ClusTrast: a short read de novo transcript isoform assembler guided by clustered contigs

Motivation: Transcriptome assembly from RNA sequencing data in species without a reliable reference genome has to be performed de novo, but studies have shown that de novo methods often have inadequate reconstruction ability of transcript isoforms. This impedes the study of alternative splicing, in particular for lowly expressed isoforms. Result: We present the de novo transcript isoform assembler ClusTrast, which clusters a set of guiding contigs by similarity, aligns short reads to the guiding contigs, and assembles each clustered set of short reads individually. We tested ClusTrast on datasets from six eukaryotic species, and showed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novo assemblers, at a moderate reduction in precision. An appreciable fraction were reconstructed to at least 95% of their length. We suggest that ClusTrast will be useful for studying alternative splicing in the absence of a reference genome. Availability and implementation: The code and usage instructions are available at https://github.com/karljohanw/clustrast.

Understanding the source of METTL3-independent m6A in mRNA

N6-methyladenosine (m6A) is a highly prevalent mRNA modification which promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30—80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we re-examined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines which continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3's role in synthesizing m6A using a genomic deletion of Mettl3, and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.

Genetic Control of Splicing at SIRPG Modulates Risk of Type 1 Diabetes

<b>Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes where it acts by engaging its ligand, CD47. <i>SIRPG,</i> which encodes SIRPγ, contains a non-synonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. <i>SIRPG</i> produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of <i>SIRPG</i> transcript isoforms. All of these transcript isoforms produced protein upon transient expression <i>in vitro</i>. However, CRISPR targeting of one of the alternatively spliced exons in <i>SIRPG</i> eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling and had significantly increased levels of cell surface CD47. In primary CD4⁺ and CD8⁺ T cells cell surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that <i>SIRPG</i> is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.</b>

Genetic Control of Splicing at SIRPG Modulates Risk of Type 1 Diabetes

<b>Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes where it acts by engaging its ligand, CD47. <i>SIRPG,</i> which encodes SIRPγ, contains a non-synonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. <i>SIRPG</i> produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of <i>SIRPG</i> transcript isoforms. All of these transcript isoforms produced protein upon transient expression <i>in vitro</i>. However, CRISPR targeting of one of the alternatively spliced exons in <i>SIRPG</i> eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling and had significantly increased levels of cell surface CD47. In primary CD4⁺ and CD8⁺ T cells cell surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that <i>SIRPG</i> is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.</b>

Alternative splicing landscape of small brown planthopper and different response of JNK2 isoforms to rice stripe virus infection

Alternative splicing (AS) is a frequent posttranscriptional regulatory event occurring in response to various endogenous and exogenous stimuli in most eukaryotic organisms. However, little is known about the effects of insect-transmitted viruses on AS events in insect vectors. The present study used third-generation sequencing technology and RNA sequencing (RNA-Seq) to evaluate the AS response in the small brown planthopper Laodelphax striatellus to rice stripe virus (RSV). The full-length transcriptome of L. striatellus was obtained using single-molecule real-time sequencing technology (SMRT). Posttranscriptional regulatory events, including AS, alternative polyadenylation, and fusion transcripts, were analyzed. A total of 28,175 nonredundant transcript isoforms included 24,950 transcripts assigned to 8,500 annotated genes of L. striatellus , and 5,000 of these genes (58.8%) had AS events. RNA-Seq of the gut samples of insects infected by RSV for 8 d identified 3,458 differentially expressed transcripts (DETs); 2,185 of these DETs were transcribed from 1,568 genes that had AS events, indicating that 31.4% of alternatively spliced genes responded to RSV infection of the gut. One of the c-Jun N-terminal kinase ( JNK ) genes, JNK2 , experienced exon skipping, resulting in three transcript isoforms. These three isoforms differentially responded to RSV infection during development and in various organs. Injection of double-stranded RNAs targeting all or two isoforms indicated that three or at least two JNK2 isoforms facilitated RSV accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors. Importance Alternative splicing (AS) is a regulatory mechanism that occurs after gene transcription. AS events can enrich protein diversity to promote the reactions of the organisms to various endogenous and exogenous stimulations. It is not known how insect vectors exploit AS events to cope with transmitted viruses. The present study used third-generation sequencing technology to obtain the profile of AS events in the small brown planthopper Laodelphax striatellus , which is an efficient vector for rice stripe virus (RSV). The results indicated that 31.4% of alternatively spliced genes responded to RSV infection in the gut of planthoppers. One of the c-Jun N-terminal kinase ( JNK ) genes, JNK2 , produced three transcript isoforms by AS. These three isoforms showed different responses to RSV infection, and at least two isoforms facilitated viral accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors.

Nanopore microscope identifies RNA isoforms with structural colors

Identifying RNA transcript isoforms requires intricate protocols that suffer from various enzymatic biases. Here we design three-dimensional molecular constructs that enable identification of transcript isoforms at the single-molecule level using solid-state nanopore microscopy. We refold target RNA into RNA identifiers (IDs) with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colors. Structural colors consist of DNA structures, protein labels, native RNA structures, or a combination of all three. The sequence of structural colors of RNA IDs enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) reveals structural arrangements in native transcripts in agreement with published variants. ARTEMIS discriminates circular and linear transcript isoforms in a one step, enzyme-free reaction in a complex human transcriptome using single-molecule readout.

Time-Course Transcriptome Profiling of a Poxvirus Using Long-Read Full-Length Assay

Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of VACV gene expression.

Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.