Complex RNA Secondary Structures Mediate Mutually Exclusive Splicing of Coleoptera Dscam1

Mutually exclusive splicing is an important mechanism for expanding protein diversity. An extreme example is the Down syndrome cell adhesion molecular (Dscam1) gene of insects, containing four clusters of variable exons (exons 4, 6, 9, and 17), which potentially generates tens of thousands of protein isoforms through mutually exclusive splicing, of which regulatory mechanisms are still elusive. Here, we systematically analyzed the variable exon 4, 6, and 9 clusters of Dscam1 in Coleoptera species. Through comparative genomics and RNA secondary structure prediction, we found apparent evidence that the evolutionarily conserved RNA base pairing mediates mutually exclusive splicing in the Dscam1 exon 4 cluster. In contrast to the fly exon 6, most exon 6 selector sequences in Coleoptera species are partially located in the variable exon region. Besides, bidirectional RNA–RNA interactions are predicted to regulate the mutually exclusive splicing of variable exon 9 of Dscam1. Although the docking sites in exon 4 and 9 clusters are clade specific, the docking sites-selector base pairing is conserved in secondary structure level. In short, our result provided a mechanistic framework for the application of long-range RNA base pairings in regulating the mutually exclusive splicing of Coleoptera Dscam1.

Accessibility Over Transposable Elements Reveals Genetic Determinants of Stemness Properties in Normal and Leukemic Hematopoiesis

Despite most acute myeloid leukemia (AML) patients achieving complete remission after induction chemotherapy, two thirds of patients will relapse with fatal disease within 5 years. AML is organized as a cellular hierarchy sustained by leukemia stem cells (LSC) at the apex, with LSC properties directly linked to tumor progression, therapy failure and disease relapse 1–5. Despite the central role of LSC in poor patient outcomes, little is known of the genetic determinants of their stemness properties 6–8. Although much AML research focuses on mutational processes and their impact on gene expression programs, the genetic determinants of cell state properties including stemness expand beyond mutations, relying on the genetic architecture captured in the chromatin of each cell 9–11. As LSCs share many functional and molecular properties with normal hematopoietic stem cells (HSC), we identified genetic determinants of primitive populations enriched for LSCs and HSCs in comparison with their downstream mature progeny by investigating their chromatin accessibility. Our work reveals how distinct transposable element (TE) subfamilies are used in primitive versus mature populations, functioning as docking sites for stem cell-associated regulators of genome topology, including CTCF, or lineage-specific transcription regulators in primitive and mature populations, respectively. We further show how TE subfamilies accessible in LSCs define docking sites for several oncogenic drivers in AML, namely FLI1, LYL1 and MEIS1. Using chromatin accessibility profiles from a cohort of AML patients, we further show the clinical utility of our TE accessibility-based LSCTE121 scoring scheme to identify patients with high rates of relapse. Collectively, our work reveals how different accessible TE subfamilies serve as genetic determinants of stemness properties in normal and leukemic hematopoietic stem cells.

Polo-like kinase 1 independently controls microtubule-nucleating capacity and size of the centrosome

Centrosomes are composed of a centriolar core surrounded by a pericentriolar material (PCM) matrix that docks microtubule-nucleating γ-tubulin complexes. During mitotic entry, the PCM matrix increases in size and nucleating capacity in a process called centrosome maturation. Polo-like kinase 1 (PLK1) is recruited to centrosomes and phosphorylates PCM matrix proteins to drive their self-assembly, which leads to PCM expansion. Here, we show that in addition to controlling PCM expansion, PLK1 independently controls the generation of binding sites for γ-tubulin complexes on the PCM matrix. Selectively preventing the generation of PLK1-dependent γ-tubulin docking sites led to spindle defects and impaired chromosome segregation without affecting PCM expansion, highlighting the importance of phospho-regulated centrosomal γ-tubulin docking sites in spindle assembly. Inhibiting both γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulated the effects of loss of centrosomal PLK1 on the ability of centrosomes to catalyze spindle assembly.

Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future.

Interactome analysis and docking sites prediction of (AtCHR8, AtCUL4 and AtERCC1/UVR7) proteins in Arabidopsis Thaliana

The UV irradiation is a major DNA damaging factor in plants. Arabidopsis thaliana uses various repair pathways for these kinds of DNA lesions. One of them is the nucleotide excision repair pathway. The AtCUL4, ERCC1/UVR7 and CHR8 are vital proteins for nucleotide excision pathway and mutations in these proteins cause flaws in the repair mechanism. Two of these proteins play crucial role during DNA damage recognition and the other is involved in the excision of damaged bases. During NER processes, Arabidopsis uses different sets of proteins during the DNA damage recognition for transcriptionally active and genomic DNA. In order to get better insight into these proteins, we used bioinformatics tools to predict, analyze, and validate 3D structures of ERCC1/UVR7, AtCUL4 and CHR8. We also predicted the subcellular and sub-nuclear localization of proteins. Subsequently, we predicted the docking sites for each individual proteins and searched for interacting residues which mediate the protein-protein interactions.