The expanding functional landscape of alternative splicing in plants.

Intron retention as a hub for splicing regulation in plant environmental adaptation and developmental programming.

Alternative splicing (AS) greatly expands transcriptome and proteome diversity in eukaryotes. Intron retention (IR) is the predominant AS type in plants, with critical roles in environmental adaptation and developmental control. Long viewed as splicing noise, IR is now established as a precisely regulated mechanism that modulates RNA stability, translation, subcellular localization, and protein function. Here, we review recent advances in plant IR research, covering technical progress in accurate IR identification using long-read and single-cell sequencing, cis- and trans-regulatory mechanisms, epigenetic coupling with transcription, and signal integration pathways. We highlight key functions of IR in flowering time regulation and stress responses, including the IR-nonsense-mediated mRNA decay (NMD) axis, functional protein isoforms, and nuclear transcript reservoirs for rapid stress memory. Finally, we discuss unresolved questions and future directions toward single-cell spatiotemporal dynamics, phase separation, and synthetic IR modules for crop improvement. This review provides an integrated framework for understanding IR as a central regulatory hub in plant post-transcriptional control.

RNA splicing in health and disease

RNA splicing expands the functional output of eukaryotic genomes by enabling individual precursor messenger RNA (pre-mRNA) to generate multiple mature transcripts with protein‑coding and regulatory properties. Its fidelity and plasticity depend on coordinated interactions among the spliceosome, trans-acting splicing factors, cis-regulatory elements, and chromatin- and RNA-associated regulatory mechanisms. However, how these components collectively generate cell- and tissue-specific splicing programs, and how their disruption drives disease, remain incompletely understood. In this review, we integrate the molecular regulation of RNA splicing with its physiological, pathological and therapeutic consequences. We describe how spliceosome assembly, splicing regulatory elements, splicing factors, epigenetic modifications, and post-transcriptional processes determine splice-site selection. We then examine how regulated isoform programs support development, tissue specialization, homeostasis, circadian timing, and stress adaptation, and how their failure contributes to cancer and diverse non-neoplastic diseases. In cancer, we highlight the bidirectional interplay between splicing dysregulation and the tumor microenvironment, through which metabolic reprogramming and immune suppression reinforce aberrant splicing. Finally, we assess strategies that modulate the spliceosome, splicing-factor activity, or disease-associated transcripts, and present a perspective on how multi-omics, artificial intelligence, targeted delivery, and combination with immunotherapy could collectively advance the discovery and precision of splicing-directed therapies. We suggest that safe clinical translation will require greater selectivity, reduced off-target toxicity, and preservation of essential physiological splicing.

Splicing genes as main targets of differential splicing.

Splicing of transcripts via the spliceosome machinery is a complex process involving a multitude of proteins and short noncoding RNAs. In addition to full-length transcripts with all exons in the genomically encoded order, alternatively spliced transcripts can also be produced through alternative splicing of pre-mRNA. We have attempted to identify which genes are most frequently targeted by differential splicing using both downloaded and original RNA-Seq data from several species and tissue types. In order to identify splicing variation on an individual-to-individual basis, random contrasting of samples within compatible sets of samples was done. Both when analyzing sequence data from similar source material (same tissue from individuals kept under similar conditions) and when contrasting samples that are more different, such as different tissue types, genes associated with the splicing process itself seem to be the most recurring targets.

Promoter identity shapes splicing outcomes and fidelity

Gene expression is a complex process subject to regulation at multiple functionally interconnected levels. One prominent example is the crosstalk between transcription and splicing regulation. Past work has shown that transcription can influence splicing in multiple ways, but a systematic investigation of this complex interplay is lacking. Here we employ massively parallel reporter assays of large combinatorial promoter-splice site libraries to dissect how promoter identity and transcription dynamics affect alternative splicing in human cells. We find that promoter identity, rather than expression level, exerts strong and highly context-specific effects on cassette exon inclusion, exceeding the effect of pharmacological inhibitors of transcription initiation or elongation. Groups of exons display coordinated promoter-dependent splicing behavior, and we identified predictive sequence and structural features underlying this sensitivity. Promoter and gene architecture also shape isoform diversity by modulating cryptic splice site usage. These findings present promoters as central regulators of splicing outcomes and fidelity. Using massively parallel reporter assays of mini-genes with variable promoters and cassette exon sequences, the authors show that promoter identity is a major determinant of alternative splicing, shaping exon inclusion levels and splicing fidelity.

Construction and Analysis of circRNA-miRNA-mRNA Regulatory Networks.

Noncanonical Functions of DICER-Mediated Pathways.

Dicer has long been viewed as the RNAse III enzyme that generates small regulatory RNAs to drive RNA interference (RNAi) machinery and posttranscriptional gene silencing. However, a growing body of evidence reveals that Dicer's functional landscape extends far beyond its canonical role in the cytoplasmic RNAi pathway. In this review, we delineate the classical, RNAse III-dependent small RNA processing activities from an expanding set of noncanonical functions in which Dicer operates independently of RNAi outputs. We highlight emerging principles that underlie these functions, including structural and regulatory principles, with a focus on nuclear functions, posttranslational regulation, and noncanonical substrates. We then explore how Dicer intersects with genome integrity pathways, transcriptional programs, and cellular response to DNA damage. Finally, we propose an integrated conceptual framework in which Dicer acts as a multifunctional regulator of chromatin architecture and genome stability, as we consider how perturbations of these pathways contribute to disease.