NEW YORK (GenomeWeb News) – Using Roche 454 and Illumina sequencing, researchers from the US and Germany catalogued the small RNA repertoire of Schmidtea mediterranea, a flatworm species capable of extensive regeneration.
The team's search yielded more than a million potential Piwi-interacting RNAs and dozens of new microRNAs, including a handful of miRNAs that are up regulated in adult stem cells. As such, the research, scheduled to appear online this week in the Proceedings of the National Academy of Sciences, provides new insights into not only the overall small RNA content of the S. mediterranea genome but also into the role that specific RNAs play in regulating body part replacement in the worms.
"[W]e present the first comprehensive small RNA analysis in animals belonging to the third animal super-phylum, the Lophatrochozoa, and single out a number of miRNAs that may function in regeneration," senior author Nikolaus Rajewsky, a researcher at the Max Delbrück Center for Molecular Medicine in Berlin, and his co-authors wrote.
S. mediterranea are free-living flatworms capable of regenerating missing body parts using adult stem cells called neoblasts. Previous research suggests this regeneration involves hundreds of genes.
Prior to the latest study, researchers had identified 61 mature miRNAs, mainly using cloning and Sanger sequencing. To look for additional miRNAs, as well as piRNAs, Rajewsky and his team used Illumina and Roche 454 platforms to sequence RNA isolated from S. mediterranea neoblasts as well as whole, untreated worms or worms that had their neoblasts removed by irradiation.
Using the two platforms, the team generated more than four million reads, which they subsequently mapped to the previously sequenced and annotated S. mediterranea genome. Their sequence data revealed some 1.2 million candidate piRNAs, each about 32 nucleotides long, and 122 potential miRNAs that were around 22 nucleotides long. Of the 61 miRNAs that hadn't been detected previously, 20 were validated in subsequent experiments.
Whereas miRNAs tended to be the most commonly detected type small RNAs in flatworms that had had their neoblasts removed, the miRNAs were somewhat less common in the untreated worms, and least common in the adult stem cell samples, the team noted.
The piRNAs showed the opposite pattern. In general, piRNAs were highly represented in the adult stem cells and depleted in the worms missing these neoblasts. The untreated worms had piRNA levels between the stem cell and treated worm samples.
Based on the sequences detected, the researchers suggested that these piRNAs may have a comparable function to fly piRNAs: curbing the expression of potentially damaging transposons by mapping to the elements in an antisense manner. About a third of S. mediterranea piRNAs mapped to known transposons in the flatworm's genome.
Nevertheless, they noted, the worm's piRNA and transposon expression and clustering patterns actually appeared to be more similar to those in mice than in flies. "Together, our data indicate that the piRNA pathway has undergone complex evolution," the authors wrote.
Meanwhile, the team's phylogenetic analysis suggested that flatworm miRNAs fall into almost 80 families, with 22 of these overlapping with mammalian families. Thirty three overlapped with fly and nematode miRNA families and more than half were distinct from any miRNAs described before.
But while the group's experiments linked more than a dozen S. mediterranea miRNAs to adult stem cell biology, they noted that more research will be needed to tease the role of these miRNAs and how the overall process compares to that observed in mammalian stem cells.
For instance, the researchers' expression experiments found one of the miRNAs that was more highly expressed in neoblast samples — let-7 — is usually down regulated in mammalian stem cells. The team speculated that this difference could be a consequence of flatworm neoblasts differentiating into other cell types. If so, they added, "[f]urther let-7 analysis … may help elucidate the specification of neoblast lineages."
The team also called for additional research into the potential role of planarian piRNAs in epigenetic control of the flatworm genome. "piRNAs have been shown selectively to silence transposons in the fly and mouse genomes and it is likely that piRNAs play such a role in planaria," they explained. "Further studies are needed to determine whether planarian piRNAs also play a critical role in epigenetic silencing through DNA/chromatin methylation like their germline homologs."