Identification of genome-wide alternative splicing events in sequential, isogenic clinical isolates of Candida albicans reveals a mechanism important for drug resistance and tolerance to cellular stress

Alternative gene splicing (AS) is a process by which a single gene can give rise to different protein isoforms, generating proteome diversity. Despite recent advances in our understanding of AS in basic cellular processes, the role of AS in drug resistance and fungal pathogenesis is poorly understood. In Candida albicans, approximately 6% of the genes contain introns. Considering this low and random distribution of introns, we focused our study on alternative splicing (AS) and its impact on the development of drug resistance, an area largely unexplored in this yeast. We performed comparative RNA sequencing of sequential isogenic azole sensitive and resistant isolates of C. albicans. The analysis revealed differential expression of splice junctions/isoforms in 14 genes, between the drug sensitive and resistant isolates. Furthermore, C. albicans WT cells exposed to antifungal drugs, heat stress or metal deficiency also showed differential expression of isoforms for the genes undergoing AS. In this study we present data on the effect of AS on the function of SOD3. The C. albicans SOD3 has a single intron and is important for the removal of superoxide radicals. The overexpression of the two isoforms of SOD3 in its null background highlighted importance of spliced isoform in complementing the susceptibility to menadione. However, the two isoforms did not differ in rescuing the susceptibility of sod3Δ/Δto Amphotericin B. Collectively, these data suggest that AS may be a novel mechanism in C. albicans for stress adaptation and overcoming drug resistance.

Evolution of the Early Spliceosomal Complex—From Constitutive to Regulated Splicing

Pre-mRNA splicing is a major process in the regulated expression of genes in eukaryotes, and alternative splicing is used to generate different proteins from the same coding gene. Splicing is a catalytic process that removes introns and ligates exons to create the RNA sequence that codifies the final protein. While this is achieved in an autocatalytic process in ancestral group II introns in prokaryotes, the spliceosome has evolved during eukaryogenesis to assist in this process and to finally provide the opportunity for intron-specific splicing. In the early stage of splicing, the RNA 5′ and 3′ splice sites must be brought within proximity to correctly assemble the active spliceosome and perform the excision and ligation reactions. The assembly of this first complex, termed E-complex, is currently the least understood process. We focused in this review on the formation of the E-complex and compared its composition and function in three different organisms. We highlight the common ancestral mechanisms in S. cerevisiae, S. pombe, and mammals and conclude with a unifying model for intron definition in constitutive and regulated co-transcriptional splicing.

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3′ UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

Improved prediction of smoking status via isoform-aware RNA-seq deep learning models

Most predictive models based on gene expression data do not leverage information related to gene splicing, despite the fact that splicing is a fundamental feature of eukaryotic gene expression. Cigarette smoking is an important environmental risk factor for many diseases, and it has profound effects on gene expression. Using smoking status as a prediction target, we developed deep neural network predictive models using gene, exon, and isoform level quantifications from RNA sequencing data in 2,557 subjects in the COPDGene Study. We observed that models using exon and isoform quantifications clearly outperformed gene-level models when using data from 5 genes from a previously published prediction model. Whereas the test set performance of the previously published model was 0.82 in the original publication, our exon-based models including an exon-to-isoform mapping layer achieved a test set AUC (area under the receiver operating characteristic) of 0.88, which improved to an AUC of 0.94 using exon quantifications from a larger set of genes. Isoform variability is an important source of latent information in RNA-seq data that can be used to improve clinical prediction models.

PAEP and cilia gene splicing changes in endometrial glands during the implantation window in women with recurrent pregnancy loss

ABSTRACTEndometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and PAEP gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.

Splicing Outcomes of 5′ Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays

Combining data derived from a meta-analysis of human disease-associated 5′ splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15–18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.

Determining the pathogenicity of MUT gene variant by mini-gene splicing assay

ObjectiveTo perform gene mutation splicing analysis on 12 suspected pathogenic MMUT/MUT gene mutation sites to verify the pathogenicity of the mutation.MethodsWild-type and mutant minigenes were inserted into pcMINI vector, and total 5 wild-type recombinant vectors and 12 mutant recombinant vectors were constructed. The total RNA in 293T cells was extracted after the recombinant vectors were transfected into 293T cell line. Then PCR products were detected by agarose gel electrophoresis and analyzed by sequencing.ResultsRT-PCR and sequencing results showed that among 12 mutations, 9 mutations (c.419T>C, 469G>T, c.470T>A, c.626dupC, c.693C>G, c.976A>G, c.1009T>C, c.1777G>T and c.1874A>C) did not affect the gene splicing, and the other 3 mutations (c.454C>T, c.421G>A and c.2125-3C>G) all affected mRNA splicing.ConclusionIn recent case reports of MMUT/MUT gene mutation sites, variant of uncertain significance (VUS) variation is very common. In this study, the pathogenicity of three mutation sites is confirmed by the mini-gene method.