containing the 3′ half of the pre-mRNA and lacking a 5′ splice site in an ATP-independent manner (Query et al. 1997). Hence, ATP is most likely used in a subsequent step to induce a conformational change that stabilizes the ATP-independent interaction with the branch-point sequence. These results support a model in which ATP is required to modulate relatively unstable, preformed interactions between particular snRNPs and the pre-mRNA. Although the precise order of events that leads to the U6 interaction with the 5′ splice site remains unclear, it is quite apparent that the U4 and U6 snRNAs are at the center of the dramatic structural rearrangements as the spliceosome is assembled into its catalytic form. U4 and U6 are associated via an extensive ∼20-nucleotide base-pairing. In a remarkable set of structural rearrangements, U6 associates with the 5′ splice site, and the base-paired interaction with U4 is replaced by base-pairing with U2, forming U2–U6 helices I and II and a U6 intramolecular stem loop (Hausner et al. 1990; Datta and Weiner 1991; Madhani and Guthrie 1992; Fortner et al. 1994; Brow and Vidaver 1995). The U2/U6 helix I is highly conserved and is proposed to be a component of the active site (Brow and Guthrie 1989; Fabrizio and Abelson 1990; Madhani and Guthrie 1992; Valadkhan and Manley 2000). U4 and U6 are completely unwound before the first step of splicing (Yean and Lin 1991), although genetic evidence indicates that U4 remains associated with U6 after recognition of the 5′ splice site (Li and Brow 1996). U6 specifically interacts with the 5′ splice site of the pre-mRNA via its conserved ACAGAG sequence (Sawa and Abelson 1992; Sawa and Shimura 1992; Wassarman and Steitz 1992; Lesser and Guthrie 1993; Sontheimer and Steitz 1993; Kandels-Lewis and Seraphin 1994; Wolff et al. 1994; Kim and Abelson 1996). Mutation of the ACAGAG box hampers the ability of U6 to bind to the 5′ splice site and cripples spliceosome assembly in vitro (Fabrizio and Abelson 1990). In vivo, mutations in any of these nucleotides result in temperature sensitivity or lethality (Madhani et al. 1990). Additionally, the U6 ACAGAG sequence–5′ splice site interaction is thought to be a prerequisite for U4/U6 separation (Li and Brow 1996), for subsequent U2–U6 interaction, and therefore for activation of the spliceosome. Recent studies of the U12-dependent spliceosome suggest that, at least in that system, U4atac and U6atac may still be partially base-paired (via stem II) even after U12 and U6atac association (via stem Ia). Quite interestingly, this U12/U6atac helix Ia can form even without U6atac replacement of U11 at the 5′ splice site (Frilander and Steitz 2001). To further define the interactions that occur at the 5′ splice site during spliceosome assembly and catalysis, we are using an in vitro trans-splicing system with nuclear extracts from the yeast Saccharomyces cerevisiae. In these reactions, the 5′ splice site is contained on a 20-nucleotide, biotinylated, synthetic RNA, and the 3′ acceptor containing the branch point and the 3′ splice site is prepared by in vitro transcription (Ghetti and Abelson 1995). The trans-splicing reaction occurs accurately, and has the same requirements for ATP, Mg2+, and splice-site consensus sequences as does cis-splicing. Using this approach, we are able to substitute photoreactive, cross-linkable groups within the 5′ splice site-containing RNA to identify the RNA–RNA interactions that occur during splicing. We have found and further characterized a previously identified cross-link between the branched intron pre-mRNA intermediate and U6 snRNA. Additionally, we have identified a novel interaction between U4 snRNA and the +2 position of the 5′ splice site. This interaction is ATP-dependent and does not require the 3′ acceptor or the 5′ end of U1, but it does require a wild-type 5′ splice site sequence. We have also identified a novel ATP-dependent, acceptor-independent U6 cross-link to the 5′ splice site. These results provide evidence of an early, ATP-dependent U4/U6 association with the 5′ splice site in the absence of the ATP-mediated association of U2 with the branch point, suggesting that there is remarkable flexibility in the order of events in spliceosome assembly.

Pre-mRNA splicing is a critical step in eukaryotic gene expression, which involves removal of noncoding intron sequences from pre-mRNA and ligation of the remaining exon sequences to make a mature message. Splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Since the first description of the pre-mRNA splicing reaction in the 1970s, elegant genetic and biochemical studies have revealed that the enzyme that catalyzes the reaction, the spliceosome, is an exquisitely dynamic macromolecular machine, and its RNA and protein components undergo highly ordered, tightly coordinated rearrangements in order to carry out intron recognition and splicing catalysis. Studies using the genetically tractable unicellular eukaryote budding yeast (Saccharomyces cerevisiae) have played an instrumental role in deciphering splicing mechanisms. In this chapter, we discuss how yeast genetics has been used to deepen our understanding of the mechanism of splicing and explore the potential for future mechanistic insights using S. cerevisiae as an experimental tool.

The discovery that many intron-containing genes can be cotranscriptionally spliced has led to an increased understanding of how splicing and transcription are intricately intertwined. Cotranscriptional splicing has been demonstrated in a number of different organisms and has been shown to play roles in coordinating both constitutive and alternative splicing. The nature of cotranscriptional splicing suggests that changes in transcription can dramatically affect splicing, and new evidence suggests that splicing can, in turn, influence transcription. In this chapter, we discuss the mechanisms and consequences of cotranscriptional splicing and introduce some of the tools used to measure this process.

Blood pressure increases with age, and dysfunction of the dopamine D3 receptor has been implicated in the pathogenesis of hypertension. To evaluate the role of the D3 receptor in aging-related hypertension, we assessed cardiac structure and function in differently aged (2 mo, 1 yr, 2 yr) wild type (WT) and young (2 mo) D3 receptor knockout mice (D3KO). In WT, systolic and diastolic blood pressures and rate-pressure product (RPP) significantly increased with age, while heart rate significantly decreased. Blood pressure values, heart rate and RPP of young D3KO were significantly elevated over age-matched WT, but similar to those of the 2 yr old WT. Echocardiography revealed that the functional measurements of ejection fraction and fractional shortening decreased significantly with age in WT and that they were significantly smaller in D3KO compared to young WT. Despite this functional change however, cardiac morphology remained similar between the age-matched WT and D3KO. Additional morphometric analyses confirmed an aging-related increase in left ventricle (LV) and myocyte cross-sectional areas in WT, but found no difference between age-matched young WT and D3KO. In contrast, interstitial fibrosis, which increased with age in WT, was significantly elevated in the D3KO over age-matched WT, and similar to 2 yr old WT. Western analyses of myocardial homogenates revealed significantly increased levels of pro- and mature collagen type I in young D3KO. Column zymography revealed that activities of myocardial MMP-2 and MMP-9 increased with age in WTs, but in D3KO, only MMP-9 activity was significantly increased over age-matched WTs. Our data provide evidence that the dopamine D3 receptor has a critical role in the emergence of aging-related cardiac fibrosis, remodeling, and dysfunction.

We have previously shown that myocardial infarct size in nonreperfused hearts of mice with a functional deletion of the circadian rhythm gene mPer2 (mPer2-M) was reduced by 43%. We hypothesized that acute ischemia-reperfusion injury (I/R = 30 min I/2 h R) would also be reduced in these mice and that ischemic preconditioning (IPC) (3 × 5 min cycles) before I/R, which enhances protection in wild-type (WT) hearts, would provide further protection in mPer2-M hearts. We observed a 69 and 75% decrease in infarct size in mPer2-M mouse hearts compared with WT following I/R and IPC, respectively. This was coincident with 67% less neutrophil infiltration and 57% less apoptotic cardiomyocytes. IPC in mPer2-M mice before I/R had 48% less neutrophil density and 46% less apoptosis than their WT counterparts. Macrophage density was not different between WT and mPer2-M I/R, but it was 45% higher in mPer2-M IPC mouse hearts compared with WT IPC. There were no baseline differences in cardiac mitochondrial function between WT and mPer2-M mice, but, following I/R, WT exhibited a marked decrease in maximal O2 consumption supported by complex I-mediated substrates, whereas mPer2-M did not, despite no difference in complex I content. Moreover, cardiac mitochondria from WT mice exhibited a very robust increase in ADP-stimulated O2 consumption in response to exogenously added cytochrome c, along with a high rate of reactive oxygen species production, none of which was exhibited by cardiac mitochondria from mPer2-M following I/R. Taken together, these findings suggest that mPer2 deletion preserves mitochondrial membrane structure and functional integrity in heart following I/R injury, the consequence of which is preservation of myocardial viability. Understanding the mechanisms connecting cardiac events, mitochondrial function, and mPer2 could lead to preventative and therapeutic strategies for at risk populations.

U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. Here, a high-throughput sequencing strategy of differentially expressed transcripts (HIDE-seq) mapped PCPA sites genome wide in divergent organisms. Surprisingly, whereas U1 depletion terminated most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifted PCPA downstream and elicited mRNA 3' UTR shortening and proximal 3' exon switching characteristic of activated immune and neuronal cells, stem cells, and cancer. Activated neurons' signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 overexpression. Importantly, we show that rapid and transient transcriptional upregulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest cotranscriptional PCPA counteracted by U1 association with nascent transcripts, a process we term telescripting, ensuring transcriptome integrity and regulating mRNA length.