RNA sequencing: new technologies and applications in cancer research

Acute myeloid leukemia (AML) is a fatal hematopoietic malignancy and has a prognosis that varies with its genetic complexity. However, there has been no appropriate integrative analysis on the hierarchy of different AML subtypes. Using Microwell-seq, a high-throughput single-cell mRNA sequencing platform, we analyzed the cellular hierarchy of bone marrow samples from 40 patients and 3 healthy donors. We also used single-cell single-molecule real-time (SMRT) sequencing to investigate the clonal heterogeneity of AML cells. From the integrative analysis of 191727 AML cells, we established a single-cell AML landscape and identified an AML progenitor cell cluster with novel AML markers. Patients with ribosomal protein high progenitor cells had a low remission rate. We deduced two types of AML with diverse clinical outcomes. We traced mitochondrial mutations in the AML landscape by combining Microwell-seq with SMRT sequencing. We propose the existence of a phenotypic “cancer attractor” that might help to define a common phenotype for AML progenitor cells. Finally, we explored the potential drug targets by making comparisons between the AML landscape and the Human Cell Landscape. We identified a key AML progenitor cell cluster. A high ribosomal protein gene level indicates the poor prognosis. We deduced two types of AML and explored the potential drug targets. Our results suggest the existence of a cancer attractor.     

Trypanosomatids: mitochondrial RNA editing

RNA editing is a genetic regulatory process that was recently discovered in the mitochondrion of trypanosomatid parasites. It alters mRNA by the addition and deletion of uridines. Much remains to be learned about this process, including identification and characterization of the macromolecules that catalyze and regulate this process and the mechanism of editing.     

Naphthalene-based RNA editing inhibitor blocks RNA editing activities and editosome assembly in Trypanosoma brucei

RNA editing, catalyzed by the multiprotein editosome complex, is an essential step for the expression of most mitochondrial genes in trypanosomatid pathogens. It has been shown previously that Trypanosoma brucei RNA editing ligase 1 (TbREL1), a core catalytic component of the editosome, is essential in the mammalian life stage of these parasitic pathogens. Because of the availability of its crystal structure and absence from human, the adenylylation domain of TbREL1 has recently become the focus of several studies for designing inhibitors that target its adenylylation pocket. Here, we have studied new and existing inhibitors of TbREL1 to better understand their mechanism of action. We found that these compounds are moderate to weak inhibitors of adenylylation of TbREL1 and in fact enhance adenylylation at higher concentrations of protein. Nevertheless, they can efficiently block deadenylylation of TbREL1 in the editosome and, consequently, result in inhibition of the ligation step of RNA editing. Further experiments directly showed that the studied compounds inhibit the interaction of the editosome with substrate RNA. This was supported by the observation that not only the ligation activity of TbREL1 but also the activities of other editosome proteins such as endoribonuclease, terminal RNA uridylyltransferase, and uridylate-specific exoribonuclease, all of which require the interaction of the editosome with the substrate RNA, are efficiently inhibited by these compounds. In addition, we found that these compounds can interfere with the integrity and/or assembly of the editosome complex, opening the exciting possibility of using them to study the mechanism of assembly of the editosome components.     

Gene editing: from technologies to applications in research and beyond

Science China Life Sciences -     

Evolutionary origin of RNA editing

The term "RNA editing" encompasses a wide variety of mechanistically and phylogenetically unrelated processes that change the nucleotide sequence of an RNA species relative to that of the encoding DNA. Two general classes of editing, substitution and insertion/deletion, have been described, with all major types of cellular RNA (messenger, ribosomal, and transfer) undergoing editing in different organisms. In cases where RNA editing is required for function (e.g., to generate a translatable open reading frame in a mRNA), editing is an obligatory step in the pathway of genetic information expression. How, when, and why individual RNA editing systems originated are intriguing biochemical and evolutionary questions. Here I review briefly what is known about the biochemistry, genetics, and phylogenetics of several very different RNA editing systems, emphasizing what we can deduce about their origin and evolution from the molecular machinery involved. An evolutionary model, centered on the concept of "constructive neutral evolution", is able to account in a general way for the origin of RNA editing systems. The model posits that the biochemical elements of an RNA editing system must be in place before there is an actual need for editing, and that RNA editing systems are inherently mutagenic because they allow potentially deleterious or lethal mutations to persist at the genome level, whereas they would otherwise be purged by purifying selection.     

Site-selective inhibition of plastid RNA editing by heat shock and antibiotics: a role for plastid translation in RNA editing.

RNA editing in higher plant plastids changes single cytidine residues to uridine through an unknown mechanism. In order to investigate the relation of editing to physiological processes and to other steps in plastid gene expression, we have tested the sensitivity of chloroplast RNA editing to heat shock and antibiotics. We show that heat shock conditions as well as treatment of plants with prokaryotic translational inhibitors can inhibit plastid RNA editing. Surprisingly, this inhibitory effect is confined to a limited number of plastid editing sites suggesting that some site-specific factor(s) but none of the general components of the plastid RNA editing machinery are compromised. Contrary to previous expectations, our results provide evidence for a role of plastid translation in RNA editing.     

Genome editing technologies and their applications in crop improvement

Crop improvement is very essential to meet the increasing global food demands and enhance food nutrition. Conventional crop-breeding methods have certain limitations such as taking lot of time and resources, and causing biosafety concerns. These limitations could be overcome by the recently emerged-genome editing technologies that can precisely modify DNA sequences at the genomic level using sequence-specific nucleases (SSNs). Among the artificially engineered SSNs, the CRISPR/Cas9 is the most recently developed targeted genome modification system and seems to be more efficient, inexpensive, easy, user-friendly and rapidly adopted genome-editing tool. Large-scale genome editing has not only improved the yield and quality but also has enhanced the disease resistance ability in several model and other major crops. Increasing case studies suggest that genome editing is an efficient, precise and powerful technology that can accelerate basic and applied research towards crop improvement. In this review, we briefly overviewed the structure and mechanism of genome editing tools and then emphatically reviewed the advances in the application of genome editing tools for crop improvement, including the most recent case studies with CRISPR/Cpf1 and base-editing technologies. We have also discussed the future prospects towards the improvement of agronomic traits in crops.     

Editing machines: the complexities of trypanosome RNA editing

The assembly and disassembly of ribonucleoprotein complexes containing substrate precursor mRNAs and guide RNAs is crucial to the initiation and propagation of RNA editing. We discuss here the composition of these complexes and how their assembly may regulate RNA editing.