microRNA in Porifera: Evolution of a Molecular Mechanism in Sponges

Sponges are a historically ambiguous taxon, often considered primitively simple representatives of early Metazoa, living fossils from the era before the complex morphological features of the bilaterian phyla appeared in the Cambrian. Yet recent findings in genetics, genomics, and molecular and cellular biology show that sponges possess most characteristic protein-coding gene families and genetic pathways necessary for establishing complex tissues in bilaterian development. Absent are substantial complements of microRNAs, endogenous small RNAs produced by activity of the Drosha/Pasha complex. miRNAs are non-coding regulators of protein-coding transcripts, found in large complements in complex bilaterians. Data from deep-sequencing and northern blot analysis, including data presented here, show that miRNAs appear as a complement of 8 conserved in Demospongia consistent with the general observation that the size of miRNA complements correllate well with developmental and organismal complexity.;miRNA complements in calcisponges and homoscleromorph sponges were tested for miRNAs shared between demosponges and bilaterians using RNA deep sequencing and bioinformatic genome searching. It was found that no conserved, expressed miRNAs exist between calcisponges and demosponges, demonstrating that miRNAs do not have an apparent evolutionarily conserved role in establishing the cell types and body plan characteristic within the Porifera.;To better understand the biological role of sponge miRNAs, miRNA expression levels were compared across four demosponge species and miRNAs were found expressed to be expressed at similar relative levels in distantly related demosponges. Expression was tested using qPCR in tissue dissociation and reaggregation in the demosponge Spongosorites, where global miRNA expression was shown to decrease in dissociated and reaggregating tissue relative to fresh tissue. Comparison of miRNA relative expression in choanoderm and exopinacoderm shows that several miRNAs exhibit increased expression in sponge cortex (exopinacoderm), the epithelia-like outer covering and differentiated tissue structure of the Porifera, relative to expression in the choanoderm. The findings of differential expression in tissues and conservation of relative expression levels between species is consistent with the hypothesis that demosponge miRNAs could play a conserved role in demosponge tissue identity and development. Further experiments will be required to determine specific evolutionary and developmental roles for miRNAs.;Comparative sequence analysis of the canonical protein components of miRNA biogenesis Drosha and Pasha shows that these factors have lost known and putative functional sequence in Amphimedon which is retained in calcisponges and eumetazoans. This may indicate a partial loss-of-function of canonical miRNA processing in the demosponge lineage. A novel, semi-automated database analysis presented here shows that DDX5 (also known as p68) (a mediator of p53 co-activation of miRNA biogenesis), is present throughout Eukaryotes and is commonly known as a spliceosomal and rRNA biogenesis factor. The role of DDX5 in metazoan miRNA biogenesis is therefore modified from deeply conserved, pre-existing function in eukaryotes.;Together data presented here provides additional understanding of miRNA function and evolution in the Porifera and direction for further studies.