Eight new mtDNA sequences of glass sponges reveal an extensive usage of +1 frameshifting in mitochondrial translation.

Three previously studied mitochondrial genomes of glass sponges (phylum Porifera, class Hexactinellida) contained single nucleotide insertions in protein coding genes inferred as sites of +1 translational frameshifting. To investigate the distribution and evolution of these sites and to help elucidate the mechanism of frameshifting, we determined eight new complete or nearly complete mtDNA sequences from glass sponges and examined individual mitochondrial genes from three others. We found nine new instances of single nucleotide insertions in these sequences and analyzed them both comparatively and phylogenetically. The base insertions appear to have been gained and lost repeatedly in hexactinellid mt protein genes, suggesting no functional significance for the frameshifting sites. A high degree of sequence conservation, the presence of unusual tRNAs, and a distinct pattern of codon usage suggest the "out-of-frame pairing" model of translational frameshifting. Additionally, we provide evidence that relaxed selection pressure on glass sponge mtDNA - possibly a result of their low growth rates and deep-water lifestyle - has allowed frameshift insertions to be tolerated for hundreds of millions of years. Our study provides the first example of a phylogenetically diverse and extensive usage of translational frameshifting in animal mitochondrial coding sequences.

RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria.

BACKGROUND: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. RESULTS: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene. CONCLUSION: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.

Parallel loss of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases and mtDNA-encoded tRNAs in Cnidaria.

Unlike most animal mitochondrial (mt) genomes, which encode a set of 22 transfer RNAs (tRNAs) sufficient for mt protein synthesis, those of cnidarians have only retained one or two tRNA genes. Whether the missing cnidarian mt-tRNA genes relocated outside the main mt chromosome or were lost remains unclear. It is also unknown what impact the loss of tRNA genes had on other components of the mt translational machinery. Here, we explored the nuclear genome of the cnidarian Nematostella vectensis for the presence of mt-tRNA genes and their corresponding mt aminoacyl-tRNA synthetases (mt-aaRS). We detected no candidates for mt-tRNA genes and only two mt-aaRS orthologs. At the same time, we found that all but one cytosolic aaRS appear to be targeted to mitochondria. These results indicate that the loss of mt-tRNAs in Cnidaria is genuine and occurred in parallel with the loss of nuclear-encoded mt-aaRS. Our phylogenetic analyses of individual aaRS revealed that although the nearly total loss of mt-aaRS is rare, aaRS gene deletion and replacement have occurred throughout the evolution of Metazoa.

Glass sponges and bilaterian animals share derived mitochondrial genomic features: a common ancestry or parallel evolution?

Glass sponges (Hexactinellida) are a group of deep-water benthic animals that have a unique syncytial organization and possess a characteristic siliceous skeleton. Although hexactinellids are traditionally grouped with calcareous and demosponges in the phylum Porifera, the monophyly of sponges and the phylogenetic position of the Hexactinellida remain contentious. We determined and analyzed the nearly complete mitochondrial genome sequences of the hexactinellid sponges Iphiteon panicea and Sympagella nux. Unexpectedly, our analysis revealed several mitochondrial genomic features shared between glass sponges and bilaterian animals, including an Arg --> Ser change in the genetic code, a characteristic secondary structure of one of the serine tRNAs, highly derived tRNA and rRNA genes, and the presence of a single large noncoding region. At the same time, glass sponge mtDNA contains atp9, a gene previously found only in the mtDNA of demosponges (among animals), and encodes a tRNA(Pro);(UGG) with an atypical A11-U24 pair that is also found in demosponges and placozoans. Most of our sequence-based phylogenetic analyses place Hexactinellida as the sister group to the Bilateria; however, these results are suspect given accelerated rates of mitochondrial sequence evolution in these groups. Thus, it remains an open question whether shared mitochondrial genomic features in glass sponges and bilaterian animals reflect their close phylogenetic affinity or provide a remarkable example of parallel evolution.