Background: With the fast advances in nextgen sequencing technology, high-throughput RNA sequencing has emerged as a powerful and cost-effective way for transcriptome study. De novo assembly of transcripts provides an important solution to transcriptome analysis for organisms with no reference genome. However, there lacked understanding on how the different variables affected assembly outcomes, and there was no consensus on how to approach an optimal solution by selecting software tool and suitable strategy based on the properties of RNA-Seq data. Results: To reveal the performance of different programs for transcriptome assembly, this work analyzed some important factors, including k-mer values, genome complexity, coverage depth, directional reads, etc. Seven program conditions, four single k-mer assemblers (SK: SOAPdenovo, ABySS, Oases and Trinity) and three multiple k-mer methods (MK: SOAPdenovo-MK, trans-ABySS and Oases-MK) were tested. While small and large k-mer values performed better for reconstructing lowly and highly expressed transcripts, respectively, MK strategy worked well for almost all ranges of expression quintiles. Among SK tools, Trinity performed well across various conditions but took the longest running time. Oases consumed the most memory whereas SOAPdenovo required the shortest runtime but worked poorly to reconstruct full-length CDS. ABySS showed some good balance between resource usage and quality of assemblies. Conclusions: Our work compared the performance of publicly available transcriptome assemblers, and analyzed important factors affecting de novo assembly. Some practical guidelines for transcript reconstruction from short-read RNA-Seq data were proposed. De novo assembly of C. sinensis transcriptome was greatly improved using some optimized methods.

Anti-melanoma differentiation-associated gene 5-positive dermatomyositis (MDA5⁺ DM) is an autoimmune condition associated with rapidly progressive interstitial lung disease and high mortality. The aetiology and pathogenesis of MDA5⁺ DM are still largely unknown. Here we describe the immune signatures of MDA5⁺ DM via single-cell RNA sequencing, flow cytometry and multiplex immunohistochemistry in peripheral B and T cells and in affected lung tissue samples from one patient. We find strong peripheral antibody-secreting cell and CD8⁺ T cell responses as cellular immune hallmarks, and over-stimulated type I interferon signaling and associated metabolic reprogramming as molecular immune signature in MDA5⁺ DM. High frequency of circulating ISG15⁺ CD8⁺ T cells at baseline predicts poor one-year survival in MDA5⁺ DM patients. In affected lungs, we find profuse immune cells infiltration, which likely contributes to the pro-fibrotic response via type I interferon production. The importance of type I interferons in MDA5⁺ DM pathology is further emphasized by our observation in a retrospective cohort of MDA5⁺ DM patients that combined calcineurin and Janus kinase inhibitor therapy show superior efficacy to calcineurin inhibitor monotherapy. In summary, this study reveals key immune-pathogenic features of MDA5⁺ DM and provides a potential basis for future tailored therapies.

The hydrolytic deamination of adenosine to inosine (A-to-I editing) in precursor mRNA induces variable gene products at the post-transcription level. How and to what extent A-to-I RNA editing diversifies transcriptome is not fully characterized in the evolution, and very little is known about the selective constraints that drive the evolution of RNA editing events. Here we present a study on A-to-I RNA editing, by generating a global profile of A-to-I editing for a phylogeny of seven Drosophila species, a model system spanning an evolutionary timeframe of approximately 45 million years. Of totally 9281 editing events identified, 5150 (55.5%) are located in the coding sequences (CDS) of 2734 genes. Phylogenetic analysis places these genes into 1,526 homologous families, about 5% of total gene families in the fly lineages. Based on conservation of the editing sites, the editing events in CDS are categorized into three distinct types, representing events on singleton genes (type I), and events not conserved (type II) or conserved (type III) within multi-gene families. While both type I and II events are subject to purifying selection, notably type III events are positively selected, and highly enriched in the components and functions of the nervous system. The tissue profiles are documented for three editing types, and their critical roles are further implicated by their shifting patterns during holometabolous development and in post-mating response. In conclusion, three A-to-I RNA editing types are found to have distinct evolutionary dynamics. It appears that nervous system functions are mainly tested to determine if an A-to-I editing is beneficial for an organism. The coding plasticity enabled by A-to-I editing creates a new class of binary variations, which is a superior alternative to maintain heterozygosity of expressed genes in a diploid mating system. One prevalent form of RNA editing is the deamination of adenosines (A-to-I editing) in the precursor mRNA molecules, pertaining to most organisms in the metazoan lineage. While examples of A-to-I editing on critical genes have been known for years, it has not been fully characterized how A-to-I editing shapes the transcriptome and proteome in the evolution. To understand how A-to-I editing affects genes’ evolution and how itself is constrained by selection, we generated a global profile of A-to-I editing for a phylogeny of seven fly species, a model system representing an evolutionary timeframe of about 45 million years. We are focused on 5150 editing sites (of totally 9281 identified) located in the coding region of 2734 genes. Our analysis revealed the evolution dynamics of A-to-I editing sites and functional specificity of targeted genes. The shifting patterns of A-to-I editing are documented during holometabolous development and in post-mating response in flies. This work points to the important roles of regulated RNA editing in animal development and offers new insight into the evolution of A-to-I editing events and their harboring genes.

Models developed using Nanopore direct RNA sequencing data from in vitro synthetic RNA with all adenosine replaced by N⁶-methyladenosine (m⁶A) are likely distorted due to superimposed signals from saturated m⁶A residues. Here, we develop a neural network, DENA, for m⁶A quantification using the sequencing data of in vivo transcripts from Arabidopsis. DENA identifies 90% of miCLIP-detected m⁶A sites in Arabidopsis and obtains modification rates in human consistent to those found by SCARLET, demonstrating its robustness across species. We sequence the transcriptome of two additional m⁶A-deficient Arabidopsis, mtb and fip37-4, using Nanopore and evaluate their single-nucleotide m⁶A profiles using DENA.

In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.