Researchers have identified a specific molecular mechanism by which the protein RALY regulates alternative splicing, a critical process in gene expression. Published in Frontiers in Genetics, the study demonstrates that RALY targets poly-uridine sequences on pre-mRNA molecules to influence how genetic information is processed. This finding provides a clearer understanding of how this member of the heterogeneous nuclear ribonucleoprotein family operates within the cell, moving beyond previous observations that were limited to a few specific genes. The work combines high-throughput sequencing with detailed biochemical assays to map the global impact of RALY on the transcriptome.
The study, led by scientists from the Gwangju Institute of Science and Technology and the National Research Council in Italy, utilized high-throughput RNA sequencing to analyze the effects of RALY depletion in human colon cancer cells. By comparing cells with normal RALY levels to those where the protein was knocked down, the team identified thousands of alternative splicing events that were significantly altered. The analysis revealed that RALY has a widespread influence on splicing, particularly affecting exon skipping, where specific segments of the genetic code are removed or retained. This global approach allowed the researchers to move past isolated examples and view the full scope of RALY’s regulatory activity.
Alternative splicing is a fundamental layer of gene regulation that allows a single gene to produce multiple protein variants. In this process, introns are removed from pre-mRNA, and exons are joined to form the final mRNA molecule. The decision to include or skip certain exons can dramatically change the function, structure, or localization of the resulting protein. The research team found that RALY depletion led to changes in 2,704 cassette-exon skipping events, representing approximately 7.4% of the exons analyzed. These changes were not limited to exon skipping; the study also detected alterations in alternative 5′ and 3′ splice site selection, as well as intron retention, indicating a multifaceted role for RALY in splicing decisions.
To determine the specific RNA sequences that RALY recognizes, the researchers employed a reporter system based on the Fas minigene. This experimental tool allowed them to test which parts of the pre-mRNA were necessary for RALY to exert its regulatory effect. Through a series of deletion and mutation experiments, the team pinpointed a region containing a sequence of consecutive uridines as the critical target. They demonstrated that RALY promotes the exclusion of a specific exon in the minigene only when this poly-uridine sequence is intact. Mutating even a single uridine to a different nucleotide disrupted RALY’s ability to regulate splicing, highlighting the precision of this interaction.
Further biochemical experiments confirmed that RALY binds directly to these poly-uridine-rich sequences. Using RNA pull-down assays, the researchers showed that RALY interacts strongly with oligonucleotides containing the identified uridine-rich motif, while showing little to no interaction with sequences lacking this feature. This direct physical binding provides a mechanistic basis for the splicing regulation observed in the cell-based assays. The findings suggest that RALY acts by physically engaging with specific RNA structures to modulate the splicing machinery, a process that is distinct from its known roles in transcription and mRNA export.
The study also investigated the structural domains of RALY required for its various functions. The team generated deletion mutants of the protein to see which parts were essential for splicing activity versus nuclear localization. They found that all major domains of RALY, including the RNA recognition motif, variable domain, basic domain, leucine zipper, and acidic domain, are required for its splicing regulatory function. However, the requirements for cellular localization were different. Only the variable and basic domains were necessary for RALY to localize to the nucleus, suggesting that the protein’s splicing activity and its positioning within the cell are governed by distinct structural elements.
Importantly, the researchers distinguished between splicing changes and general gene expression changes. While RALY depletion also caused widespread changes in the expression levels of thousands of genes, the splicing alterations were largely independent of these transcriptional effects. Statistical analysis showed no significant correlation between the magnitude of splicing changes and changes in gene expression levels. This separation indicates that RALY regulates splicing through a direct mechanism that is separate from its role in controlling transcription. The distinct nature of these two regulatory pathways underscores the complexity of RALY’s function in cellular biology.
The relevance of these findings is highlighted by the observation that RALY is frequently upregulated in various cancer types. By analyzing data from the Cancer Genome Atlas, the team confirmed that RALY mRNA and protein levels are significantly higher in tumor tissues compared to normal tissues across multiple cancer subtypes. This upregulation is often associated with poor prognosis and therapeutic resistance in patients. The study specifically examined the splicing of the UPF3A gene in colon cancer, a gene known to be involved in nonsense-mediated mRNA decay. The researchers found a positive correlation between RALY expression and the inclusion of a specific exon in UPF3A, a splicing event that is linked to cancer cell proliferation.
Despite the robust findings, the authors emphasize several limitations of the study. The RNA-seq analysis was based on only two biological replicates per condition, which is a technical constraint that requires careful interpretation of the statistical results. Additionally, the splicing changes were observed following chronic knockdown of RALY, which can trigger secondary cellular responses that are not direct effects of the protein. Therefore, while the study provides strong evidence for RALY’s role in splicing regulation, the full extent of its direct targets in living cells requires further investigation. The authors note that motif enrichment in the genome is a correlative feature and does not independently prove direct binding in all endogenous contexts.
Future research will likely focus on using more rapid protein degradation systems to isolate the immediate effects of RALY depletion. Such approaches could help distinguish primary splicing responses from downstream cellular adaptations. Additionally, high-resolution binding studies will be necessary to map RALY’s interactions across the entire genome. Understanding how RALY’s splicing regulation contributes to cancer progression could open new avenues for therapeutic intervention. Identifying novel RALY-regulated alternative splicing events represents a potential source for future prognostic and therapeutic targets. These findings highlight RALY as a promising candidate for further investigation in cancer-cell biology and therapeutic development.
The study utilized HCT116 cells, a human colon cancer cell line that expresses relatively high levels of RALY protein. To assess the impact of RALY depletion, the researchers treated these cells with specific short hairpin RNA (shRNA) constructs. This intervention reduced both RALY mRNA and protein levels by more than 90%, while having minimal effect on the expression of its homolog, hnRNP C. The resulting RNA sequencing data, deposited in the NCBI GEO repository under accession number GSE141283, provided the foundation for the genome-wide analysis. By mapping reads to the Ensembl GRCh38 human reference genome, the team was able to quantify the extent of splicing alterations with high precision. This experimental setup allowed for a direct comparison between cells with normal RALY function and those with significantly reduced levels, isolating the specific contributions of the protein to the splicing landscape.
Functional analysis of the RALY protein revealed that its splicing activity is dependent on the integrity of its structural domains. The researchers generated deletion mutants lacking the RNA recognition motif, variable domain, basic domain, leucine zipper, or acidic domain. While the variable domain mutant showed a comparatively milder defect in splicing regulation, the removal of the other domains severely disrupted the protein’s ability to modulate exon skipping. The leucine zipper domain, in particular, is required for splicing activity, potentially indicating its involvement in RNA binding or protein oligomerization. These findings highlight the complex structural requirements for RALY to function effectively in the splicing machinery.
In the context of cancer biology, the study examined the relationship between RALY expression and the splicing of the UPF3A gene in colon cancer. UPF3A is involved in nonsense-mediated mRNA decay, and its alternative splicing produces two isoforms with different functional consequences. The long isoform, which includes exon 4, is promoted by RALY and sustains colon cancer cell proliferation, whereas the short isoform is ineffective as an NMD inhibitor. Analysis of TCGA data confirmed that the inclusion of exon 4 is significantly increased in tumor samples compared to normal tissues. Furthermore, a significant positive correlation was observed between RALY mRNA expression and UPF3A exon-4 inclusion in patient specimens. This patient-cohort association complements the cell-culture-based findings, suggesting that RALY-mediated splicing regulation may contribute to the aggressive phenotype of colon cancer cells.
The authors caution that the observed splicing alterations may include indirect secondary cellular outcomes resulting from chronic RALY knockdown. To better distinguish direct RALY-dependent responses from downstream adaptations, future studies could utilize rapid protein-degradation systems such as dTAG-mediated depletion. Additionally, while the study identified poly-uridine-rich motifs near regulated exons, these computationally derived signatures represent candidate sequence features rather than experimentally verified binding sites. Definitive mapping of RALY interactions in living cells will require high-resolution CLIP-seq datasets. Until such data are available, the extent to which the poly-uridine-dependent mechanism applies to endogenous splicing events genome-wide remains an area for further investigation. These limitations underscore the need for continued research to fully elucidate the role of RALY in cancer-associated splicing programs.
Subject of Research: RALY regulates alternative splicing through targeting poly-uridine sequences
Article Title: RALY regulates alternative splicing through targeting poly-uridine sequences
Article References: Liu, Y., Jang, H. N., Pradella, D., Hong, J.-U., Moon, H., Ashraf, H., Zheng, X., Ghigna, C., & Shen, H. (2026). RALY regulates alternative splicing through targeting poly-uridine sequences. Frontiers in Genetics, 17, Article 1965445. https://doi.org/10.3389/fgene.2026.1965445
Image Credits: AI Generated
DOI: 10.3389/fgene.2026.1965445
Keywords: RALY, regulates, alternative, splicing, targeting, poly-uridine, sequences, scientific research
News Source: Drew Townsend. (November 10, 2026). RALY Protein Targets Poly-Uridine Sequences to Control Alternative Splicing. Scienmag.
