Open research questions in RNA Research and Splicing

What the literature leaves open

• Long, dosage-sensitive mRNAs encoding neurodevelopmental regulators are selectively dependent on fragile X messenger ribonucleoprotein (FMRP) for efficient translation across animal systems, but the mechanism underlying this length-dependent requirement remains unclear.

  FMRP prevents Me31B/DDX6-associated repression of long neurodevelopmental mRNAs · 2026 · DOI

• Although the molecular functions and protein interactions of SNRNP70 in pre-mRNA splicing are well established, the mechanisms underlying its cytoplasmic functions remain poorly understood.

  SNRNP70 interacts with TDP-43 to promote RNP granule localisation and regulate motor neuron development · 2026 · DOI

• However, the regulatory logic that specifies neuronal RT and the structural and cellular consequences of the resulting C-terminal protein extensions remain poorly understood.

  Neuronal stop-codon readthrough is associated with ribosome pausing and alters protein localization in Drosophila · 2026 · DOI

• Although transcription by Pol II is intensively studied, how this central multi-subunit enzyme is made and the role of dedicated assembly factors remains unclear.

  Structure of cytoplasmic RNA polymerase II · 2026 · DOI

• Further investigation of the interaction networks between SNR17 and protein-coding genes revealed the emergence of paralog-specific interactions under stress conditions.

  Genome-wide analysis of the non-coding RNA synthetic genetic network reveals extensive plasticity and distinct environmental dependent roles for U3 snoRNA paralogs. · 2026 · DOI

• This observation suggests the possibility of PTC detection at the transcription site, which has not been thoroughly investigated with high temporal and spatial resolution.

  Real-time imaging of transcriptional feedback in nonsense-mediated mRNA decay · 2026 · DOI

• Removal of the 5' mG cap by the Dcp1/Dcp2 complex commits mRNAs to degradation, yet the mechanisms regulating decapping remain incompletely understood.

  Sequence-encoded autoinhibition couples mRNA decapping activity to phase separation · 2026 · DOI

• Previous work shows that PABPC can straddle the junction between the poly(A) tail and the 3' untranslated region (UTR), but whether this conformation influences deadenylation has not been tested.

  The molecular determinants of PABPC-mediated deadenylation rate · 2026 · DOI

• Microexons are highly conserved fragments of exons ranging from 3 to 51 nucleotides (nts), representing a precise but poorly understood layer of post-transcriptional regulation outside the central nervous system.

  In silico investigation of alternative splicing of microexons in human peripheral tissues · 2026 · DOI

• Splicing outcomes are controlled by combinatorial RNA-binding protein (RBP) interactions and positional context, but how these features are integrated at the transcriptome scale remains unclear.

  A positional and combinatorial regulatory code for alternative splicing · 2026 · DOI

• increasingly addressed by simultaneous quantification of splicing changes across thousands of genes (Jaganathan et al., 2019; Zhang et al., 2019). However, short-read sequencing is inherently limited in resolving complex isoforms. Specifically, short reads often fail to resolve long-range exon connectivity (phasing) between distant exons, leading to potential inaccuracies in full-length transcript assembly (Jin et al., 2025; Zhang et al., 2023). is long-read sequencing technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), which sequence full-length transcripts from the 5 cap to the 3 poly(A) tail (Kabza et al., 2024). Notably, Nanopore sequencing oers the distinct advantage of direct native RNA sequencing, allowing for the simultaneous detection of RNA modifications alongside splicing patterns, thereby revealing crucial layers of post-transcriptional regulation (Jenjaroenpun et al., 2021; Liu et al., 2019). Despite its transformative potential, long-read sequencing currently faces several notable limitations that must be critically considered. First, platforms like Oxford Nanopore inherently exhibit higher per-base error rates compared to short-read sequencing, often necessitating hybrid error-correction strategies combined with highly accurate Illumina reads.

  Molecular arms races at the virus-host splicing interface and their pathogenic implications · 2026 · DOI

• The interplay between viral pathogens and the host RNA splicing machinery represents a sophisticated evolutionary adaptation, driven by the stringent constraints of compact viral genomes. As highlighted herein, viruses ranging from the segmented Influenza A Virus to DNA viruses such as HBV and HPV have evolved convergent strategies to exploit the host spliceosome (Ashraf et al., 2019; Schwartz and Ast, 2010). By hijacking this cellular machinery, viruses achieve two critical objectives: maximizing the coding potential of restricted genetic templates and ensuring the precise spatiotemporal regulation of viral gene expression. However, this absolute dependency constitutes a strategic vulnerability. While alternative splicing endows viruses with the proteomic plasticity required for replication and persistence, their reliance on suboptimal splice sites and specific host splicing factors exposes an exploitable bottleneck that is highly amenable to therapeutic intervention. Furthermore, the discovery of vcircRNAs has fundamentally expanded our understanding of viral RNA processing. The recognition that diverse viruses utilize non-canonical backsplicing to generate highly stable, immunomodulatory transcripts challenges the traditional dogma that viral transcriptomes are exclusively linear. These covalently closed circular molecules, functioning as potent miRNA sponges or protein decoys, add an unprecedented layer of complexity to the virus-host interactome, particularly in the context of innate immune evasion. Significantly, the inherent biostability that renders these molecules eective viral eectors is now being actively harnessed to develop next-generation circular RNA vaccines, exemplifying the rapid translation of fundamental viral RNA biology into transformative biomedical innovation (Qu et al., 2022). Looking forward, the integration of long-read nanopore sequencing with advanced targeted proteomic approaches is poised to unveil the full, unannotated spectrum of viral splicing isoform diversity and their distinct functional roles in pathogenesis (Depledge et al., 2019). Future research must prioritize dissecting the highly specific molecular crosstalk between cis-acting viral RNA elements and trans-acting host splicing factors to identify novel, druggable interfaces. From a translational perspective, this virushost splicing axis oers a compelling avenue for the development of splicing-targeted antivirals. Several small-molecule splicing modulators, such as inhibitors targeting the SF3b complex or SR protein kinases (SRPKs), have already demonstrated potent broadspectrum antiviral eÿcacies in vitro (Fukuhara et al., 2006; Kyei et al., 2018; Li et al., 2022).

  Molecular arms races at the virus-host splicing interface and their pathogenic implications · 2026 · DOI

• LLPS is a common cellular behavior that regulates DNA synthesis, RNA synthesis, and processing, and signal trans- duction [1]. In various cancer types, LLPS can promote the proliferation, metastasis, and drug resistance of cancer cells by affecting BRD4 [6–9] and p53 [8, 9]. LLPS is regulated by several mechanisms, especially epigenetic modification. Therefore, targeting LLPS may facilitate the development of effective and promising anticancer strategies. Nowadays, increasingly more studies have revealed the critical regula- tory roles of LLPS during tumor progression, but multiple problems remain to be solved. First, we know little about the exact and detailed mechanisms of LLPS-mediated tumorigenesis. Second, there is still a poor understanding of more epigenetic mechanisms underlying protein modifica- tion-caused LLPS in cancer. Third, further investigation is needed to develop LLPS-based anticancer methods. Against this background, protein lactylation has emerged as a newly identified and important epigenetic post-translational modi- fication [79]. However, its biological link with LLPS in can- cer remains unclear. Nevertheless, emerging studies suggest that lactylation may affect downstream gene transcription and cancer-related phenotypes in specific contexts [80]. Separately, limited evidence has hinted at a possible con- nection between lactylation-associated changes and LLPS in tumors [81]. Overall, exploring the regulatory connection between protein lactylation and phase separation in can- cer is quite important. Additionally, given that lactylation is associated with tumor drug resistance, these findings may provide a rationale for future therapeutic exploration aimed at overcoming drug resistance, for example through glycolysis inhibition or modulation of histone lactylation, such as H3K18 lactylation [82, 83]. In summary, unraveling the oncogenic roles, therapeutic potentials, and epigenetic Molecular Biology Reports (2026) 53:663 1 3 663 Page 10 of 12 regulatory mechanisms of liquid-liquid phase separation will help develop novel strategies for cancer control and to overcome drug resistance. Acknowledgements We gratefully acknowledge the use of BioRender (https://www.biorender.com/) for the creation of scientific illustrations in this study. Author contributions ZZH wrote the manuscript. ZZH and LT pre- pared the figures. SZZ revised the manuscript. All authors reviewed the manuscript. Funding This study was funded by the National Natural Science Foundation of China (No. 82160585), Joint Medical Program of Kun- ming University of Science and Technology (KUST-KH2022001Z and KUST-PE2022002Z), and the innovation team of oxidative stress and defense of Yunnan Province (202305AS350011). Data availability No datasets were generated or analysed during the current study.

  Liquid-liquid phase separation in cancer: oncogenic roles, therapeutic potential, and epigenetic regulation · 2026 · DOI

• In this review, we have summarized and discussed multiple forms of non-canonical alternative splicing, outlining their principal structures, current landscapes, challenges, and prospective directions for future research (Table 3). Non-canonical splicing comprises a broad and mechanistically diverse set of RNA-processing events. Accordingly, not all forms of non-canonical splicing should be discussed at the same analytical level. A useful organizing principle is to classify these events according to their degree of deviation from spliceosome-dependent splicing. For example, many cases of splicing with non-canonical splice sites, particularly the U2/U12-like subset, can still be tolerated by the spliceosome and are best regarded as spliceosomemediated. Events that depart from canonical splicing in certain mechanistic aspects but still remain partially compatible with spliceosomal recognition may be more appropriately grouped as spliceosome-compatible, including microexons, recursive splicing, and trans-splicing. By contrast, forms of non-canonical splicing that operate through fundamentally distinct mechanisms, such as tRNA splicing or the splicing of XBP1 and HAC1, are more appropriately classified as spliceosome-independent. These three categories differ substantially in mechanism and should therefore be analyzed separately. At the same time, this framework is not absolute. Some reported subclasses of non-canonical splicing may span conceptual boundaries; for instance, certain minor non-canonical splice junctions that do not conform to current U2/U12-like models may nonetheless rely on as-yet-uncharacterized mechanisms, potentially placing them closer to the spliceosome-compatible or even spliceosome-independent end of the spectrum. In such cases, individual events may require case-by-case discussion, as is already common for XBP1 and HAC1. A second general conclusion is that reported prevalence is highly method dependent. Because published studies differ substantially in scope, period, and analytical design, the resulting prevalence estimates and reporting frameworks are often difficult to compare directly. Two issues are particularly important. First, the increasing use of long-read sequencing has not fundamentally overturned the overall view of non-canonical splicing, but it has revealed a large number of junctions that were not detectable in the shortread era, thereby exposing a substantial and still underexplored landscape. These newly detected long-read junctions represent an important resource for future investigation. Second, studies often differ markedly in pipeline stringency and filtering strategy, because different analytical aims naturally lead to different dataprocessing workflows and parameter choices. As a result, cross-study comparisons of non-canonical splicing require careful attention to methodology. When necessary, reanalysis of public raw datasets under a consistent filtering framework may provide a more reliable basis for direct comparison. Species specificity and tissue specificity also emerge as important dimensions of non-canonical splicing. For example, trans-splicing is almost a dominant mode of transcript processing in C. elegans, whereas microexons, recursive splicing, and noncanonical splice sites have all been repeatedly linked to the nervous system in multiple studies. More broadly, these forms of potentially neural-associated non-canonical splicing may contribute to a deeper understanding of neuronal RNA processing and of neurological disease. Finally, mechanistic to change stratification interpretation. In the earlier RNA-seq reanalysis presented in this review, non-canonical introns were compared with canonical introns only as a single combined category.

  The “cutting edge” of non-canonical RNA splicing · 2026 · DOI

• G-patch proteins are important cofactors of RNA helicases, orchestrating dynamic RNA-processing events including pre- mRNA splicing, ribosome biogenesis, transcription, and genome maintenance. Through these multifunctional roles, they integrate diverse RNA metabolic pathways, and their dysregulation can result in aberrant splicing, defective ribosome assembly, altered transcription, or genomic instability, with clear implications for human disease. Despite their evolutionary conservation, many G-patch proteins remain only partially characterized, and the regulatory their helicase principles governing mechanisms, still poorly functional outcomes understood. Key open questions include how G-patch proteins in different cellular contexts, select specific RNA helicases specificity, are and Frontiers in Cell and Developmental Biology 08 frontiersin.org Karika et al. 10.3389/fcell.2026.1750689 FIGURE 2 Roles of human G-patch proteins in RNA metabolism and cellular function. G-patch proteins interact with RNA helicases to coordinate pre-mRNA splicing and ribosome biogenesis. Dysregulation of these proteins can disrupt pre-mRNA splicing, ribosome biogenesis, transcription regulation, genome stability, and overall cellular homeostasis and development. factors are whether and how multiple G-patch proteins compete for the same RNA helicase, how individual proteins coordinate distinct RNA- processing pathways simultaneously, and which additional regulated by G-patch proteins. spliceosomal Additional questions concern the molecular basis by which G-patch protein dysfunctions contribute to disease pathogenesis, particularly in cancer and disorders linked to splicing, ribosome biogenesis defects or genome instability. Addressing these questions will require integrated structural, biochemical, and cellular approaches to define G-patch interactomes, resolve mechanisms of RNA helicase recruitment and activation, and map their roles in regulating RNA metabolism. Ultimately, a deeper understanding of G-patch proteins promises to reveal fundamental principles of RNA metabolism and identify potential novel therapeutic targets.

  G-patch proteins: important regulators of pre-mRNA splicing and ribosome biogenesis · 2026 · DOI

• EXOSC2, a core component of the RNA exosome, directly participates in mRNA metabolism, but the functional roles of its alternatively spliced isoforms remain unclear [53,54].

  Casein kinase 2-mediated phosphorylation of the splicing factor SF3B3 plays a key role in esophageal squamous cell carcinoma progression · 2026 · DOI

• Positional enrichment of eCLIP peaks was calculated for RBPs with consistent effects in only K562 and HepG2 cells; validation in additional cell types is needed.

  Alu-mediated RNA duplexes are associated with widespread exon skipping across primate transcriptomes · 2026 · DOI

• Analysis focused on only two RBPs (HNRNPC and ILF3) with differential effects on splicing of exons flanked by inverted Alu repeats; other RBPs remain unexplored.

  Alu-mediated RNA duplexes are associated with widespread exon skipping across primate transcriptomes · 2026 · DOI

• Only eight exons were selected for minigene experiments from an initial set of 205 that meet the criteria, limiting the experimental validation scope.

  Alu-mediated RNA duplexes are associated with widespread exon skipping across primate transcriptomes · 2026 · DOI

• While SATB1 role in T cells is well-established, its regulation and function in B cells remain underexplored.

  Lineage-specific regulation and RNA-associated functions of SATB1 in mature B-cells · 2026 · DOI

• Amyotrophic lateral sclerosis (ALS) is categorized by TDP-43 proteinopathy, however, the nuclear pathological events remain poorly defined.

  HSC70 prevents TDP-43 nuclear puncta formation and toxicity in a novel nuclear puncta cell model for ALS · 2026 · DOI

• Despite this, the precise executors of intronic premature termination under CDK12-inactivation-induced elongation stress remain poorly understood.

  Termination dynamics set RNAPII elongation rate and gate the response to CDK12 inactivation · 2026 · DOI

• RNA viruses form membraneless condensates in host cells to drive replication, but whether these compartments also regulate host RNAs remains unclear.

  Aberrant host mRNA partitioning in Ebola virus condensates driven by RNA folding perpetuates species-dependent interferon response · 2026 · DOI

• The mechanism(s) that governs the liquid-to-solid phase transition of TDP-43 is poorly defined.

  Cellular modifiers of TDP-43 phase transition and cytoplasmic aggregation · 2026 · DOI

• DSIF and NELF, however, are insufficient to fully reconstitute the degree of promoter-proximal pausing observed in cells.

  ELOF1 is a core component of the promoter-proximal paused RNA polymerase II complex · 2026 · DOI

Most-cited papers in RNA Research and Splicing

• rMATS-turbo: an efficient and flexible computational tool for alternative splicing analysis of large-scale RNA-seq data · Nature Protocols · 2024 · 253 citations
• Possibilities and limitations of antisense oligonucleotide therapies for the treatment of monogenic disorders · Communications Medicine · 2024 · 234 citations
• Expanding the molecular language of protein liquid–liquid phase separation · Nature Chemistry · 2024 · 191 citations
• Genome organization around nuclear speckles drives mRNA splicing efficiency · Nature · 2024 · 161 citations
• De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome · Nature · 2024 · 123 citations
• On the genetic basis of tail-loss evolution in humans and apes · Nature · 2024 · 112 citations
• Determinants that enable disordered protein assembly into discrete condensed phases · Nature Chemistry · 2024 · 91 citations
• Mutations in the U4 snRNA gene RNU4-2 cause one of the most prevalent monogenic neurodevelopmental disorders · Nature Medicine · 2024 · 87 citations
• A solid beta-sheet structure is formed at the surface of FUS droplets during aging · Nature Chemical Biology · 2024 · 86 citations
• Transcriptome-wide splicing network reveals specialized regulatory functions of the core spliceosome · Science · 2024 · 84 citations

Most recent work

• Susceptibility and Regulation of Biomolecular Condensates by Solutes · bioRxiv · 2026
• RNA editing controls gene drive by a Neurospora Spore killer · bioRxiv · 2026
• Phase separation behavior of TDP-43 governs its protein interactome and regulation of alternative splicing · bioRxiv · 2026
• Nonsense mutations can increase mRNA levels · Biology Open · 2026
• TENT5C extends Odf1 poly(A) tail to sustain sperm morphogenesis and fertility · Nature Communications · 2026
• HELIX: a scalable model for predicting context-dependent regulation of RNA splicing and isoform usage · Nature Computational Science · 2026
• RNA Selectively Modulates Activity of Virulent Amyloid PSMα3 and Host Defense LL-37 via Phase Separation and Aggregation Dynamics · bioRxiv · 2026
• Argonaute-2 slicing of Rtl1 promotes skeletal muscle development and postnatal survival · bioRxiv · 2026
• Poison exon splicing in the human brain: a new paradigm for understanding and targeting neurological disorders · bioRxiv · 2026
• The Function of LncRNA DRIR in Freezing Tolerance by Promoting Autophagic Degradation of CP29A and CP29B to Alter Alternative Splicing Patterns of Pre-mRNAs · bioRxiv · 2026

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