Chimeric RNAs (chRNAs) expand transcriptomic and proteomic diversity by joining sequences from distinct genomic loci, yet their physiological functions remain poorly understood. In a recent paper published in Nature, Venezia et al. show that inflammation induces CTCF-dependent chRNA formation in mammalian immune cells and that a Gsdmd-Tmem106a chRNA encodes a functional fusion protein that modulates pyroptosis and the balance between antibacterial defense and inflammatory pathology.The mammalian genome contains a surprisingly limited number of protein-coding genes relative to the complexity of mammalian organisms. Alternative splicing, RNA editing, non-canonical translation and post-translational modification expand the functional output of this finite genome. Chimeric RNAs (chRNAs), in which sequences from distinct genomic loci are joined within a single transcript, may represent another layer of this regulatory complexity.1,2,3 Historically, chimeric transcripts have been closely associated with cancer-associated chromosomal rearrangements. Recent work demonstrated that RNA-level trans-splicing can generate chimeric RNAs and functional fusion proteins without corresponding genomic rearrangements, including in normal physiological and developmental contexts.4,5,6 However, because of technical limitation and lack of functional evidence, only a few isolated trans-splicing chimera have been reported, including JAZF1-JJAZ1 in endometrium fibroblast4 and PAX3-FOXO1 in muscle differentiation.5,6 These findings raised a broader question: are there other trans-spliced chimeric RNAs and can they generate functional proteins in normal mammalian tissues?Venezia et al.7 address this question in mammalian immune cells. Using long-read direct RNA sequencing together with complementary computational and experimental approaches, they identified a large and dynamically regulated landscape of chRNAs in mouse macrophages. More than 30,000 candidate chRNAs were detected, including numerous transcripts joining genes located on different chromosomes or separated by large genomic distances. Many were altered following inflammatory stimulation, suggesting that chRNA production is not simply a consequence of transcriptional noise but may be responsive to cellular state (Fig. 1).Fig. 1: Inflammation-induced chimeric RNA Gsdmd-Tmem106a links 3D genome organization and fusion protein active GSDMD-NT to immune regulation.Full size imageInflammatory stimulation promotes CTCF-dependent interactions between the Gsdmd and Tmem106a loci, facilitating formation of a Gsdmd-Tmem106a chimeric RNA. The resulting GSDMD-TMEM106A fusion protein cooperates with activated GSDMD-NT to enhance membrane pore formation and IL-1β release, thereby influencing the balance between antibacterial defense and inflammatory pathology. This figure is created with BioRender.A central challenge in chRNA research is distinguishing genuine RNA-fusion events from technical artifacts. Conventional short-read RNA sequencing relies on reverse transcription, raising the possibility that template switching during reverse transcription or library preparation can produce artificial junctions. The authors here combined Oxford Nanopore direct RNA sequencing with short-read sequencing, NanoString junction-specific profiling, PCR and Sanger sequencing. Direct RNA sequencing avoids reverse transcription, whereas NanoString provides an independent hybridization-based strategy for validating predefined junctions. The agreement among orthogonal approaches provides substantially stronger evidence for endogenous chRNA formation. The different platforms did not identify identical sets of chRNAs, highlighting an important technical limitation. Differences in transcript abundance, isoform structure, sequencing chemistry and assay sensitivity may influence which chRNAs are detected. Establishing rigorous criteria for validating low-abundance chRNAs will therefore remain important as the field expands.How can transcripts originating from distant genomic loci become joined? Venezia et al. found that inflammatory stimulation increased interactions between several parental genomic loci associated with chRNA formation. Depletion of CTCF reduced these chromosomal interactions and decreased chRNA production without substantially changing expression of the parental genes. These observations support a model in which three-dimensional (3D) genome organization facilitates RNA-level gene joining. Together with previous evidence that CTCF regulates SLC45A3-ELK4 chimeric RNA,8 the findings suggest that genomic architecture may influence not only which genes are transcribed, but also which independently transcribed RNAs can subsequently become connected. Spatial proximity, however, is unlikely to be sufficient for trans-splicing. The molecular mechanisms that select two RNAs, bring them together and direct the splicing machinery to a specific junction remain unresolved. Transcriptional timing, splice-site compatibility, RNA sequence elements, RNA-binding proteins and nuclear organization may all contribute.The most compelling evidence for biological relevance comes from the inflammation-induced Gsdmd-Tmem106a chRNA. This transcript joins Gsdmd exons 1–2 with Tmem106a exons 6–9 without an accompanying chromosomal rearrangement. Importantly, the transcript is translated into a previously unannotated GSDMD-TMEM106A fusion protein. The fusion retains the membrane-associated N-terminal (NT) region of GSDMD while acquiring a shifted reading frame C-terminal region derived from TMEM106A. GSDMD-NT is a well-established executor of pyroptosis: following inflammatory caspase-mediated cleavage, GSDMD-NT oligomerizes at cellular membranes to form pores that promote membrane permeabilization and IL-1β release.9,10 Venezia et al. showed that GSDMD-TMEM106A adds another regulatory layer to this pathway. The fusion protein localizes to the plasma membrane and associates with activated GSDMD-NT domains, accelerating pore formation.7 Strikingly, the chRNA is expressed at less than 10% of parental Gsdmd levels, yet supplementing GSDMD-encoding mRNA with only 10% GSDMD-TMEM106A-encoding mRNA nearly doubled IL-1β release, whereas an equivalent increase in GSDMD had little effect. Deletion of the TMEM106A-derived C-terminal region abolished this enhancement, demonstrating that the fusion protein has a specific function that cannot be explained simply by increased GSDMD expression. The authors further established physiological relevance using complementary genetic mouse models. A GSDMD-TMEM106A-MYC knock-in model enabled direct detection of the fusion protein, whereas a Gsdmd-Tmem106aStop model selectively prevented fusion-protein translation while preserving the parental genes. Loss of GSDMD-TMEM106A increased bacterial burden following Salmonella Typhimurium infection but protected mice from lethal endotoxemia. Conversely, increased fusion activity enhanced bacterial clearance while exacerbating inflammatory pathology. Thus, this chimeric protein appears to contribute to the balance between effective antimicrobial defense and excessive inflammation.An intriguing feature of the study is the apparent species specificity of GSDMD-TMEM106A. Although more than 900 chRNAs were identified in human monocyte-derived macrophages and chRNAs involving the same parental genes were detected in both mouse and human cells, GSDMD-TMEM106A itself was not detected in the human macrophages examined. This observation illustrates that conservation of parental genes does not necessarily imply conservation of their RNA-level interactions. Species-specific exon architecture, splice-site compatibility, regulatory sequences, RNA-binding factors and 3D genome organization may determine which individual chRNAs are generated.The significance of this work extends beyond the discovery of one fusion protein. Conventional transcriptomic analyses generally focus on changes in parental gene expression. Yet a chRNA may be relatively rare while its encoded protein has a disproportionately large biological effect. Thus, understanding cellular responses may require asking not only how much of each gene is expressed, but also which transcripts become connected and whether those new RNAs encode functional proteins. Venezia et al. move the field beyond the question of whether chRNAs exist to the more consequential questions of how they are regulated, when they are translated and what biological information they encode. Their findings connect 3D genome organization, RNA processing and proteome diversification, suggesting that chRNAs may constitute a previously underappreciated layer of mammalian gene regulation. Future studies will determine whether stimulus-responsive chRNAs form broader regulatory networks in development, neurobiology, tissue repair, aging and disease, and how frequently such transcripts are translated into functional proteins. The work also raises a fundamental question: to what extent can the 3D organization of the genome determine which genes become functionally connected at the RNA level?ReferencesLicatalosi, D. D. & Darnell, R. B. Nat. Rev. Genet. 11, 75–87 (2010).Article PubMed PubMed Central Google Scholar Sibley, C. R., Blazquez, L. & Ule, J. Nat. Rev. Genet. 17, 407–421 (2016).Article PubMed PubMed Central Google Scholar Konarska, M. M., Padgett, R. A. & Sharp, P. A. Cell 42, 165–171 (1985).Article PubMed Google Scholar Li, H., Wang, J., Mor, G. & Sklar, J. Science 321, 1357–1361 (2008).Article PubMed Google Scholar Yuan, H. et al. Cancer Discov. 3, 1394–1403 (2013).Article PubMed Google Scholar Xie, Z. et al. Proc. Natl. Acad. Sci. USA 113, 13126–13131 (2016).Article PubMed PubMed Central Google Scholar Venezia, O. et al. Nature https://doi.org/10.1038/s41586-026-10982-x (2026).Article PubMed Google Scholar Qin, F. et al. PLoS ONE 11, e0153986 (2016).Article Google Scholar Kayagaki, N. et al. Nature 526, 666–671 (2015).Article PubMed Google Scholar Liu, X. et al. Nature 535, 153–158 (2016).Article PubMed PubMed Central Google Scholar Download referencesAuthor informationAuthors and AffiliationsDepartment of Pathology, School of Medicine, University of Virginia, Charlottesville, VA, USAZhongqiu Xie & Hui LiCenter for RNA Science and Medicine, University of Virginia, Charlottesville, VA, USAHui LiUniversity of Virginia Comprehensive Cancer Center, University of Virginia, Charlottesville, VA, USAHui LiAuthorsZhongqiu XieView author publicationsSearch author on:PubMed Google ScholarHui LiView author publicationsSearch author on:PubMed Google ScholarCorresponding authorCorrespondence to Hui Li.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Rights and permissionsReprints and permissionsAbout this article