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Sunday, September 22, 2019

The landscape of SNCA transcripts across synucleinopathies: New insights from long reads sequencing analysis

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both…

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Sunday, September 22, 2019

A comparative transcriptional landscape of maize and sorghum obtained by single-molecule sequencing.

Maize and sorghum are both important crops with similar overall plant architectures, but they have key differences, especially in regard to their inflorescences. To better understand these two organisms at the molecular level, we compared expression profiles of both protein-coding and noncoding transcripts in 11 matched tissues using single-molecule, long-read, deep RNA sequencing. This comparative analysis revealed large numbers of novel isoforms in both species. Evolutionarily young genes were likely to be generated in reproductive tissues and usually had fewer isoforms than old genes. We also observed similarities and differences in alternative splicing patterns and activities, both among tissues and…

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Sunday, September 22, 2019

Targeted combinatorial alternative splicing generates brain region-specific repertoires of neurexins.

Molecular diversity of surface receptors has been hypothesized to provide a mechanism for selective synaptic connectivity. Neurexins are highly diversified receptors that drive the morphological and functional differentiation of synapses. Using a single cDNA sequencing approach, we detected 1,364 unique neurexin-a and 37 neurexin-ß mRNAs produced by alternative splicing of neurexin pre-mRNAs. This molecular diversity results from near-exhaustive combinatorial use of alternative splice insertions in Nrxn1a and Nrxn2a. By contrast, Nrxn3a exhibits several highly stereotyped exon selections that incorporate novel elements for posttranscriptional regulation of a subset of transcripts. Complexity of Nrxn1a repertoires correlates with the cellular complexity of neuronal…

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Sunday, September 22, 2019

A transcriptome atlas of rabbit revealed by PacBio single-molecule long-read sequencing.

It is widely acknowledged that transcriptional diversity largely contributes to biological regulation in eukaryotes. Since the advent of second-generation sequencing technologies, a large number of RNA sequencing studies have considerably improved our understanding of transcriptome complexity. However, it still remains a huge challenge for obtaining full-length transcripts because of difficulties in the short read-based assembly. In the present study we employ PacBio single-molecule long-read sequencing technology for whole-transcriptome profiling in rabbit (Oryctolagus cuniculus). We totally obtain 36,186 high-confidence transcripts from 14,474 genic loci, among which more than 23% of genic loci and 66% of isoforms have not been annotated yet…

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Sunday, September 22, 2019

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein…

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Sunday, September 22, 2019

Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure.

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first…

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Sunday, September 22, 2019

Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing.

Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1,…

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Sunday, September 22, 2019

Universal alternative splicing of noncoding exons.

The human transcriptome is so large, diverse, and dynamic that, even after a decade of investigation by RNA sequencing (RNA-seq), we have yet to resolve its true dimensions. RNA-seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed targeted single-molecule and short-read RNA-seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome, identifying a fundamental distinction between protein-coding and noncoding gene content: almost every noncoding exon undergoes alternative splicing, producing a seemingly limitless variety of isoforms. Analysis of syntenic regions of the…

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Sunday, September 22, 2019

Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

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Sunday, September 22, 2019

Alternative isoform analysis of Ttc8 expression in the rat pineal gland using a multi-platform sequencing approach reveals neural regulation.

Alternative isoform regulation (AIR) vastly increases transcriptome diversity and plays an important role in numerous biological processes and pathologies. However, the detection and analysis of isoform-level differential regulation is difficult, particularly in the face of complex and incompletely-annotated transcriptomes. Here we have used Illumina short-read/high-throughput RNA-Seq to identify 55 genes that exhibit neurally-regulated AIR in the pineal gland, and then used two other complementary experimental platforms to further study and characterize the Ttc8 gene, which is involved in Bardet-Biedl syndrome and non-syndromic retinitis pigmentosa. Use of the JunctionSeq analysis tool led to the detection of several novel exons and splice…

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Sunday, September 22, 2019

Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1a, Nrxn1ß, Nrxn2ß, Nrxn3a, and Nrxn3ß mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs,…

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Sunday, September 22, 2019

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study.

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for…

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Sunday, September 22, 2019

Transcriptome-wide investigation of circular RNAs in rice.

Various stable circular RNAs (circRNAs) are newly identified to be the abundance of noncoding RNAs in Archaea, Caenorhabditis elegans, mice, and humans through high-throughput deep sequencing coupled with analysis of massive transcriptional data. CircRNAs play important roles in miRNA function and transcriptional controlling by acting as competing endogenous RNAs or positive regulators on their parent coding genes. However, little is known regarding circRNAs in plants. Here, we report 2354 rice circRNAs that were identified through deep sequencing and computational analysis of ssRNA-seq data. Among them, 1356 are exonic circRNAs. Some circRNAs exhibit tissue-specific expression. Rice circRNAs have a considerable number…

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Sunday, September 22, 2019

Tracking alternatively spliced isoforms from long reads by SpliceHunter.

Alternative splicing increases the functional complexity of a genome by generating multiple isoforms and potentially proteins from the same gene. Vast amounts of alternative splicing events are routinely detected by short read deep sequencing technologies but their functional interpretation is hampered by an uncertain transcript context. Emerging long-read sequencing technologies provide a more complete picture of full-length transcript sequences. We introduce SpliceHunter, a tool for the computational interpretation of long reads generated by for example Pacific Biosciences instruments. SpliceHunter defines and tracks isoforms and novel transcription units across time points, compares their splicing pattern to a reference annotation, and translates…

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Sunday, September 22, 2019

Unveiling novel targets of paclitaxel resistance by single molecule long-read RNA sequencing in breast cancer.

RNA sequencing has become one of the most common technology to study transcriptomes in cancer, whereas its length limits its application on alternative splicing (AS) events and novel isoforms. Firstly, we applied single molecule long-read RNA sequencing (Iso-seq) and de novo assembly with short-read RNA sequencing (RNA-seq) in both wild type (231-WT) and paclitaxel resistant type (231-PTX) of human breast cancer cell MDA-MBA-231. The two sequencing technology provide both the accurate transcript sequences and the deep transcript coverage. Then we combined shor-read and long-read RNA-seq to analyze alternative events and novel isoforms. Last but not the least, we selected BAK1…

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