Transcript Isoforms Research Articles

Isoform-Level Transcriptome Analysis of Peripheral Blood Mononuclear Cells from Breast Cancer Patients Identifies a Disease-Associated RASGEF1A Isoform.

Background: Breast cancer (BC) comprises multiple subtypes with distinct molecular features, which differ in their interplay with host immunity, prognosis, and treatment. Non-invasive blood analyses can provide valuable insights into systemic immunity during cancer. The aim of this study was to analyze the expression of transcriptional isoforms in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women to identify potential BC immune biomarkers. Methods: RNA sequencing and isoform-level bioinformatics were performed on PBMCs from 12 triple-negative and 13 luminal A patients. Isoform expression validation by qRT-PCR and clinicopathological correlations were performed in a larger cohort (156 BC patients and 32 healthy women). Results: Transcriptional analyses showed a significant (p < 0.001) decrease in the ENST00000374459 RASGEF1A isoform in PBMCs of BC compared to healthy subjects, indicating disease-related expression changes. The decrease was associated with higher ctDNA and Ki-67 values. Conclusions: The levels of the RASGEF1A transcriptional isoform ENST00000374459 may have the potential to distinguish between BC and healthy subjects. The downregulation of ENST00000374459 in breast cancer is associated with higher proliferation and ctDNA shedding. Specialized bioinformatics analyses such as isoform analyses hold significant promise in the detection of biomarkers, since standard RNA sequencing analyses may overlook specific transcriptional changes that may be disease-associated and biologically important.

NMDtxDB: data-driven identification and annotation of human NMD target transcripts.

The nonsense-mediated RNA decay (NMD) pathway is a crucial mechanism of mRNA quality control. Current annotations of NMD substrate RNAs are rarely data-driven, but use generally established rules. We present a data set with four cell lines and combinations for SMG5, SMG6, and SMG7 knockdowns or SMG7 knockout. Based on this data set, we implemented a workflow that combines Nanopore and Illumina sequencing to assemble a transcriptome, which is enriched for NMD target transcripts. Moreover, we use coding sequence information (CDS) from Ensembl, Gencode consensus Ribo-seq ORFs, and OpenProt to enhance the CDS annotation of novel transcript isoforms. In summary, 302,889 transcripts were obtained from the transcriptome assembly process, out of which 24% are absent from Ensembl database annotations, 48,213 contain a premature stop codon, and 6433 are significantly upregulated in three or more comparisons of NMD active versus deficient cell lines. We present an in-depth view of these results through the NMDtxDB database, which is available at https://shiny.dieterichlab.org/app/NMDtxDB, and supports the study of NMD-sensitive transcripts. We open sourced our implementation of the respective web-application and analysis workflow at https://github.com/dieterich-lab/NMDtxDB and https://github.com/dieterich-lab/nmd-wf.

Long-read sequencing transcriptome quantification with lr-kallisto.

RNA abundance quantification has become routine and affordable thanks to high-throughput "short-read" technologies that provide accurate molecule counts at the gene level. Similarly accurate and affordable quantification of definitive full-length, transcript isoforms has remained a stubborn challenge, despite its obvious biological significance across a wide range of problems. "Long-read" sequencing platforms now produce data-types that can, in principle, drive routine definitive isoform quantification. However some particulars of contemporary long-read datatypes, together with isoform complexity and genetic variation, present bioinformatic challenges. We show here, using ONT data, that fast and accurate quantification of long-read data is possible and that it is improved by exome capture. To perform quantifications we developed lr-kallisto, which adapts the kallisto bulk and single-cell RNA-seq quantification methods for long-read technologies.

Progerin mRNA expression in non-HGPS patients is correlated with widespread shifts in transcript isoforms.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease caused primarily by a C1824T mutation in LMNA. This mutation activates a cryptic splice donor site, producing a lamin variant called progerin. Interestingly, progerin has also been detected in cells and tissues of non-HGPS patients. Here, we investigated progerin expression using publicly available RNA-seq data from non-HGPS patients in the GTEx project. We found that progerin expression is present across all tissue types in non-HGPS patients and correlated with telomere shortening in the skin. Transcriptome-wide correlation analyses suggest that the level of progerin expression is correlated with switches in gene isoform expression patterns. Differential expression analyses show that progerin expression is correlated with significant changes in genes involved in splicing regulation and mitochondrial function. Interestingly, 5' splice sites whose use is correlated with progerin expression have significantly altered frequencies of consensus trinucleotides within the core 5' splice site. Furthermore, introns whose alternative splicing correlates with progerin have reduced GC content. Our study suggests that progerin expression in non-HGPS patients is part of a global shift in splicing patterns.

Genomic analyses of intricate interaction of TE-lncRNA overlapping genes with miRNAs in human diseases.

Transposable elements (TEs) are known to be inserted into genome to create transcript isoforms or to generate long non-coding RNA (lncRNA) sequences. The insertion of TEs generates a gene protein sequence within the genome, but also provides a microRNA (miRNA) regulatory region. To determine the effect of gene sequence changes caused by TE insertion on miRNA binding and to investigate the formation of an overlapping lncRNA that represses it. The distribution of overlapping regions between exons and TE regions with lncRNA was examined using the Bedtools. miRNAs that can bind to those overlapping regions were identified through the miRDB web program. For TE-lncRNA overlapping genes, bioinformatic analysis was conducted using DAVID web database. Differential expression analysis was conducted using data from the GEO dataset and TCGA. Most TEs were distributed more frequently in untranslated regions than open reading frames. There were 30 annotated TE-lncRNA overlapping genes with same strand that could bind to the same miRNA. As a result of identifying the association between these 30 genes and diseases, TGFB2, FCGR2A, DCTN5, and IFI6 were associated with breast cancer, and HMGCS1, FRMD4A, EDNRB, and SNCA were associated with Alzheimer's disease. Analysis of the GEO and TCGA data showed that the relevant expression of miR-891a and miR-28, which bind to the TE overlapping region of DCTN5 and HMGCS1, decreased. This study indicates that the interaction between TE-lncRNA overlapping genes and miRNAs can affect disease progression.

Upstream alternative polyadenylation in SCN5A produces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration.

SCN5A encodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials. SCN5A gene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the first SCN5A coding exon. This yields a SCN5A-short transcript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-length SCN5A mRNA ratios in failing hearts. Knock-in of the human SCN5A APA sequence into mice is sufficient to enable expression of SCN5A -short transcript, while significantly decreasing expression of full-length SCN5A mRNA. Notably, SCN5A -short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation of SCN5A produces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.

Sleep and diurnal alternative polyadenylation sites associated with human APA-linked brain disorders.

Disruption of sleep and circadian rhythms are a comorbid feature of many pathologies, and can negatively influence many health conditions, including neurodegenerative disease, metabolic illness, cancer, and various neurological disorders. Genetic association studies linking sleep and circadian disturbances with disease susceptibility have mainly focused on changes in gene expression due to mutations, such as single-nucleotide polymorphisms. The interaction between sleep and/or circadian rhythms with the use of Alternative Polyadenylation (APA) has been largely undescribed, particularly in the context of other disorders. APA is a process that generates various transcript isoforms of the same gene affecting its mRNA translation, stability, localization, and subsequent function. Here we identified unique APAs expressed in rat brain over time-of-day, immediately following sleep deprivation, and the subsequent recovery period. From these data, we performed a secondary analysis of these sleep- or time-of-day associated PASs with recently described APA-linked human brain disorder susceptibility genes.

R2Dtool: integration and visualization of isoform-resolved RNA features.

Long-read RNA sequencing enables the mapping of RNA modifications, structures, and protein-interaction sites at the resolution of individual transcript isoforms. To understand the functions of these RNA features, it is critical to analyze them in the context of transcriptomic and genomic annotations, such as open reading frames and splice junctions. We have developed R2Dtool, a bioinformatics tool that integrates transcript-mapped information with transcript and genome annotations, allowing for the isoform-resolved analytics and graphical representation of RNA features in their genomic context. We illustrate R2Dtool's capability to integrate and expedite RNA feature analysis using epitranscriptomics data. R2Dtool facilitates the comprehensive analysis and interpretation of alternative transcript isoforms. R2Dtool is freely available under the MIT license at github.com/comprna/R2Dtool.

Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease

A major fraction of loci identified by genome-wide association studies (GWASs) mediate alternative splicing, but mechanistic interpretation is hindered by the technical limitations of short-read RNA sequencing (RNA-seq), which cannot directly link splicing events to full-length protein isoforms. Long-read RNA-seq represents a powerful tool to characterize transcript isoforms, and recently, infer protein isoform existence. Here, we present an approach that integrates information from GWASs, splicing quantitative trait loci (sQTLs), and PacBio long-read RNA-seq in a disease-relevant model to infer the effects of sQTLs on the ultimate protein isoform products they encode. We demonstrate the utility of our approach using bone mineral density (BMD) GWAS data. We identified 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes that colocalized with BMD associations (H4PP ≥ 0.75). We generated PacBio Iso-Seq data (N= ∼22 million full-length reads) on human osteoblasts, identifying 68,326 protein-coding isoforms, of which 17,375 (25%) were unannotated. By casting the sQTLs onto protein isoforms, we connected 809 sQTLs to 2,029 protein isoforms from 441 genes expressed in osteoblasts. Overall, we found that 74 sQTLs influenced isoforms likely impacted by nonsense-mediated decay and 190 that potentially resulted in the expression of unannotated protein isoforms. Finally, we functionally validated colocalizing sQTLs in TPM2, in which siRNA-mediated knockdown in osteoblasts showed two TPM2 isoforms with opposing effects on mineralization but exhibited no effect upon knockdown of the entire gene. Our approach should be to generalize across diverse clinical traits and to provide insights into protein isoform activities modulated by GWAS loci.

Transcriptional Isoforms of NAD+ kinase regulate oxidative stress resistance and melanoma metastasis

Metastasizing cancer cells encounter a multitude of stresses throughout the metastatic cascade. Oxidative stress is known to be a major barrier for metastatic colonization, such that metastasizing cancer cells must rewire their metabolic pathways to increase their antioxidant capacity. NADPH is essential for regeneration of cellular antioxidants and several NADPH-regenerating pathways have been shown to play a role in metastasis. We have found that metastatic melanoma cells have increased levels of both NADPH and NADP+ suggesting increased de novo biosynthesis of NADP+. De novo biosynthesis of NADP+ occurs through a single enzymatic reaction catalyzed by NAD+ kinase (NADK). Here we show that different NADK isoforms are differentially expressed in metastatic melanoma cells, with Isoform 3 being specifically upregulated in metastasis. We find that Isoform 3 is more potent in expanding the NADP(H) pools, increasing oxidative stress resistance and promoting metastatic colonization compared to Isoform 1. We have found that Isoform 3 is transcriptionally upregulated by oxidative stress through the action of NRF2. Together, our work presents a previously uncharacterized role of NADK isoforms in oxidative stress resistance and metastasis and suggests that NADK Isoform 3 is a potential therapeutic target in metastatic disease.

Get Spliced: Uniting Alternative Splicing and Arthritis.

Immune responses demand the rapid and precise regulation of gene protein expression. Splicing is a crucial step in this process; ~95% of protein-coding gene transcripts are spliced during mRNA maturation. Alternative splicing allows for distinct functional regulation, as it can affect transcript degradation and can lead to alternative functional protein isoforms. There is increasing evidence that splicing can directly regulate immune responses. For several genes, immune cells display dramatic changes in isoform-level transcript expression patterns upon activation. Recent advances in long-read RNA sequencing assays have enabled an unbiased and complete description of transcript isoform expression patterns. With an increasing amount of cell types and conditions that have been analyzed with such assays, thousands of novel transcript isoforms have been identified. Alternative splicing has been associated with autoimmune diseases, including arthritis. Here, GWASs revealed that SNPs associated with arthritis are enriched in splice sites. In this review, we will discuss how alternative splicing is involved in immune responses and how the dysregulation of alternative splicing can contribute to arthritis pathogenesis. In addition, we will discuss the therapeutic potential of modulating alternative splicing, which includes examples of spliceform-based biomarkers for disease severity or disease subtype, splicing manipulation using antisense oligonucleotides, and the targeting of specific immune-related spliceforms using antibodies.

Swi/Snf chromatin remodeling regulates transcriptional interference and gene repression

Alternative transcription start sites can affect transcript isoform diversity and translation levels. In a recently described form of gene regulation, coordinated transcriptional and translational interference results in transcript isoform-dependent changes in protein expression. Specifically, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, interfering with expression of the gene-proximal promoter. Although transcriptional and chromatin features associated with LUTI expression have been described, the mechanism underlying LUTI-based transcriptional interference is not well understood. Using an unbiased genetic approach followed by functional genomics, we uncovered that the Swi/Snf chromatin remodeling complex is required for co-transcriptional nucleosome remodeling that leads to LUTI-based repression. We identified genes with tandem promoters that rely on Swi/Snf function for transcriptional interference during protein folding stress, including LUTI-regulated genes. This study provides clear evidence for Swi/Snf playing a direct role in gene repression via a cis transcriptional interference mechanism.

MiRglmm: a generalized linear mixed model of isomiR-level counts improves estimation of miRNA-level differential expression and uncovers variable differential expression between isomiRs.

MicroRNA-seq data is produced by aligning small RNA sequencing reads of different miRNA transcript isoforms, called isomiRs, to known microRNAs. Aggregation to microRNA-level counts discards information and violates core assumptions of differential expression (DE) methods developed for mRNA-seq data. We establish miRglmm, a DE method for microRNA-seq data, that uses a generalized linear mixed model of isomiR-level counts, facilitating detection of miRNA with differential expression or differential isomiR usage. We demonstrate that miRglmm outperforms current DE methods in estimating DE for miRNA, whether or not there is significant isomiR variability, and simultaneously provides estimates of isomiR-level DE.

IS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection.

Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of predefined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (lrRNA-seq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNaseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This lrRNA-seq-informed Tomahto targeted approach is a new modality for generating protein-level evidence of alternative isoforms─a critical first step in designing functional studies and eventually clinical assays.

Accurate isoform quantification by joint short- and long-read RNA-sequencing.

Accurate quantification of transcript isoforms is crucial for understanding gene regulation, functional diversity, and cellular behavior. Existing RNA sequencing methods have significant limitations: short-read (SR) sequencing provides high depth but struggles with isoform deconvolution, whereas long-read (LR) sequencing offers isoform resolution at the cost of lower depth, higher noise, and technical biases. Addressing this gap, we introduce Multi-Platform Aggregation and Quantification of Transcripts (MPAQT), a generative model that combines the complementary strengths of different sequencing platforms to achieve state-of-the-art isoform-resolved transcript quantification, as demonstrated by extensive simulations and experimental benchmarks. By applying MPAQT to an in vitro model of human embryonic stem cell differentiation into cortical neurons, followed by machine learning-based modeling of transcript abundances, we show that untranslated regions (UTRs) are major determinants of isoform proportion and exon usage; this effect is mediated through isoform-specific sequence features embedded in UTRs, which likely interact with RNA-binding proteins that modulate mRNA stability. These findings highlight MPAQT's potential to enhance our understanding of transcriptomic complexity and underline the role of splicing-independent post-transcriptional mechanisms in shaping the isoform and exon usage landscape of the cell.

Expanding the phenotypic spectrum of CC2D2A-related ciliopathies: a rare homozygous nonsense variant in a patient with suspected nephronophthisis.

Biallelic pathogenic variants in the gene CC2D2A cause a spectrum of ciliopathies, including Joubert and Meckel syndrome, which frequently involve the kidney; however, no cases of isolated renal disease (i.e., nephronophthisis) have yet been reported. In an adult with a clinical presentation consistent with nephronophthisis, next-generation sequencing identified a rare homozygous nonsense variant in CC2D2A (c.100 C > T; p.(Arg34*)). Tissue-specific expression data and promoter activity analysis demonstrates that this variant primarily affects a transcript isoform predominant in the kidneys but does not affect the transcript isoforms predominant in other tissues typically involved in CC2D2A-related ciliopathies (e.g., cerebellum, liver). Expression analysis of patient-specific cDNA in MDCK cells demonstrates partial translation re-initiation downstream of p.(Arg34*) as a possible escape mechanism from nonsense mediated decay. These data provide mechanistic insights in support of this novel genotype-phenotype association.

ESRP2-microRNA-122 axis directs the postnatal onset of liver polyploidization and maturation.

Hepatocyte polyploidy and maturity are critical to acquiring specialized liver functions. Multiple intra- and extracellular factors influence ploidy, but how they cooperate temporally to steer liver polyploidization and maturation or how post-transcriptional mechanisms integrate into these paradigms is unknown. Here, we identified an important regulatory hierarchy in which postnatal activation of Epithelial-Splicing-Regulatory-Protein-2 (ESRP2) stimulates biogenesis of liver-specific microRNA (miR-122), thereby facilitating polyploidization, maturation, and functional competence of hepatocytes. By determining transcriptome-wide protein-RNA interactions in vivo and integrating them with single-cell and bulk hepatocyte RNA-seq datasets, we delineate an ESRP2-driven RNA processing program that drives sequential replacement of fetal-to-adult transcript isoforms. Specifically, ESRP2 binds the primary miR-122 host gene transcript to promote its processing/biogenesis. Combining constitutive and inducible ESRP2 gain- and loss-of-function mice models with miR-122 rescue experiments, we demonstrate that timed activation of ESRP2 augments miR-122-driven program of cytokinesis failure, ensuring proper onset and extent of hepatocyte polyploidization.

Alternative splicing of a potato disease resistance gene maintains homeostasis between growth and immunity.

Plants possess a robust and sophisticated innate immune system against pathogens and must balance growth with rapid pathogen detection and defense. The intracellular receptors with nucleotide-binding leucine-rich repeat (NLR) motifs recognize pathogen-derived effector proteins and thereby trigger the immune response. The expression of genes encoding NLR receptors is precisely controlled in multifaceted ways. The alternative splicing (AS) of introns in response to infection is recurrently observed but poorly understood. Here we report that the potato (Solanum tuberosum) NLR gene RB undergoes AS of its intron, resulting in 2 transcriptional isoforms, which coordinately regulate plant immunity and growth homeostasis. During normal growth, RB predominantly exists as an intron-retained isoform RB_IR, encoding a truncated protein containing only the N-terminus of the NLR. Upon late blight infection, the pathogen induces intron splicing of RB, increasing the abundance of RB_CDS, which encodes a full-length and active R protein. By deploying the RB splicing isoforms fused with a luciferase reporter system, we identified IPI-O1 (also known as Avrblb1), the RB cognate effector, as a facilitator of RB AS. IPI-O1 directly interacts with potato splicing factor StCWC15, resulting in altered localization of StCWC15 from the nucleoplasm to the nucleolus and nuclear speckles. Mutations in IPI-O1 that eliminate StCWC15 binding also disrupt StCWC15 re-localization and RB intron splicing. Thus, our study reveals that StCWC15 serves as a surveillance facilitator that senses the pathogen-secreted effector and regulates the trade-off between RB-mediated plant immunity and growth, expanding our understanding of molecular plant-microbe interactions.

High-density resolution of the Kaposi's sarcoma associated herpesvirus transcriptome identifies novel transcript isoforms generated by long-range transcription and alternative splicing.

Kaposi's sarcoma-associated herpesvirus is the etiologic agent of Kaposi's sarcoma and two B-cell malignancies. Recent advancements in sequencing technologies have led to high resolution transcriptomes for several human herpesviruses that densely encode genes on both strands. However, for KSHV progress remained limited due to the overall low percentage of KSHV transcripts, even during lytic replication. To address this challenge, we have developed a target enrichment method to increase the KSHV-specific reads for both short- and long-read sequencing platforms. Furthermore, we combined this approach with the Transcriptome Resolution through Integration of Multi-platform Data (TRIMD) pipeline developed previously to annotate transcript structures. TRIMD first builds a scaffold based on long-read sequencing and validates each transcript feature with supporting evidence from Illumina RNA-Seq and deepCAGE sequencing data. Our stringent innovative approach identified 994 unique KSHV transcripts, thus providing the first high-density KSHV lytic transcriptome. We describe a plethora of novel coding and non-coding KSHV transcript isoforms with alternative untranslated regions, splice junctions and open-reading frames, thus providing deeper insights on gene expression regulation of KSHV. Interestingly, as described for Epstein-Barr virus, we identified transcription start sites that augment long-range transcription and may increase the number of latency-associated genes potentially expressed in KS tumors.

Population-level exploration of alternative splicing and its unique role in controlling agronomic traits of rice.

Alternative splicing (AS) plays crucial roles in regulating various biological processes in plants. However, the genetic mechanisms underlying AS and its role in controlling important agronomic traits in rice (Oryza sativa) remain poorly understood. In this study, we explored AS in rice leaves and panicles using the rice minicore collection. Our analysis revealed a high level of transcript isoform diversity, with approximately one-fifth of the potential isoforms acting as major transcripts in both tissues. Regarding the genetic mechanism of AS, we found that the splicing of 833 genes in the leaf and 1,230 genes in the panicle was affected by cis-genetic variation. Twenty-one percent of these AS events could only be explained by large structural variations. Approximately 77.5% of genes with significant splicing quantitative trait loci (sGenes) exhibited tissue-specific regulation, and AS can cause 26.9% (leaf) and 23.6% (panicle) of sGenes to have altered, lost, or gained functional domains. Additionally, through splicing-phenotype association analysis, we identified phosphate-starvation-induced RING-type E3 ligase (OsPIE1; LOC_Os01g72480), whose splicing ratio was significantly associated with plant height. In summary, this study provides an understanding of AS in rice and its contribution to the regulation of important agronomic traits.