Highly disordered histone H1-DNA model complexes and their condensates.

Disordered proteins play an essential role in a wide variety of biological processes, and are often posttranslationally modified. One such protein is histone H1; its highly disordered C-terminal tail (CH1) condenses internucleosomal linker DNA in chromatin in a way that is still poorly understood. Moreover, CH1 is phosphorylated in a cell cycle-dependent manner that correlates with changes in the chromatin condensation level. Here we present a model system that recapitulates key aspects of the in vivo process, and also allows a detailed structural and biophysical analysis of the stages before and after condensation. CH1 remains disordered in the DNA-bound state, despite its nanomolar affinity. Phase-separated droplets (coacervates) form, containing higher-order assemblies of CH1/DNA complexes. Phosphorylation at three serine residues, spaced along the length of the tail, has little effect on the local properties of the condensate. However, it dramatically alters higher-order structure in the coacervate and reduces partitioning to the coacervate phase. These observations show that disordered proteins can bind tightly to DNA without a disorder-to-order transition. Importantly, they also provide mechanistic insights into how higher-order structures can be exquisitely sensitive to perturbation by posttranslational modifications, thus broadening the repertoire of mechanisms that might regulate chromatin and other macromolecular assemblies

Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD. METHODOLOGY: Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. PRINCIPAL FINDINGS: Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients. CONCLUSIONS: The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies

Identifying and rectifying aberrant RNA metabolism in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a devastating and incurable disease. Despite decades of work, its cause is yet to be fully understood, and we lack effective treatments. There is therefore a great need for further research on the mechanisms which drive disease to help guide new drug development. In this thesis, I describe work spanning from the development of new techniques and software to aid the fundamental study of RNA biology, to the identification of a novel disease mechanism in ALS that should be straightforward to target therapeutically, and the development of a new precision medicine approach that could enable safer and more efficacious gene therapy for this disease. In the first chapter, I outline three short projects: 1. The development of software for the processing of fastq files with inline barcodes and UMIs; 2. A streamlined computational and experimental method for the removal of rRNA sequences from sequencing libraries, and 3. Several related protocols for the production of various RNA sequencing library types, all of which involve template-switching. In the second chapter, I describe how I helped characterise and validate a novel cryptic exon in UNC13A using a variety of wet- and dry-lab-based approaches. Furthermore, I describe how I designed and tested candidate therapeutic antisense oligonucleotides to rescue this splicing abnormality, and how I developed a protocol for detecting large numbers of cryptic exons in parallel. Finally, in the third chapter, I describe the development of software to design expression vectors which are activated by the depletion of TDP-43, potentially paving the way towards safer and more efficacious gene therapy for ALS and related diseases

Identifying ribosome heterogeneity using ribosome profiling

Recent studies have revealed multiple mechanisms that can lead to heterogeneity in ribosomal composition. This heterogeneity can lead to preferential translation of specific panels of mRNAs, and is defined in large part by the ribosomal protein (RP) content, amongst other things. However, it is currently unknown to what extent ribosomal composition is heterogeneous across tissues, which is compounded by a lack of tools available to study it. Here we present dripARF, a method for detecting differential RP incorporation into the ribosome using Ribosome Profiling (Ribo-seq) data. We combine the ‘waste’ rRNA fragment data generated in Ribo-seq with the known 3D structure of the human ribosome to predict differences in the composition of ribosomes in the material being studied. We have validated this approach using publicly available data, and have revealed a potential role for eS25/RPS25 in development. Our results indicate that ribosome heterogeneity can be detected in Ribo-seq data, providing a new method to study this phenomenon. Furthermore, with dripARF, previously published Ribo-seq data provides a wealth of new information, allowing the identification of RPs of interest in many disease and normal contexts. dripARF is available as part of the ARF R package and can be accessed through https://github.com/fallerlab/ARF

Letras, 1999-2000, nº 40-41 (número completo)

Contenido: Nota preliminar – Poesía mariana en Gonzalo de Berceo / Lía N. Uriarte Rebaudi – El judío y el diablo como “otro” en los Milagros de Nuestra Señora de Berceo / Heanon M. Wilkins – “Intellectum tibi dabo et instruam te” / Eric Naylor – Descripción, narración, argumentación y crisis de la oralidad en el Libro del Buen Amor / Sofía M. Carrizo Rueda – El poema de Alfonso XI: ¿Un eco priscilianista, sabeliano, patripasiano? / Jorge Norberto Ferro – El vino en el refranero español; concordancias / Dolly M. Lucero Ontiveros – Una propuesta de clasificación de la Literatura Sapiencial Hispánica en el siglo XIII / Alicia E. Ramadori – Fray Juan García de Castrojeriz receptor de Aristóteles / Hugo Oscar Bizzarri – La especificidad de la prosa alfonsí; el caso del Lapidario / Mirta G. Aguayo – Alfonso el Sabio y Brunetto Latini: convergencias / Gretchem Arnstedt de Magneres – La figura autoral de Alfonso X en las Cantigas de Santa María / Santiago Aníbal Disalvo – La historia del rey Guillermo de Inglaterra: del relato del siglo XIV a la crónica quinientista / Carina Alejandra Zubillaga – El prólogo de “El Victorial”: heterogeneidad y orden a favor de una adecuada recepción / María Mercedes Rodríguez Temperley – A propósito del prólogo de la Confesión del amante / María Cristina Balestrini – Función de la “ekprhasis” en los relatos caballerescos / Lilia Elda Ferrario de Orduna – Una nueva fuente del Amadís de Gaula primitivo: la Waltbarii poesis del abad Ekkehard I. de Saint Gall / Aquilino Suárez Pallasá – Profecía mesiánica y profecía apocalíptica: la cuestión constantinopolitana en las Sergas de Esplandián y Primaleón / Javier Roberto González – Dos romances sefardíes de incógnito en una colección de romances. Aporte de nuevas versiones / Eleonora Angélica Alberti de Kleinbort – Conjeturas acerca del original manuscrito de la Comedia de Fernando Rojas / Germán Orduna – Fernando de Rojas, ¿autor? / Joseph Thomas Snow – El fondo medieval de la colección Foulché-Delbosc / Georgina Olivetto – Nómina de todos los congresista

Letras, 1999-2000, nº 40-41 (número completo)

Contenido: Nota preliminar – Poesía mariana en Gonzalo de Berceo / Lía N. Uriarte Rebaudi – El judío y el diablo como “otro” en los Milagros de Nuestra Señora de Berceo / Heanon M. Wilkins – “Intellectum tibi dabo et instruam te” / Eric Naylor – Descripción, narración, argumentación y crisis de la oralidad en el Libro del Buen Amor / Sofía M. Carrizo Rueda – El poema de Alfonso XI: ¿Un eco priscilianista, sabeliano, patripasiano? / Jorge Norberto Ferro – El vino en el refranero español; concordancias / Dolly M. Lucero Ontiveros – Una propuesta de clasificación de la Literatura Sapiencial Hispánica en el siglo XIII / Alicia E. Ramadori – Fray Juan García de Castrojeriz receptor de Aristóteles / Hugo Oscar Bizzarri – La especificidad de la prosa alfonsí; el caso del Lapidario / Mirta G. Aguayo – Alfonso el Sabio y Brunetto Latini: convergencias / Gretchem Arnstedt de Magneres – La figura autoral de Alfonso X en las Cantigas de Santa María / Santiago Aníbal Disalvo – La historia del rey Guillermo de Inglaterra: del relato del siglo XIV a la crónica quinientista / Carina Alejandra Zubillaga – El prólogo de “El Victorial”: heterogeneidad y orden a favor de una adecuada recepción / María Mercedes Rodríguez Temperley – A propósito del prólogo de la Confesión del amante / María Cristina Balestrini – Función de la “ekprhasis” en los relatos caballerescos / Lilia Elda Ferrario de Orduna – Una nueva fuente del Amadís de Gaula primitivo: la Waltbarii poesis del abad Ekkehard I. de Saint Gall / Aquilino Suárez Pallasá – Profecía mesiánica y profecía apocalíptica: la cuestión constantinopolitana en las Sergas de Esplandián y Primaleón / Javier Roberto González – Dos romances sefardíes de incógnito en una colección de romances. Aporte de nuevas versiones / Eleonora Angélica Alberti de Kleinbort – Conjeturas acerca del original manuscrito de la Comedia de Fernando Rojas / Germán Orduna – Fernando de Rojas, ¿autor? / Joseph Thomas Snow – El fondo medieval de la colección Foulché-Delbosc / Georgina Olivetto – Nómina de todos los congresista

Extreme drought impacts have been underestimated in grasslands and shrublands globally.

Climate change is increasing the frequency and severity of short-term (~1 y) drought events-the most common duration of drought-globally. Yet the impact of this intensification of drought on ecosystem functioning remains poorly resolved. This is due in part to the widely disparate approaches ecologists have employed to study drought, variation in the severity and duration of drought studied, and differences among ecosystems in vegetation, edaphic and climatic attributes that can mediate drought impacts. To overcome these problems and better identify the factors that modulate drought responses, we used a coordinated distributed experiment to quantify the impact of short-term drought on grassland and shrubland ecosystems. With a standardized approach, we imposed ~a single year of drought at 100 sites on six continents. Here we show that loss of a foundational ecosystem function-aboveground net primary production (ANPP)-was 60% greater at sites that experienced statistically extreme drought (1-in-100-y event) vs. those sites where drought was nominal (historically more common) in magnitude (35% vs. 21%, respectively). This reduction in a key carbon cycle process with a single year of extreme drought greatly exceeds previously reported losses for grasslands and shrublands. Our global experiment also revealed high variability in drought response but that relative reductions in ANPP were greater in drier ecosystems and those with fewer plant species. Overall, our results demonstrate with unprecedented rigor that the global impacts of projected increases in drought severity have been significantly underestimated and that drier and less diverse sites are likely to be most vulnerable to extreme drought

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A.

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies