Molecular Mechanisms Underlying the Biosynthesis of Melatonin and Its Isomer in Mulberry

Mulberry (Morus alba L.) leaves and fruit are traditional Chinese medicinal materials with anti-inflammatory, immune regulatory, antiviral and anti-diabetic properties. Melatonin performs important roles in the regulation of circadian rhythms and immune activities. We detected, identified and quantitatively analyzed the melatonin contents in leaves and mature fruit from different mulberry varieties. Melatonin and three novel isoforms were found in the Morus plants. Therefore, we conducted an expression analysis of melatonin and its isomer biosynthetic genes and in vitro enzymatic synthesis of melatonin and its isomer to clarify their biosynthetic pathway in mulberry leaves. MaASMT4 and MaASMT20, belonging to class II of the ASMT gene family, were expressed selectively in mulberry leaves, and two recombinant proteins that they expressed catalyzed the conversion of N-acetylserotonin to melatonin and one of three isomers in vitro. Unlike the ASMTs of Arabidopsis and rice, members of the three ASMT gene families in mulberry can catalyze the conversion of N-acetylserotonin to melatonin. This study provides new insights into the molecular mechanisms underlying melatonin and its isomers biosynthesis and expands our knowledge of melatonin isomer biosynthesis.

High-throughput RNA isoform sequencing using programmable cDNA concatenation

Alternative splicing is a core biological process that enables profound and essential diversification of gene function. Short-read RNA sequencing approaches fail to resolve RNA isoforms and therefore primarily enable gene expression measurements - an isoform unaware representation of the transcriptome. Conversely, full-length RNA sequencing using long-read technologies are able to capture complete transcript isoforms, but their utility is deeply constrained due to throughput limitations. Here, we introduce MAS-ISO-seq, a technique for programmably concatenating cDNAs into single molecules optimal for long-read sequencing, boosting the throughput >15 fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. We validated unambiguous isoform assignment with MAS-ISO-seq using a synthetic RNA isoform library and applied this approach to single-cell RNA sequencing of tumor-infiltrating T cells. Results demonstrated a >30 fold boosted discovery of differentially spliced genes and robust cell clustering, as well as canonical PTPRC splicing patterns across T cell subpopulations and the concerted expression of the associated hnRNPLL splicing factor. Methods such as MAS-ISO-seq will drive discovery of novel isoforms and the transition from gene expression to transcript isoform expression analyses.

Intraspecific de novo gene birth revealed by presence absence variant genes in Caenorhabditis elegans

Genes embed their evolutionary history in the form of various alleles. Presence absence variants (PAVs) are extreme cases of such alleles, where a gene present in one haplotype does not exist in another. Since PAVs may result from either birth or death of a gene, PAV genes and their alternative alleles, if available, can represent a basis for rapid intraspecific gene evolution. Here, we traced a possible evolution of PAV genes in the PD1074 and CB4856 C. elegans strains as well as their alternative alleles found in other 14 wild strains, using long-read sequencing technologies. We updated the CB4856 genome by filling 18 gaps and identified 50 novel genes and 7,460 novel isoforms from both strains. We verified 328 PAV genes, out of which 48 were C. elegans-specific. Among these possible newly-born genes, 13 had alternative alleles in other wild strains and, in particular, alternative alleles of three genes showed signatures active transposons. Alternative alleles of four other genes showed another type of signature reflected in accumulation of small insertions or deletions. Our results exemplify that research on gene evolution using both species-specific PAV genes and their alternative alleles is expected to provide new perspectives for how genes evolve.

Full-length, single-cell RNA-sequencing of human bone marrow subpopulations reveals hidden complexity

Bone marrow progenitor cell differentiation has frequently been used as a model for studying cellular plasticity and cell-fate decisions. Recent analysis at the level of single-cells has expanded knowledge of the transcriptional landscape of human hematopoietic cell lineages. Using single-molecule real-time (SMRT) full-length RNA sequencing, we have previously shown that human bone marrow lineage-negative (Lin-neg) cell populations contain a surprisingly diverse set of mRNA isoforms. Here, we report from single cell, full-length RNA sequencing that this diversity is also reflected at the single-cell level. From fresh human bone marrow unselected and lineage-negative progenitor cells were isolated by droplet-based single-cell selection (10xGenomics). The single cell-derived mRNAs were analyzed by full-length SMRT and short-read sequencing. In both samples we detected an average of 8000 different genes using short-read sequencing. Differential expression analysis arranged the single-cells of the total bone marrow into only four clusters whereas the Lin-neg population was much more diverse with nine clusters. mRNA isoform analysis of the single-cell populations using full-length sequencing revealed that Lin-neg cells contain on average 24% more novel splice variants than the total bone marrow cells. Interestingly, among the most frequent genes expressing novel isoforms were members of the spliceosome, e.g. HNRNPs, DEAD box helicases and SRSFs. Mapping the isoforms from all genes to the cell type clusters revealed that total bone marrow cells express novel isoforms only in a small subset of clusters. On the other hand, lineage-negative progenitor cells expressing novel isoforms were present in nearly all subpopulations. In conclusion, on a single-cell level lineage-negative cells express a higher diversity of genes and more alternatively spliced novel isoforms suggesting that cells in this subpopulation are poised for different fates.

Bruton’s Tyrosine Kinase and Its Isoforms in Cancer

Bruton’s tyrosine kinase (BTK) is a soluble tyrosine kinase with central roles in the development, maturation, and signaling of B cells. BTK has been found to regulate cell proliferation, survival, and migration in various B-cell malignancies. Targeting BTK with recently developed BTK inhibitors has been approved by the Food and Drug Administration (FDA) for the treatment of several hematological malignancies and has transformed the treatment of several B-cell malignancies. The roles that BTK plays in B cells have been appreciated for some time. Recent studies have established that BTK is expressed and plays pro-tumorigenic roles in several epithelial cancers. In this review, we focus on novel isoforms of the BTK protein expressed in epithelial cancers. We review recent work on the expression, function, and signaling of these isoforms and their value as potential therapeutic targets in epithelial tumors.

Full-length Transcriptome Analysis of Pecan (Carya illinoinensis) Kernels

Pecan is rich in bioactive components such as fatty acids and flavonoids and is an important nut type worldwide. Therefore, the molecular mechanisms of phytochemical biosynthesis in pecan are a focus of research. Recently, a draft genome and several transcriptomes have been published. However, the full-length mRNA transcripts remain unclear, and the regulatory mechanisms behind the quality components biosynthesis and accumulation have not been fully investigated. In this study, single-molecule long read sequencing technology was used to obtain full-length transcripts of pecan kernels. In total, 37 504 isoforms of 16 702 genes were mapped to the reference genome. The numbers of known isoforms, new isoforms, and novel isoforms were 9013 (24.03%), 26 080 (69.54%), and 2411 (6.51%), respectively. Over 80% of the transcripts (30 751, 81.99%) had functional annotations. A total of 15 465 alternative splicing (AS) events and 65 761 alternative polyadenylation events were detected; wherein, the retained intron was the predominant type (5652, 36.55%) of AS. Furthermore, 1894 long non-coding RNAs and 1643 transcription factors were predicted using bioinformatics methods. Finally, the structural genes associated with fatty acid (FA) and flavonoid biosynthesis were characterized. A high frequency of AS accuracy (70.31%) was observed in FA synthesis-associated genes. The present study provides a full-length transcriptome dataset of pecan kernels, which will significantly enhance the understanding of the regulatory basis of phytochemical biosynthesis during pecan kernel maturation.

Long-read transcriptome sequencing reveals abundant promoter diversity in distinct molecular subtypes of gastric cancer

Background Deregulated gene expression is a hallmark of cancer; however, most studies to date have analyzed short-read RNA sequencing data with inherent limitations. Here, we combine PacBio long-read isoform sequencing (Iso-Seq) and Illumina paired-end short-read RNA sequencing to comprehensively survey the transcriptome of gastric cancer (GC), a leading cause of global cancer mortality. Results We performed full-length transcriptome analysis across 10 GC cell lines covering four major GC molecular subtypes (chromosomal unstable, Epstein-Barr positive, genome stable and microsatellite unstable). We identify 60,239 non-redundant full-length transcripts, of which > 66% are novel compared to current transcriptome databases. Novel isoforms are more likely to be cell line and subtype specific, expressed at lower levels with larger number of exons, with longer isoform/coding sequence lengths. Most novel isoforms utilize an alternate first exon, and compared to other alternative splicing categories, are expressed at higher levels and exhibit higher variability. Collectively, we observe alternate promoter usage in 25% of detected genes, with the majority (84.2%) of known/novel promoter pairs exhibiting potential changes in their coding sequences. Mapping these alternate promoters to TCGA GC samples, we identify several cancer-associated isoforms, including novel variants of oncogenes. Tumor-specific transcript isoforms tend to alter protein coding sequences to a larger extent than other isoforms. Analysis of outcome data suggests that novel isoforms may impart additional prognostic information. Conclusions Our results provide a rich resource of full-length transcriptome data for deeper studies of GC and other gastrointestinal malignancies.

Global tissue transcriptomic analysis to improve genome annotation and unravel skin pigmentation in goldfish

Goldfish is an ornamental fish with diverse phenotypes. However, the limited genomic resources of goldfish hamper our understanding of the genetic basis for its phenotypic diversity. To provide enriched genomic resources and infer possible mechanisms underlying skin pigmentation, we performed a large-scale transcriptomic sequencing on 13 adult goldfish tissues, larvae at one- and three-days post hatch, and skin tissues with four different color pigmentation. A total of 25.52 Gb and 149.80 Gb clean data were obtained using the PacBio and Illumina platforms, respectively. Onto the goldfish reference genome, we mapped 137,674 non-redundant transcripts, of which 5.54% was known isoforms and 78.53% was novel isoforms of the reference genes, and the remaining 21,926 isoforms are novel isoforms of additional new genes. Both skin-specific and color-specific transcriptomic analyses showed that several significantly enriched genes were known to be involved in melanogenesis, tyrosine metabolism, PPAR signaling pathway, folate biosynthesis metabolism and so on. Thirteen differentially expressed genes across different color skins were associated with melanogenesis and pteridine synthesis including mitf, ednrb, mc1r, tyr, mlph and gch1, and xanthophore differentiation such as pax7, slc2a11 and slc2a15. These transcriptomic data revealed pathways involved in goldfish pigmentation and improved the gene annotation of the reference genome.

Long-read isoform sequencing reveals survival-associated splicing in breast cancer

SummaryTumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is not known. We developed a long-read RNA sequencing and analytical platform that identifies and annotates full-length isoforms, and infers tumor-specific splicing events. Application of this platform to breast cancer samples vastly expands the known isoform landscape of breast cancer, identifying thousands of previously unannotated isoforms of which ~30% impact protein coding exons and are predicted to alter protein localization and function, including of the breast cancer-associated genes ESR1 and ERBB2. We performed extensive cross-validation with -omics data sets to support transcription and translation of novel isoforms. We identified 3,059 breast tumor-specific splicing events, including 35 that are significantly associated with patient survival. Together, our results demonstrate the complexity, cancer subtype-specificity, and clinical relevance of novel isoforms in breast cancer that are only annotatable by LR-seq, and provide a rich resource of immuno-oncology therapeutic targets.