Diego A. R. Zorio

Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans.

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3′ splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed ‘AG-dependent introns’, the molecule responsible for AG recognition has never been identified. A key player in 3′ splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabiditis elegans introns have a highly conserved U4CAG/R at their 3′ splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF (refs 10, 12). Here we show that both U2AF subunits crosslink to the 3′ splice site. Our results suggest that the U2AF65–U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.

RNA polymerase II carboxy-terminal domain phosphorylation is required for cotranscriptional pre-mRNA splicing and 3'-end formation.

ABSTRACT We investigated the role of RNA polymerase II (pol II) carboxy-terminal domain (CTD) phosphorylation in pre-mRNA processing coupled and uncoupled from transcription in Xenopus oocytes. Inhibition of CTD phosphorylation by the kinase inhibitors 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole and H8 blocked transcription-coupled splicing and poly(A) site cleavage. These experiments suggest that pol II CTD phosphorylation is required for efficient pre-mRNA splicing and 3′-end formation in vivo. In contrast, processing of injected pre-mRNA was unaffected by either kinase inhibitors or α-amanitin-induced depletion of pol II. pol II therefore does not appear to participate directly in posttranscriptional processing, at least in frog oocytes. Together these experiments show that the influence of the phosphorylated CTD on pre-mRNA splicing and 3′-end processing is mediated by transcriptional coupling.

Transcription elongation: The ‘Foggy’ is lifting…

Abstract Transcription can be controlled by regulating either the initiation or elongation of RNA chains. Recent studies highlight the importance of an elongation regulator, Spt5, in controlling gene expression in yeast, fruit fly and zebrafish; Spt5 may provide a link between transcriptional elongation and cell fate.

Fine-tuning alkyne cycloadditions: Insights into photochemistry responsible for the double-strand DNA cleavage via structural perturbations in diaryl alkyne conjugates

Summary Hybrid molecules combining photoactivated aryl acetylenes and a dicationic lysine moiety cause the most efficient double-strand (ds) DNA cleavage known to date for a small molecule. In order to test the connection between the alkylating ability and the DNA-damaging properties of these compounds, we investigated the photoreactivity of three isomeric aryl–tetrafluoropyridinyl (TFP) alkynes with amide substituents in different positions (o-, m-, and p-) toward a model π-system. Reactions with 1,4-cyclohexadiene (1,4-CHD) were used to probe the alkylating properties of the triplet excited states in these three isomers whilst Stern–Volmer quenching experiments were used to investigate the kinetics of photoinduced electron transfer (PET). The three analogous isomeric lysine conjugates cleaved DNA with different efficiencies (34, 15, and 0% of ds DNA cleavage for p-, m-, and o-substituted lysine conjugates, respectively) consistent with the alkylating ability of the respective acetamides. The significant protecting effect of the hydroxyl radical and singlet oxygen scavengers to DNA cleavage was shown only with m-lysine conjugate. All three isomeric lysine conjugates inhibited human melanoma cell growth under photoactivation: The p-conjugate had the lowest CC50 (50% cell cytotoxicity) value of 1.49 × 10−7 M.

In Vivo Analysis of Troponin C Knock-In (A8V) Mice: Evidence that TNNC1 Is a Hypertrophic Cardiomyopathy Susceptibility Gene

Background— Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results— The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions— The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.Background—Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results—The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions—The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.

Early growth response 1 (EGR1): a gene with as many names as biological functions.

Although most researchers in biology tend to focus on very specific issues and questions about their preferred gene or pathway, sometimes we face situations in which nature presents us with a remarkable example of a gene with multiple functions. Since the discovery of the Early Growth Response 1 (EGR1) gene in the mid eighties, several independent groups attributed its activation as an immediate early response gene to extracellular stimuli such as environmental cues, growth factors, irradiation and small molecules. Even twenty-plus years after its initial cloning and characterization, EGR1 continues to attract considerable attention among biological circles. Recently, work by Wagers and colleagues reported yet another novel and rather promising function for EGR1. They demonstrated that EGR1 is involved in regulating homeostasis of hematopoietic stem cells (HSC) by coordinating proliferation and migration. Importantly, this finding allows a conduit by which manipulation of this pathway will elucidate further understanding of the biology driving HSC mobilization and transplantation in a clinical setting.

Can the Status of the Breast and Ovarian Cancer Susceptibility Gene 1 Product (BRCA1) Predict Response to Taxane-Based Cancer Therapy?

Taxanes (paclitaxel and docetaxel) are currently used to treat ovarian, breast, lung, and head and neck cancers. Despite its clinical success taxane-based treatment could be significantly improved by identifying those patients whose tumors are more likely to present a clinical response. In this mini-review we discuss the accumulating evidence indicating that the breast and ovarian cancer susceptibility gene product BRCA1 mediates cellular response to taxanes. We review data from in vitro, animal, and clinical studies, and discuss them in context of response to therapy. We argue that levels of BRCA1 in tumors may provide a predictive marker for the response to treatment with taxanes. In addition, the study of the role of BRCA1 in the mechanism of action of taxanes might reveal alternative approaches to avoid resistance.

Human Mesenchymal Stem Cells Are Resistant to Paclitaxel by Adopting a Non-Proliferative Fibroblastic State

Human mesenchymal stem cell (hMSC) resistance to the apoptotic effects of chemotherapeutic drugs has been of major interest, as these cells can confer this resistance to tumor microenvironments. However, the effects of internalized chemotherapeutics upon hMSCs remain largely unexplored. In this study, cellular viability and proliferation assays, combined with different biochemical approaches, were used to investigate the effects of Paclitaxel exposure upon hMSCs. Our results indicate that hMSCs are highly resistant to the cytotoxic effects of Paclitaxel treatment, even though there was no detectable expression of the efflux pump P-glycoprotein, the usual means by which a cell resists Paclitaxel treatment. Moreover, Paclitaxel treatment induces hMSCs to adopt a non-proliferative fibroblastic state, as evidenced by changes to morphology, cellular markers, and a reduction in differentiation potential that is not directly coupled to the cytoskeletal effects of Paclitaxel. Taken together, our results show that Paclitaxel treatment does not induce apoptosis in hMSCs, but does induce quiescence and phenotypic changes.

Cellular Distribution of the Fragile X Mental Retardation Protein in the Mouse Brain

The fragile X mental retardation protein (FMRP) plays an important role in normal brain development. Absence of FMRP results in abnormal neuronal morphologies in a selected manner throughout the brain, leading to intellectual deficits and sensory dysfunction in the fragile X syndrome (FXS). Despite FMRP importance for proper brain function, its overall expression pattern in the mammalian brain at the resolution of individual neuronal cell groups is not known. In this study we used FMR1 knockout and isogenic wildtype mice to systematically map the distribution of FMRP expression in the entire mouse brain. Using immunocytochemistry and cellular quantification analyses, we identified a large number of prominent cell groups expressing high levels of FMRP at the subcortical levels, in particular sensory and motor neurons in the brainstem and thalamus. In contrast, many cell groups in the midbrain and hypothalamus exhibit low FMRP levels. More important, we describe differential patterns of FMRP distribution in both cortical and subcortical brain regions. Almost all major brain areas contain high and low levels of FMRP cell groups adjacent to each other or between layers of the same cortical areas. These differential patterns indicate that FMRP expression appears to be specific to individual neuronal cell groups instead of being associated with all neurons in distinct brain regions, as previously considered. Taken together, these findings support the notion of FMRP differential neuronal regulation and strongly implicate the contribution of fundamental sensory and motor processing at subcortical levels to FXS pathology. J. Comp. Neurol. 525:818–849, 2017.

Optimizing Protonation States for Selective Double-Strand DNA Photocleavage in Hypoxic Tumors: pH-Gated Transitions of Lysine Dipeptides

We report pH-switching properties of the new family of dipeptide-acetylene conjugates where pH-gated light-activated double-strand (ds) DNA cleavage is controlled by variations in electronic and geometric parameters. The conjugates have higher activities at the slightly acidic pH values that separate normal and cancerous tissue (pH < 7). This favorable pH dependence originates from several elements of structural design. Basicities of the two amines determine the threshold pH range where the changes in binding and reactivity are observed, whereas the distance between the two amino groups and the hydrophobic aryl alkyne moiety can further modulate DNA binding. The changes of the protonation state from a neutral molecule to a dication results in dramatically increased efficiency of ds DNA photocleavage, the most therapeutically valuable type of DNA cleavage.