BRIE2: computational identification of splicing phenotypes from single-cell transcriptomic experiments

RNA splicing is an important driver of heterogeneity in single cells through the expression of alternative transcripts and as a determinant of transcriptional kinetics. However, the intrinsic coverage limitations of scRNA-seq technologies make it challenging to associate specific splicing events to cell-level phenotypes. BRIE2 is a scalable computational method that resolves these issues by regressing single-cell transcriptomic data against cell-level features. We show that BRIE2 effectively identifies differential disease-associated alternative splicing events and allows a principled selection of genes that capture heterogeneity in transcriptional kinetics and improve RNA velocity analyses, enabling the identification of splicing phenotypes associated with biological changes.

Human STAT3 variants underlie autosomal dominant hyper-IgE syndrome by negative dominance

Most patients with autosomal dominant hyper-IgE syndrome (AD-HIES) carry rare heterozygous STAT3 variants. Only six of the 135 in-frame variants reported have been experimentally shown to be dominant negative (DN), and it has been recently suggested that eight out-of-frame variants operate by haploinsufficiency. We experimentally tested these 143 variants, 7 novel out-of-frame variants found in HIES patients, and other STAT3 variants from the general population. Strikingly, all 15 out-of-frame variants were DN via their encoded (1) truncated proteins, (2) neoproteins generated from a translation reinitiation codon, and (3) isoforms from alternative transcripts or a combination thereof. Moreover, 128 of the 135 in-frame variants (95%) were also DN. The patients carrying the seven non-DN STAT3 in-frame variants have not been studied for other genetic etiologies. Finally, none of the variants from the general population tested, including an out-of-frame variant, were DN. Overall, our findings show that heterozygous STAT3 variants, whether in or out of frame, underlie AD-HIES through negative dominance rather than haploinsufficiency.

The Alter Retina: Alternative Splicing of Retinal Genes in Health and Disease

Alternative splicing of mRNA is an essential mechanism to regulate and increase the diversity of the transcriptome and proteome. Alternative splicing frequently occurs in a tissue- or time-specific manner, contributing to differential gene expression between cell types during development. Neural tissues present extremely complex splicing programs and display the highest number of alternative splicing events. As an extension of the central nervous system, the retina constitutes an excellent system to illustrate the high diversity of neural transcripts. The retina expresses retinal specific splicing factors and produces a large number of alternative transcripts, including exclusive tissue-specific exons, which require an exquisite regulation. In fact, a current challenge in the genetic diagnosis of inherited retinal diseases stems from the lack of information regarding alternative splicing of retinal genes, as a considerable percentage of mutations alter splicing or the relative production of alternative transcripts. Modulation of alternative splicing in the retina is also instrumental in the design of novel therapeutic approaches for retinal dystrophies, since it enables precision medicine for specific mutations.

THE EXPRESSION OF THE MAIN AND ALTERNATIVE TRANSCRIPT (SORL1-Δ2) OF THE SORL1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS AFFECTED BY THE CHORNOBYL ACCIDENT

Objective: to study clinical-hematological data and expression of the main and alternative transcripts of SORL1 gene in chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophe. Methods. Analysis was performed in the main group of 34 CLL patients irradiated due to the Chornobyl NPP accident (30 clean-up workers, and 4 evacuees) and in the control group of 27 non-irradiated CLL patients. Groups of patients were comparable by age, sex, stage of disease, mutational status of IGHV genes. Expression of the main and alternative transcripts of SORL1 gene was evaluated by Quantitative Real-time polymerase chain reaction (PCR). The IGHV gene mutational status, TP53 and SF3B1 mutations were studied by PCR followed by direct sequencing. Data were analyzed with the SPSS software package, version 20.0. Results. Relative expression level of the main transcript of SORL1 gene was low (mean 1.71 ± 0.55, median 0.57), did not correlate with the IGHV gene mutational status, TP53 and SF3B1 mutations, stage of disease. The expression of B transcript was not detected, F transcript was expressed at a very low level in 9 patients. The average relative expression level of SORL1-Δ2 transcript was 14.1 ± 6.04 (median 3.48; range 0.01–90.51). The expression of SORL1-Δ2 transcript above the median was more frequent among patients on C stage (p = 0.001), and in patients with unmutated IGHV genes was associated with an extremely negative course of CLL (median of overall survival 9 months vs 61 months at low expression). Relative expression levels of the main and alternative transcripts of SORL1 gene in patients of the main and the control groups did not differ. Conclusions. Our preliminary data suggest that increased expression of SORL1-Δ2 transcript in CLL patients with unmutated IGHV genes can be considered as a negative prognostic marker. Key words: chronic lymphocytic leukemia, SORL1, SORL1-Δ2, Chornobyl NPP accident.

Ranked Choice Voting for Representative Transcripts with TRaCE

Genome sequencing projects annotate protein-coding gene models with multiple transcripts, aiming to represent all of the available transcript evidence. However, downstream analyses often operate on only one representative transcript per gene locus, sometimes known as the canonical transcript. To choose canonical transcripts, TRaCE (Transcript Ranking and Canonical Election) holds an 'election' in which a set of RNA-seq samples rank transcripts by annotation edit distance. These sample-specific votes are tallied along with other criteria such as protein length and InterPro domain coverage. The winner is selected as the canonical transcript, but the election proceeds through multiple rounds of voting to order all the transcripts by relevance. Based on the set of expression data provided, TRaCE can identify the most common isoforms from a broad expression atlas or prioritize alternative transcripts expressed in specific contexts.

Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish

Alternate isoforms contribute immensely to phenotypic diversity across eukaryotes. While short read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length transcripts, preventing the identification of many alternate isoforms. Long-read sequencing technologies have made it possible to sequence full length alternative transcripts, accurately characterizing alternative splicing events, alternate transcription start and end sites, and differences in UTR regions. Here, we utilize PacBio long read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five tissues in threespine stickleback fish (Gasterosteus aculeatus), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly with gene annotations that are based on short-read RNA sequencing and predictions from coding sequence of other species. This suggests some of the existing annotations may be inaccurate or alternative transcripts may not be fully characterized. Using Iso-Seq we detected thousands of novel isoforms, indicating many isoforms are absent in the current Ensembl gene annotations. In addition, we refined many of the existing annotations within the genome. We noted many improperly positioned transcription start sites that were refined with long-read sequencing. The Iso-Seq predicted transcription start sites were more accurate, verified through ATAC-seq. We were also able to detect many alternative splicing events between sexes and across tissues. We found a substantial number of genes in both somatic and gonad tissue that had sex-specific isoforms. Our study highlights the power of long-read sequencing to study the complexity of transcriptomes, greatly improving genomic resources for the threespine stickleback fish.

F-Box Gene D5RF Is Regulated by Agrobacterium Virulence Protein VirD5 and Essential for Agrobacterium-Mediated Plant Transformation

We previously reported that the Agrobacterium virulence protein VirD5 possesses transcriptional activation activity, binds to a specific DNA element D5RE, and is required for Agrobacterium-mediated stable transformation, but not for transient transformation. However, direct evidence for a role of VirD5 in plant transcriptional regulation has been lacking. In this study, we found that the Arabidopsis gene D5RF (coding for VirD5 response F-box protein, At3G49480) is regulated by VirD5. D5RF has two alternative transcripts of 930 bp and 1594 bp that encode F-box proteins of 309 and 449 amino acids, designated as D5RF.1 and D5RF.2, respectively. D5RF.2 has a N-terminal extension of 140 amino acids compared to D5RF.1, and both of them are located in the plant cell nucleus. The promoter of the D5RF.1 contains two D5RE elements and can be activated by VirD5. The expression of D5RF is downregulated when the host plant is infected with virD5 deleted Agrobacterium. Similar to VirD5, D5RF also affects the stable but not transient transformation efficiency of Agrobacterium. Some pathogen-responsive genes are downregulated in the d5rf mutant. In conclusion, this study further confirmed Agrobacterium VirD5 as the plant transcription activator and identified Arabidopsis thalianaD5RF.1 as the first target gene of VirD5 in regulation.