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Laboratory of RNA Biology
              
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2. REPAG_GenomesR38R91

hREPAG
(Consensus of 1000 human REPAG intron ends. Credit: Aydan Wang)
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Faculty of Graduate Studies
College of Medicine
Faculty of Health Sciences
University of Manitoba


CONTACT:
Prof. Jiuyong Xie
Department of Physiology
University of Manitoba
440 Basic Medical Sciences Bldg
745 Bannatyne Avenue
Winnipeg, MB  CANADA
R3E 0J9
Tel  +1-204 975-7774 (O),
       +1-204 975 7740 (lab)
Email: xiej@umanitoba.ca

ZOOM Meeting ID: 626 2950 9516
(by email appointment only)   

 

 


        







RESEARCH:
      We are interested in understanding how the different fragments of a gene are selectively combined (spliced) to produce multiple products in biology and diseases. This process, called pre-mRNA splicing, has profound implications in the evolution and functional complexity of genes/gene products.  Its disruption is a cause of human genetic diseases by many mutations and also of the aberrant splicing in cancers. Our past research has focused on the molecular basis of adaptive splicing, likely involved in cell adaptation, neuron homeostasis and neurological diseases. This has also allowed us to discover and extend the scope to a novel class of introns/splice sites (REPA-harbouring) contributing to the evolution of alternative splicing, gene product diversity or to the prevention of aberrant splicing in human health/diseases. Particularly welcome to join us are students with backgrounds in biochemistry, genetics, computer science or statistics.

CaRRE
(Right-click image to view details)
The CaRRE element among vertebrates
(Liu GD, et al., J. Biol. Chem. 2012, 287:22709–22716)
REPAG
(Right-click image to view details)
The REPAG element specifically among mammals

(Sohail M., and Xie J. Molecular & Cellular Biology, 2015, 35(12): 2203-2214)


Control of alternative pre-mRNA splicing in biology and diseases

Alternative splicing is a common way of gene regulation that allows the generation of multiple mRNA and often protein isoforms from a single gene. Almost all human gene transcripts are alternatively spliced and some are known to generate extremely diverse protein isoforms. This greatly contributes to the proteomic complexity, particularly in neurons and endocrine cells.  In experimental animals, genetic deficiencies in alternative splicing factors result in developmental defects or embryonic-lethal phenotypes. In humans, aberrant splicing accounts for the defect of at least 30% of genetic mutations that cause diseases. Thus, it is imperative to understand alternative splicing and its regulation in  biology and diseases in the omics era.

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 Slo1BK 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) 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. 1) 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. Further detailed characterization of this molecular process and its role in homeostatic or adaptive splicing, 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, Fig. 3). 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. Moreover, a REPAG prevents aberrant splicing of the CBS gene, mutation of which causes the human genetic disease homocystinuria.

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.

We hope these studies will provide molecular details of splicing changes in cell physiology, as well as knowledge for cancer diagnosis/therapy or the correction of aberrant splicing that causes human genetic diseases.

Keywords: molecular biology, RNA, processing, alternative splicing, cryptic splicing, transcriptome integrity, hnRNP, gene regulation, gene expression, evolution, proteomic diversity, complexity, splicing factors, protein kinases, cell signaling, neurons, endocrine cells, SNP, mutation, cancer, GWAS, human health/genetic diseases


PUBLICATIONS:

Ogunsola S, Liu L, Das U, Xie J. 5-Aza-Cytidine Enhances Terminal Polyadenylation Site Usage for Full-Length Transcripts in Cells. Genes to Cells (2024) In revision. bioRxiv: 2024.2002.2022.581641.

2.     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) In revision. bioRxiv 2024:2024.02.23.581772. https://doi.org/10.1101/2024.02.23.581772

3.     Joyce W, He K, Zhang M, Ogunsola S, Wu X, Joseph KT, Bogomolny D, Yu W, Springer MS, Xie J, Signore AV, Campbell KL: Parallel genetic excisions of the cardiac troponin I N-terminal extension in tachycardic mammals. bioRxiv 2024:2023.05.19.541292. https://doi.org/10.1101/2023.05.19.541292

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 complexityRNA 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 plantsFrontiers 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 hnRNPsRNA 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 cellsMol. Cell. Biol., 2018 May 29;38(12):e00651-17.(MCB Most-Read'04-05'18). |PDF|

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



Research in this lab has been supported by CIHR , NCIC, CBCF, NSERC, MMSF, MHRC and CFI.
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STREX model

Fig. 1. Molecular basis of the depolarization-regulated alternative splicing of the STREX exon of the Slo1 gene.

hnrnpl-prolactin
Fig. 2. Importance of proper splicing control in hormone production: aberrant splice variant of prolactin (right) due to the loss of hnRNP L (image by Lei Lei).


REPA

Fig. 3. A new group of introns: REPA element 'inserted' between the Py and 3'AG and its effects on alternative splicing.
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