High-resolution profiling and discovery of planarian small RNAs

Abstract

Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are downregulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.microRNAs ͉ miRNAs ͉ piRNAs ͉ regeneration ͉ stem cells P lanarians have become a molecularly tractable model system in which to study regeneration, tissue homeostasis, and stem-cell biology (1). Planaria are free-living, triploblastic flatworms of the phylum Platyhelminthes, which is presently considered to belong to the superphylum Lophotrochozoa. Model systems for modern molecular and developmental biology have almost exclusively focused on the other 2 superphyla, i.e., the Deuterostomes (which includes vertebrates) and the Ecdysozoa (e.g., Caenorhabditis elegans and Drosophila melanogaster). Unlike these model systems, planarians possess remarkable regeneration abilities. Decapitation, for example, results in the complete regeneration of the head within 7 days after amputation. Such robust restoration of missing body parts is mediated by adult stem cells known as neoblasts (2). Of the thousands of known planarian species, Schmidtea mediterranea is arguably the species of choice for modern molecular biology and high-throughput, genome-wide approaches because it is diploid, it exists in sexual and asexual strains, and its genome has recently been sequenced and annotated (3). The size of its genome is roughly a third of the human genome, and Ϸ80% of the Ϸ20,000 annotated planarian genes have orthologs in humans. Moreover, by morphology alone, neoblasts and their immediate division progeny comprise Ϸ25% of all cells in the adult animal (4). In addition, RNAi screens have identified hundreds of genes specifically linked to planarian regeneration and stem-cell biology (5). Many of these genes are conserved in humans, and thus understanding planarian ...

Small RNA pathways in Schmidtea mediterranea

“…Deep sequencing of small RNA libraries from non-irradiated and irradiated animals and neoblast cells identified several miRNAs that were irradiation-sensitive and expressed in the neoblast cell population. In planarians, let-7a miRNA expression showed a marked decrease upon irradiation (Lu et al, 2009, Friedländer et al, 2009. In contrast, let-7a expression in mammals is suppressed in mammalian embryonic stem (ES) cells and expressed upon differentiation.…”

“…Interestingly, lin-28, a suppressor of pre-let-7a processing, was expressed in mammalian ES cells, whereas in planarians lin-28 was expressed in differentiated tissues, indicating a reversal in function and expression for let-7a and lin-28 during evolution. Deep sequencing of small RNA libraries also revealed that miRNAs belonging to the major miRNA cluster mir71a/2d/13/752 were sensitive to irradiation and expressed in the neoblast population, suggesting that miRNAs belonging to the same cluster may be co-regulated (Lu et al, 2009, Friedländer et al, 2009. Nonetheless, it is important to consider the expression of individual miRNAs from the same cluster within different neoblast populations (i.e.…”

“…As with mammals, planarian piRNAs that map uniquely to the planarian genome show a similar distribution to pachytene piRNAs and are highly abundant compared to the piRNAs that map to the transposable elements. These piRNAs might have similar functional roles and it will be interesting to identify the targets that are regulated by these small RNA molecules (Palakdoeti et al, 2008;Friedländer et al, 2009). The fact that planarian and other basal metazoan piRNAs are abundant in adult stem cells, unlike mammalian piRNAs, indicates that piRNA pathways may have initially evolved in adult stem cells and subsequently localized to germ line tissues as complex metazoans evolved.…”

“…Knocking down either DGCR8 or Drosha, both of which are involved in miRNA biogenesis, will be helpful in establishing the exact nature of miRNA function in the regeneration process. Several microRNAs were recently identified in Schmidtea mediterranea; many of these are highly conserved across the animal kingdom and a few are planarian specific (Palakodeti et al, 2006;Lu et al, 2009;Friedländer et al, 2009). In addition, many miRNA clusters were identified in Schmidtea mediterranea.…”

“…It was recently shown that planarian miRNAs are expressed in strain-specific and neoblast-specific fashion. Deep sequencing of small RNA libraries from irradiated and non-irradiated sexual and asexual strains and adult neoblast cell populations from Schmidtea mediterranea revealed numerous examples of strain-specific and neoblast-specific miRNA expression (Lu et al, 2009;Friedländer et al, 2009). …”