Crabs encompass the infra-orders Brachyura and Anomura, collectively constitute the clade Meiura within order Decapoda. Despite their considerable diversity, genomic resources for crabs remain scarce, hindering our understanding of their phylogeny and genetic mechanisms underlying such unique traits as carcinization. To address these questions, here we sequenced genomes of 10 crab species covering all currently controversial taxonomies at section level. Our whole-genome phylogenetic results support Raninoida is closer to Eubrachyura rather than Dromiacea, challenging the traditional classifications. Notably, the freshwater crab subsection Potamoida, represented by S. planum, is found to be more closely related to subsection Thoracotremata than to Heterotremata as previously suggested, indicating that crab classification based solely on morphology may be misleading. Our results also clarify that the family Gecarcinidae should be classified into Thoracotremata, contrary to previous placements in Heterotremata. Comparative genomic analyses identified lineage-specific families related to crab traits, including ionotropic glutamate receptors, neurotransmitters, and energy metabolism. Additionally, transcriptomic studies of Chinese mitten crab larval stages suggest that some lineage-specific genes such as ghrA, and TCB2, may account for the prominent carcinization in brachyurans. This study not only significantly expands the genomic repository for crabs, but also provides insights into the phylogeny and trait evolution of crabs.

Functional studies of long noncoding RNAs (lncRNAs) have been hindered by the lack of methods to assess their evolution. Here we present lncRNA Homology Explorer (lncHOME), a computational pipeline that identifies a unique class of long noncoding RNAs (lncRNAs) with conserved genomic locations and patterns of RNA-binding protein (RBP) binding sites (coPARSE-lncRNAs). Remarkably, several hundred human coPARSE-lncRNAs can be evolutionarily traced to zebrafish. Using CRISPR–Cas12a knockout and rescue assays, we found that knocking out many human coPARSE-lncRNAs led to cell proliferation defects, which were subsequently rescued by predicted zebrafish homologs. Knocking down coPARSE-lncRNAs in zebrafish embryos caused severe developmental delays that were rescued by human homologs. Furthermore, we verified that human, mouse and zebrafish coPARSE-lncRNA homologs tend to bind similar RBPs with their conserved functions relying on specific RBP-binding sites. Overall, our study demonstrates a comprehensive approach for studying the functional conservation of lncRNAs and implicates numerous lncRNAs in regulating vertebrate physiology.

RNA excited states represent a class of high-energy-level and thus low-populated conformational states of RNAs that are sequestered within the free energy landscape until being activated by cellular cues. In recent years, there has been growing interest in structural and functional studies of these transient states, but the rational design of excited states remains unexplored. Here we developed a method to design small hairpin RNAs with predefined excited states that exchange with ground states through base pair reshuffling, and verified these transient states by combining NMR relaxation dispersion technique and imino chemical shift prediction. Using van’t Hoff analysis and accelerated molecular dynamics simulations, a mechanism of multi-step sequential transition has been revealed. The efforts made in this study will expand the scope of RNA rational design, and also contribute towards improved predictions of RNA secondary structure.

Abstract Advances in RNA-seq studies have enabled transcriptome-scale annotation of long noncoding RNAs (lncRNAs) across vertebrates, with many of them having been implicated as regulators in diverse cellular processes1–3. However, our understanding of lncRNA function has long been hindered by the lack of methods to assess lncRNA evolution across species4–6. Here, we develop a computational pipeline, lncHOME (lncRNA Homology Explorer), to identify a special class of lncRNAs with conserved genomic locations and patterns of RNA binding protein (RBP) binding sites (coPARSE-lncRNAs). Strikingly, lncHOME identifies several hundred human coPARSE-lncRNAs that can be evolutionarily traced to zebrafish. We further develop a CRISPR-Cas12a (Cpf1) knockout screen system and find that the knockout of many of these human coPARSE-lncRNAs leads to cell proliferation defects, which could be rescued by the predicted lncRNA homologs from zebrafish. Finally, for two coPARSE-lncRNAs, we verified that their homologs from human, mouse, and zebrafish tend to bind a similar set of RBPs with cell proliferation functions. Thus, our study illustrates a general approach for studying lncRNA functional conservation and implicates numerous lncRNAs in regulating cellular physiology.

NH groups in proteins or nucleic acids are the most challenging target for chemical shift prediction. Here we show that the RNA base pair triplet motif dictates imino chemical shifts in its central base pair. A lookup table is established that links each type of base pair triplet to experimental chemical shifts of the central base pair, and can be used to predict imino chemical shifts of RNAs to remarkable accuracy. Strikingly, the semiempirical method can well interpret the variations of chemical shifts for different base pair triplets, and is even applicable to non-canonical motifs. This finding opens an avenue for predicting chemical shifts of more complicated RNA motifs. Furthermore, we combine the imino chemical shift prediction with NMR relaxation dispersion experiments targeting both 15 N and 1 H N of the imino group, and verify a previously characterized excited state of P5abc subdomain including an earlier speculated non-native G•G mismatch.