Isolation and Library Preparation of Planarian piRNAs.

In planarian flatworms, piRNAs and SMEDWI (Schmidtea mediterranea PIWI) proteins are both essential for the animals' impressive regenerative ability and for their survival. A knockdown of SMEDWI proteins disrupts the specification of the planarian germline and impairs stem cell differentiation, resulting in lethal phenotypes. As the molecular targets of PIWI proteins and thus their biological function are determined by PIWI-bound small RNAs, termed piRNAs (for PIWI-interacting RNAs), it is imperative to study the wealth of PIWI-bound piRNAs using next-generation sequencing-based techniques. Prior to sequencing, piRNAs bound to individual SMEDWI proteins must be isolated. To that end, we established an immunoprecipitation protocol that can be applied to all planarian SMEDWI proteins. Co-immunoprecipitated piRNAs are visualized by using qualitative radioactive 5'-end labeling, which detects even trace amounts of small RNAs. Next, isolated piRNAs are subjected to a library preparation protocol that has been optimized for the efficient capture of piRNAs, whose 3'-ends carry a 2'-O-methyl modification. Successfully prepared piRNA libraries are subjected to Illumina-based next-generation sequencing. Obtained data are analyzed as presented in the accompanying manuscript.

Genome-Wide Analysis of Planarian piRNAs.

In planarian flatworms, the piRNA pathway is operated by three PIWI proteins, termed SMEDWI-1, SMEDWI-2, and SMEDWI-3 (SMEDWI = Schmidtea mediterranea PIWI). The interplay between these three PIWI proteins and their associated small noncoding RNAs, termed piRNAs, fuels the outstanding regenerative abilities of planarians, enables tissue homeostasis, and, ultimately, ensures animal survival. As the molecular targets of PIWI proteins are determined by the sequences of their co-bound piRNAs, it is imperative to identify these sequences by next-generation sequencing applications. Following sequencing, the genomic targets and the regulatory potential of the isolated piRNA populations need to be uncovered. To that end, here we present a bioinformatics analysis pipeline for processing and systematic characterization of planarian piRNAs. The pipeline includes steps for the removal of PCR duplicates based on unique molecular identifier (UMI) sequences, and it accounts for piRNA multimapping to different loci in the genome. Importantly, our protocol also includes a fully automated pipeline that is freely available at GitHub. Together with the piRNA isolation and library preparation protocol (see accompanying chapter), the presented computational pipeline enables researchers to explore the functional role of the piRNA pathway in flatworm biology.

The piRNA pathway in planarian flatworms: new model, new insights.

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that associate with members of the PIWI clade of the Argonaute superfamily of proteins. piRNAs are predominantly found in animal gonads. There they silence transposable elements (TEs), regulate gene expression and participate in DNA methylation, thus orchestrating proper germline development. Furthermore, PIWI proteins are also indispensable for the maintenance and differentiation capabilities of pluripotent stem cells in free-living invertebrate species with regenerative potential. Thus, PIWI proteins and piRNAs seem to constitute an essential molecular feature of somatic pluripotent stem cells and the germline. In keeping with this hypothesis, both PIWI proteins and piRNAs are enriched in neoblasts, the adult stem cells of planarian flatworms, and their presence is a prerequisite for the proper regeneration and perpetual tissue homeostasis of these animals. The piRNA pathway is required to maintain the unique biology of planarians because, in analogy to the animal germline, planarian piRNAs silence TEs and ensure stable genome inheritance. Moreover, planarian piRNAs also contribute to the degradation of numerous protein-coding transcripts, a function that may be critical for neoblast differentiation. This review gives an overview of the planarian piRNA pathway and of its crucial function in neoblast biology.

A precisely positioned MED12 activation helix stimulates CDK8 kinase activity.

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.

Efficient depletion of ribosomal RNA for RNA sequencing in planarians.

BACKGROUND: The astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed. RESULTS: In this study, we describe a workflow for the efficient depletion of rRNA in the planarian model species S. mediterranea. Our protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. We tested our approach on total RNA isolated from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium. CONCLUSIONS: The ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications our procedure might be adapted to any prokaryotic or eukaryotic species of choice.

Planarians recruit piRNAs for mRNA turnover in adult stem cells.

PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Thus, PIWI proteins enable planarians to repurpose piRNAs for potentially critical roles in neoblast mRNA turnover.