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1. Identification of cell-type specific alternative transcripts in the multicellular alga Volvox carteri

   https://doi.org/10.1186/s12864-023-09558-0  

   Balasubramanian, Ravi Nicholas ; Gao, Minglu ; Umen, James ( October 2023 , BMC Genomics)

   Cell type specialization is a hallmark of complex multicellular organisms and is usually established through implementation of cell-type-specific gene expression programs. The multicellular green algaVolvox carterihas just two cell types, germ and soma, that have previously been shown to have very different transcriptome compositions which match their specialized roles. Here we interrogated another potential mechanism for differentiation inV. carteri, cell type specific alternative transcript isoforms (CTSAI).

   We used pre-existing predictions of alternative transcripts and de novo transcript assembly with HISAT2 and Ballgown software to compile a list of loci with two or more transcript isoforms, identified a small subset that were candidates for CTSAI, and manually curated this subset of genes to remove false positives. We experimentally verified three candidates using semi-quantitative RT-PCR to assess relative isoform abundance in each cell type.

   Of the 1978 loci with two or more predicted transcript isoforms 67 of these also showed cell type isoform expression biases. After curation 15 strong candidates for CTSAI were identified, three of which were experimentally verified, and their predicted gene product functions were evaluated in light of potential cell type specific roles. A comparison of genes with predicted alternative splicing fromChlamydomonas reinhardtii, a unicellular relative ofV. carteri, identified little overlap between ortholog pairs with alternative splicing in both species. Finally, we interrogated cell type expression patterns of 126 V. carteripredicted RNA binding protein (RBP) encoding genes and found 40 that showed either somatic or germ cell expression bias. These RBPs are potential mediators of CTSAI inV. carteriand suggest possible pre-adaptation for cell type specific RNA processing and a potential path for generating CTSAI in the early ancestors of metazoans and plants.

   We predicted numerous instances of alternative transcript isoforms in Volvox, only a small subset of which showed cell type specific isoform expression bias. However, the validated examples of CTSAI supported existing hypotheses about cell type specialization inV. carteri,and also suggested new hypotheses about mechanisms of functional specialization for their gene products. Our data imply that CTSAI operates as a minor but important component ofV. cartericellular differentiation and could be used as a model for how alternative isoforms emerge and co-evolve with cell type specialization.

    

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2. Alternative splicing preferentially increases transcript diversity associated with stress responses in the extremophyte Schrenkiella parvula

   Wijesinghege, Chathura ; Tran, Kieu-Nga ; Dassanayake, Maheshi ( October 2022 , bioRxiv)

   Alternative splicing extends the coding potential of genomes by creating multiple isoforms from one gene. Isoforms can render transcript specificity and diversity to initiate multiple responses required during transcriptome adjustments in stressed environments. Although the prevalence of alternative splicing is widely recognized, how diverse isoforms facilitate stress adaptation in plants that thrive in extreme environments are unexplored. Here we examine how an extremophyte model, Schrenkiella parvula, coordinates alternative splicing in response to high salinity compared to a salt-stress sensitive model, Arabidopsis thaliana. We use Iso-Seq to generate full length reference transcripts and RNA-seq to quantify differential isoform usage in response to salinity changes. We find that single-copy orthologs where S. parvula has a higher number of isoforms than A. thaliana as well as S. parvula genes observed and predicted using machine learning to have multiple isoforms are enriched in stress associated functions. Genes that showed differential isoform usage were largely mutually exclusive from genes that were differentially expressed in response to salt. S. parvula transcriptomes maintained specificity in isoform usage assessed via a measure of expression disorderdness during transcriptome reprogramming under salt. Our study adds a novel resource and insight to study plant stress tolerance evolved in extreme environments. 

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

   https://doi.org/10.1101/gr.274282.120  

   Naftaly, Alice S. ; Pau, Shana ; White, Michael A. ( August 2021 , Genome Research)

   Alternate isoforms are important contributors to phenotypic diversity across eukaryotes. Although 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 use Pacific Biosciences (PacBio) long-read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five organs in threespine stickleback fish ( Gasterosteus aculeatus ), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly in which gene annotations 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 and verified through ATAC-seq. We also detected many alternative splicing events between sexes and across organs. We found a substantial number of genes in both somatic and gonadal samples 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. 

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4. DIFFUSE: predicting isoform functions from sequences and expression profiles via deep learning

   https://doi.org/10.1093/bioinformatics/btz367  

   Chen, Hao ; Shaw, Dipan ; Zeng, Jianyang ; Bu, Dongbo ; Jiang, Tao ( July 2019 , Bioinformatics)

   Alternative splicing generates multiple isoforms from a single gene, greatly increasing the functional diversity of a genome. Although gene functions have been well studied, little is known about the specific functions of isoforms, making accurate prediction of isoform functions highly desirable. However, the existing approaches to predicting isoform functions are far from satisfactory due to at least two reasons: (i) unlike genes, isoform-level functional annotations are scarce. (ii) The information of isoform functions is concealed in various types of data including isoform sequences, co-expression relationship among isoforms, etc.

   In this study, we present a novel approach, DIFFUSE (Deep learning-based prediction of IsoForm FUnctions from Sequences and Expression), to predict isoform functions. To integrate various types of data, our approach adopts a hybrid framework by first using a deep neural network (DNN) to predict the functions of isoforms from their genomic sequences and then refining the prediction using a conditional random field (CRF) based on co-expression relationship. To overcome the lack of isoform-level ground truth labels, we further propose an iterative semi-supervised learning algorithm to train both the DNN and CRF together. Our extensive computational experiments demonstrate that DIFFUSE could effectively predict the functions of isoforms and genes. It achieves an average area under the receiver operating characteristics curve of 0.840 and area under the precision–recall curve of 0.581 over 4184 GO functional categories, which are significantly higher than the state-of-the-art methods. We further validate the prediction results by analyzing the correlation between functional similarity, sequence similarity, expression similarity and structural similarity, as well as the consistency between the predicted functions and some well-studied functional features of isoform sequences.

   https://github.com/haochenucr/DIFFUSE.

   Supplementary data are available at Bioinformatics online.

    

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5. The Genetics of Fitness Reorganization during the Transition to Multicellularity: The Volvocine regA-like Family as a Model

   https://doi.org/10.3390/genes14040941  

   Grochau-Wright, Zachariah I. ; Nedelcu, Aurora M. ; Michod, Richard E. ( April 2023 , Genes)

   The evolutionary transition from single-celled to multicellular individuality requires organismal fitness to shift from the cell level to a cell group. This reorganization of fitness occurs by re-allocating the two components of fitness, survival and reproduction, between two specialized cell types in the multicellular group: soma and germ, respectively. How does the genetic basis for such fitness reorganization evolve? One possible mechanism is the co-option of life history genes present in the unicellular ancestors of a multicellular lineage. For instance, single-celled organisms must regulate their investment in survival and reproduction in response to environmental changes, particularly decreasing reproduction to ensure survival under stress. Such stress response life history genes can provide the genetic basis for the evolution of cellular differentiation in multicellular lineages. The regA-like gene family in the volvocine green algal lineage provides an excellent model system to study how this co-option can occur. We discuss the origin and evolution of the volvocine regA-like gene family, including regA—the gene that controls somatic cell development in the model organism Volvox carteri. We hypothesize that the co-option of life history trade-off genes is a general mechanism involved in the transition to multicellular individuality, making volvocine algae and the regA-like family a useful template for similar investigations in other lineages. 

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