RESUMEN
Salamanders have a remarkable capacity to regenerate complex tissues, such as limbs and brain, and are therefore an important comparative model system for regenerative medicine. Despite these unique properties among adult vertebrates, the genomic information for amphibians in general, and salamanders in particular, is scarce. Here, we used massive parallel sequencing to reconstruct a de novo reference transcriptome of the red spotted newt (Notophthalmus viridescens) containing 118,893 transcripts with a N50 length of 2016 nts. Comparisons to other vertebrates revealed a newt transcriptome that is comparable in size and characteristics to well-annotated vertebrate transcriptomes. Identification of putative open reading frames (ORFs) enabled us to infer a comprehensive proteome, including the annotation of 19,903 newt proteins. We used the identified domain architectures (DAs) to assign ORFs phylogenetic positions, which also revealed putative salamander specific proteins. The reference transcriptome and inferred proteome of the red spotted newt will facilitate the use of systematic genomic technologies for regeneration studies in salamanders and enable evolutionary analyses of vertebrate regeneration at the molecular level.
Asunto(s)
Perfilación de la Expresión Génica/normas , Notophthalmus viridescens/genética , Notophthalmus viridescens/metabolismo , Proteoma/análisis , Transcriptoma/fisiología , Animales , Análisis por Conglomerados , Biología Computacional/métodos , Evolución Molecular , Anotación de Secuencia Molecular , Notophthalmus viridescens/fisiología , Sistemas de Lectura Abierta/genética , Proteoma/metabolismo , Proteómica/métodos , Estándares de Referencia , Regeneración/genética , Urodelos/genética , Urodelos/metabolismo , Urodelos/fisiología , Estudios de Validación como AsuntoRESUMEN
Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a ligation-based method that enables the capture, sequencing, and molecular counting of small RNAs from individual mammalian cells. Here, we provide a detailed protocol for this approach that relies on standard reagents and instruments. The standard protocol captures a complex set of small RNAs, including microRNAs (miRNAs), fragments of tRNAs and small nucleolar RNAs (snoRNAs); however, miRNAs can be enriched through the addition of a size-selection step. Ready-to-sequence libraries can be generated in 2-3 d, starting from cell collection, with additional days needed to computationally map the sequence reads and calculate molecular counts.
Asunto(s)
ARN Pequeño no Traducido/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , ADN Complementario/genética , Citometría de Flujo/métodos , Biblioteca de Genes , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa/métodos , ARN Pequeño no Traducido/aislamiento & purificaciónRESUMEN
Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates.
Asunto(s)
Extremidades/fisiología , Genoma/genética , MicroARNs/genética , Pleurodeles/genética , Regeneración/genética , Ambystoma mexicanum/genética , Animales , Sistemas CRISPR-Cas , Elementos Transponibles de ADN/genética , Células Madre Embrionarias/metabolismo , Edición Génica , Perfilación de la Expresión Génica , Genómica , Músculo Esquelético/fisiología , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX7/genética , Proto-Oncogenes/genética , Regeneración/fisiologíaRESUMEN
Little is known about the heterogeneity of small-RNA expression as small-RNA profiling has so far required large numbers of cells. Here we present a single-cell method for small-RNA sequencing and apply it to naive and primed human embryonic stem cells and cancer cells. Analysis of microRNAs and fragments of tRNAs and small nucleolar RNAs (snoRNAs) reveals the potential of microRNAs as markers for different cell types and states.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Células Madre Embrionarias Humanas/fisiología , MicroARNs/genética , Neoplasias Experimentales/genética , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética , Línea Celular Tumoral , Células Cultivadas , HumanosRESUMEN
Although the locations of promoters and enhancers have been identified in several cell types, we still have limited information on their connectivity. We developed HiCap, which combines a 4-cutter restriction enzyme Hi-C with sequence capture of promoter regions. Applying the method to mouse embryonic stem cells, we identified promoter-anchored interactions involving 15,905 promoters and 71,984 distal regions. The distal regions were enriched for enhancer marks and transcription, and had a mean fragment size of only 699 bp--close to single-enhancer resolution. High-resolution maps of promoter-anchored interactions with HiCap will be important for detailed characterizations of chromatin interaction landscapes.