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        For example, in electrically excitable cells such as neurons, endocrine and muscle cells, ion channels allow ions in/out of the cell membranes to generate electrical firing patterns that are important for cell functions including memory, hormone secretion and muscle contraction. These processes are believed to be critical for higher order phenotype such as learning, behavior, metabolism and heart beating. How these processes are finely tuned during development and in adult life is still a mystery to researchers. Alternative splicing provides a unique way to diversify proteins and may play a critical role here. A number of studies have indicated that alternative splicing is involved in adaptive or addictive changes in neurons by neuronal activity or  alcohol stimulation.

        Interestingly, alternative splicing of some ion channel genes is regulated by membrane depolarization, the first part of an action potential, implying a gene expression change related to  the electrophysiological memory observed in neurons, or hormone production in response to experience in life (e.g. exercise or stress).  However, the molecular basis of the splicing regulation, particularly after recurrent stimulation, remains largely unclear.

        We have used the STREX (stress axis-regulated exon) variant of the Slo1 BK potassium channel gene as a model to study how cell signals regulate the choice of alternative splice sites in pre-mRNA transcripts. Inclusion of the STREX exon enhances the calcium sensitivity of BK channels and likely modulates cellular electrical properties related to hearing frequency tuning or adaptive changes in learning and memory or tolerance to alcohol. Its regulation by stress hormones and the calcium/calmodulin-dependent protein kinase IV (CaMK IV) makes it an interesting target for dissecting the components regulating alternative splicing as well as understanding the impact of splicing regulation on neuronal electrical properties. A first step toward this goal was made by coupling CaMK IV with a pre-mRNA element (CaRRE1, Fig. 1) sufficient to confer CaMK IV response to an otherwise non-responsive exon. We have recently identified the splicing factors hnRNP L and L-Like (LL) as essential components of the CaMKIV-regulated splicing of STREX by inhibiting U2AF65 binding to the upstream 3' splice site. Particularly for hnRNP L, its Ser513 is phosphorylated and essential for the regulation.(Fig. 2) Other factors including PTB and hnRNP K are involved in the regulation as well.

        In particular, hnRNP L and LL are required for the differential regulation and protection of the hormone gene expression programs for producing prolactin and growth hormones (Fig. 3). Further detailed characterization of this molecular process and its role in homeostatic or adaptive splicing (Fig 4), hormone production and stress response is ongoing .

        In recent years, reports by several labs have also demonstrated that alternative splicing of the BK channel and AMPA receptor genes after chronic inactivities or of the neurexin gene upon depolarization/activities of neurons plays a critical role in the homeostasis of cellular electrical properties or synaptic formation. Moreover, the regulation is mediated by the Ca++/calmodulin-dependent protein kinase IV (CaMKIV) and its downstream splicing factors Sam68 or Nova-2, depending on the target exons. Together, these observations support a critical role of depolarization-regulated splicing in hormone production, neuronal homeostasis or development.

       CaRRE1-like RNA elements, even G tracts, have been found to act similarly as CaRRE1 in hundreds of human genes. Together we call these groups of RNA sequences REPA (regulatory elements between the polypyrimidine tract and 3' AG (Figs. 5 & 6). Most of the REPA G tracts (REPAG) appear to have emerged in the ancestors of mammals, likely contributing to the higher abundance of alternative splicing and proteomic diversity. We have demonstrated with the REPAG of PRMT5 exon 3 that it contributes to the evolutionary emergence of a novel splice variant with an opposite effect on cell cycle. The REPAGs are widespread and highly enriched in metazoa and plants, with the highest abundance in mammals. They are also enriched in the aberrant 3' splice sites of cancer patients mutated of the 3' splicing factors SF3B1 or U2AF35. In the CBS gene, mutation of which causes the human genetic disease homocystinuria, a REPAG prevents its aberrant splicing.

        Other exons studied include those involved in neuronal function, human genetic disease or cell growth/apoptosis, or splicing regulations involving other signaling pathways including protein acetylation and methylation.

        Analyses of these signal-responsive RNA elements indicate that they are mostly mammalian-specific, likely contributing to the more delicate and dynamic control of alternative splicing  and higher proteomic complexity.

1.     Chunyu Liu, Roby Joehanes, Jiantao Ma, Jiuyong Xie, Jian Yang, Mengyao Wang, TianXiao Huan, Shih-Jen Hwang, Jia Wen, Quan Sun, Cumhur Demirkale, Nancy Heard-Costa, Peter Orchar, April P. Carson, Laura M. Raffield, Alexander P. Reiner, Yun Li, George O'Connor, Joanne M Murabito, Peter Munson, Daniel Levy: Integrating Whole Genome and Transcriptome Sequencing to Characterize the Genetic Architecture of Isoform Variation and its Implications for Health and Disease, MedRxiv, 2024, doi: https://doi.org/10.1101/2024.12.04.24318434  

2.     Joyce W, He K, Zhang M, Ogunsola S, Wu X, Joseph KT, Bogomolny D, Yu W, Springer MS, Xie J, Signore AV, Campbell KL: Genetic excision of the regulatory cardiac troponin I extension in high heart rate mammal clades. Science, (2024); 385(6716) 1466-1471, https://www.science.org/doi/10.1126/science.adi8146.

·       Related Perspective/Commentary: Can evolution-based studies inform modern medicine? https://www.science.org/doi/10.1126/science.ads2585

3.     Liu L, Nguyen H, Das U, Ogunsola S, Yu J, Lei L, Kung M, Pejhan S, Rastegar M, Xie J: Epigenetic Control of Adaptive or Homeostatic Splicing During Interval-Training Activities. Nucleic Acids Res., (2024); 52(12)7211–7224, https://doi.org/10.1093/nar/gkae311.

4.     Xie, J., Wang, L. & Lin, RJ. Variations of intronic branchpoint motif: identification and functional implications in splicing and disease. Commun. Biol. 6, 1142 (2023). https://doi.org/10.1038/s42003-023-05513-7.

5.     Xie J and Friedman R, Editorial: Evolution in Neurogenomics. Front. Genet. (2023)14:1220750. doi: 10.3389/fgene.2023.1220750.

6.     Liu L, Das U, Ogunsola S, Xie J. Transcriptome-Wide Detection of Intron/Exon Definition in the Endogenous Pre-mRNA Transcripts of Mammalian Cells and its Regulation by Depolarization, Int. J. Mol. Sci. (2022) 23, 10157. https:// doi.org/10.3390/ijms231710157

7.     Tian L, Xie X, Das U, Chen Y, Sun Y, Liu F, Lu H, Peng N, Zhu Y, Gu X, Deng H, Xie J, Zhao X. Forming cytoplasmic stress granules PURα suppresses mRNA translation initiation of IGFBP3 to promote esophageal squamous cell carcinoma progression, Oncogene, 41, 4336–4348 (2022), https://www.nature.com/articles/s41388-022-02426-3.

8.     Ling Liu, Jinghua Feng, Julian Polimeni, Manli Zhang, Hai Nguyen, Urmi Das, Xu Zhang, Harminder Singh, Xiao-Jian Yao, Etienne Leygue, Sam K.P. Kung, and Xie J. Characterization of cell free plasma methyl-DNA from xenografted tumours to guide the selection of diagnostic markers for early-stage cancers. Frontiers in Oncology, 11:615821 (2021) Feb. 5. DOI: 10.3389/fonc.2021.615821.

9.     Xie J, Weiskirchen R. What does the 'AKT' stand for in the name 'AKT kinase'?: some historical comments. Frontiers in Oncology, 10:1329. June, 2020, https://www.frontiersin.org/articles/10.3389/fonc.2020.01329/full.

10.   Nguyen H, Das U, Xie J. Genome-wide evolution of wobble base-pairing nucleotides of branchpoint motifs with increasing organismal complexity. RNA Biology, 17:3, 311-324, Dec. 2019, http://dx.doi.org/10.1080/15476286.2019.1697548. 

11.   Nguyen H, Xie J. Widespread separation of the polypyrimidine tract from 3' AG by G tracts in association with alternative exons in metazoa and plants. Frontiers in Genetics, 9:741, published: 14 January 2019, doi: 10.3389/fgene.2018.00741. |PDF|

12.   Das U, Nguyen H, Xie J. Transcriptome protection by the expanded family of hnRNPs. RNA Biology, 16:2, 155-159, 2018 Dec 30. PMID: 30596342 DOI: 10.1080/15476286.2018.1564617.

13.   Lei L, Cao W, Liu L, Das U, Wu Y, Liu G, Sohail M, Chen Y, Xie J. Multi-level differential control of hormone gene expression programs by hnRNP L and LL in pituitary cells. Mol. Cell. Biol., 2018 May 29;38(12):e00651-17.(MCB Most-Read'04-05'18). |PDF|

Structural changes of a novel PROLACTIN hormone variant (cyan) upon loss of hnRNP L (Using Google AlphaFold3.0)

14.   Nguyen H, Das U, Wang B, Xie J. The matrices and constraints of GT/AG splice sites of more than 1000 species/lineages. Gene, 2018, 660:92-101.

• *UPDATEd splice site matrices/constraints from recent Ensembl releases. **A list of alternative exons differentially spliced between male and hermaphrodite ('female') C. elegans ('sex'-specific alternative splicing in C. elegans)  from this study (Raw RNA-seq reads from 'Kramer et al, Genetics 204 (2016) 355-69' in the NCBI SRA database)

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College of Medicine

Faculty of Health Sciences

University of Manitoba