RESUMEN
Voltage-sensing (VSD) and cyclic nucleotide-binding domains (CNBD) gate ion channels for rapid electrical signaling. By contrast, solute carriers (SLCs) that passively redistribute substrates are gated by their substrates themselves. Here, we study the orphan sperm-specific solute carriers SLC9C1 that feature a unique tripartite structure: an exchanger domain, a VSD, and a CNBD. Voltage-clamp fluorimetry shows that SLC9C1 is a genuine Na+/H+ exchanger gated by voltage. The cellular messenger cAMP shifts the voltage range of activation. Mutations in the transport domain, the VSD, or the CNBD strongly affect Na+/H+ exchange, voltage gating, or cAMP sensitivity, respectively. Our results establish SLC9C1 as a phylogenetic chimaera that combines the ion-exchange mechanism of solute carriers with the gating mechanism of ion channels. Classic SLCs slowly readjust changes in the intra- and extracellular milieu, whereas voltage gating endows the Na+/H+ exchanger with the ability to produce a rapid pH response that enables downstream signaling events.
Asunto(s)
AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Espermatozoides/metabolismo , Strongylocentrotus purpuratus/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Clonación Molecular , Cricetulus , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hemaglutininas/genética , Hemaglutininas/metabolismo , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Cinética , Masculino , Mutación , Filogenia , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/genética , Espermatozoides/citología , Strongylocentrotus purpuratus/clasificación , Strongylocentrotus purpuratus/genéticaRESUMEN
Telomerase is a ribonucleoprotein (RNP) enzyme essential for telomere maintenance and chromosome stability. While the catalytic telomerase reverse transcriptase (TERT) protein is well conserved across eukaryotes, telomerase RNA (TR) is extensively divergent in size, sequence, and structure. This diversity prohibits TR identification from many important organisms. Here we report a novel approach for TR discovery that combines in vitro TR enrichment from total RNA, next-generation sequencing, and a computational screening pipeline. With this approach, we have successfully identified TR from Strongylocentrotus purpuratus (purple sea urchin) from the phylum Echinodermata. Reconstitution of activity in vitro confirmed that this RNA is an integral component of sea urchin telomerase. Comparative phylogenetic analysis against vertebrate TR sequences revealed that the purple sea urchin TR contains vertebrate-like template-pseudoknot and H/ACA domains. While lacking a vertebrate-like CR4/5 domain, sea urchin TR has a unique central domain critical for telomerase activity. This is the first TR identified from the previously unexplored invertebrate clade and provides the first glimpse of TR evolution in the deuterostome lineage. Moreover, our TR discovery approach is a significant step toward the comprehensive understanding of telomerase RNP evolution.
Asunto(s)
Biología Computacional/métodos , ARN/genética , Strongylocentrotus purpuratus/genética , Telomerasa/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Activación Enzimática , Pruebas de Enzimas , Evolución Molecular , Biblioteca de Genes , Sitios Genéticos , Gónadas/citología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Estructura Terciaria de Proteína , ARN/clasificación , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Strongylocentrotus purpuratus/clasificación , Strongylocentrotus purpuratus/enzimología , Telomerasa/clasificación , Telomerasa/metabolismoRESUMEN
An evolutionary analysis of transcriptional regulation is essential to understanding the molecular basis of phenotypic diversity. The sea urchin is an ideal system in which to explore the functional consequence of variation in cis-regulatory sequences. We are particularly interested in the evolution of genes involved in the patterning and synthesis of its larval skeleton. This study focuses on the cis-regulatory region of SM50, which has already been characterized to a considerable extent in the purple sea urchin, Strongylocentrotus purpuratus. We have isolated the cis-regulatory region from 15 individuals of S. purpuratus as well as seven closely related species in the family Strongylocentrotidae. We have performed a variety of statistical tests and present evidence that the cis-regulatory elements upstream of the SM50 gene have been subject to positive selection along the lineage leading to S. purpuratus. In addition, we have performed electrophoretic mobility shift assays (EMSAs) and demonstrate that nucleotide substitutions within Element C affect the ability of nuclear proteins to bind to this cis-regulatory element among members of the family Strongylocentrotidae. We speculate that such changes in SM50 and other genes could accumulate to produce altered patterns of gene expression with functional consequences during skeleton formation.
Asunto(s)
Evolución Molecular , Proteínas de la Matriz Extracelular/genética , Erizos de Mar/crecimiento & desarrollo , Erizos de Mar/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/metabolismo , Cartilla de ADN/genética , Proteínas de la Matriz Extracelular/metabolismo , Datos de Secuencia Molecular , Filogenia , Polimorfismo Genético , Unión Proteica , Erizos de Mar/clasificación , Erizos de Mar/metabolismo , Selección Genética , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Strongylocentrotus purpuratus/clasificación , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/crecimiento & desarrollo , Strongylocentrotus purpuratus/metabolismoRESUMEN
Paleogenomics propels the meaning of genomic studies back through hundreds of millions of years of deep time. Now that the genome of the echinoid Strongylocentrotus purpuratus is sequenced, the operation of its genes can be interpreted in light of the well-understood echinoderm fossil record. Characters that first appear in Early Cambrian forms are still characteristic of echinoderms today. Key genes for one of these characters, the biomineralized tissue stereom, can be identified in the S. purpuratus genome and are likely to be the same genes that were involved with stereom formation in the earliest echinoderms some 520 million years ago.
