We show that regenerating planarians' normal anterior-posterior pattern can be permanently rewritten by a brief perturbation of endogenous bioelectrical networks. Temporary modulation of regenerative bioelectric dynamics in amputated trunk fragments of planaria stochastically results in a constant ratio of regenerates with two heads to regenerates with normal morphology. Remarkably, this is shown to be due not to partial penetrance of treatment, but a profound yet hidden alteration to the animals' patterning circuitry. Subsequent amputations of the morphologically normal regenerates in water result in the same ratio of double-headed to normal morphology, revealing a cryptic phenotype that is not apparent unless the animals are cut. These animals do not differ from wild-type worms in histology, expression of key polarity genes, or neoblast distribution. Instead, the altered regenerative bodyplan is stored in seemingly normal planaria via global patterns of cellular resting potential. This gradient is functionally instructive, and represents a multistable, epigenetic anatomical switch: experimental reversals of bioelectric state reset subsequent regenerative morphology back to wild-type. Hence, bioelectric properties can stably override genome-default target morphology, and provide a tractable control point for investigating cryptic phenotypes and the stochasticity of large-scale epigenetic controls.

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are ∼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5′ 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5′ extensions up to 39 nt long. The 5′-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5′ extension does not block Dicer processing. Rather, Dicer directly cleaves 5′-extended pre-miRNAs by recognizing its 3′ end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5′-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA–target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3′ end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive thanP-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.