Center for Molecular Medicine Cologne

Mirka Uhlirova - A 12

Cutting through the tumor complexity: Role of splicing in epithelial homeostasis and tumorigenesis

Pre-mRNA splicing regulates gene expression and contributes to the maintenance of genome stabilityOur previous work defined the evolutionarily conserved protein Ecdysoneless (Ecd) as a new component of the splicing machinery that interacts with the core components of the spliceosome and is required for cell survival. The primary aim of our project has been to elucidate the mechanisms underlying the role of Ecd in pre-mRNA splicing and its link to the maintenance of transcriptome, genome and tissue integrity.

Introduction

Proper gene expression in eukaryotes requires precise excision of introns and fusion of exons. This multi-step reaction is catalyzed by a multi-protein/RNA complex - the spliceosome. The pre-mRNA splicing occurs co-transcriptionally and is among the first steps towards maturation of the mRNA molecule. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNP) and more than a hundred proteins that dynamically associate with the complex. Accuracy and tight regulation of this machinery must be maintained at all times, as aberrant RNA splicing has detrimental consequences for cell and tissue functions. Importantly, mutations or dysregulation of specific splicing factors have been linked to tumor development and progression. Moreover, mounting evidence shows that cancer cells often display “spliceosome addiction” to maintain high mitotic activity. The spliceosome thus represents a key machine supporting tumorigenesis but also a potential Achilles’ heel of tumor cells, manipulation of which might be exploited to combat cancer.

The contribution of spliceosome malfunction to tumorigenesis is manifold, including aberrant processing of transcripts encoding proteins required for the maintenance of cell and tissue homeostasis or generating cancer specific mRNAs. However, splicing factor dysregulation can also promote genome instability. 

Ecd promotes U5 snRNP biogenesis

Previous work from our lab identified a new role for the evolutionarily conserved proteinEcd in pre-mRNA splicing. We showed that Ecd interacts with the core components of the U5 snRNP, including Aar2 and Prp8 (Claudius et al., 2014). Combining genetic and proteomic approaches, we have identified novel interaction partners of Ecd and defined Ecd as a critical regulator of U5 snRNP assembly and Prp8 stability. We show that Ecd loss impacts the cytoplasmic steps of U5 snRNP biogenesis, causing scarcity of functional spliceosomes in the nucleus. The spliceosome shortage promotes the formation of highly mutagenic RNA•DNA hybrids called R-Loops. Exploiting the genetic amenability of the Drosophilamodel, our forward genetic screen identified suppressors as well as enhancers of R-loop formation and other ecd-deficient phenotypes (Figure 1).

Ecd loss results in a genome-wide intron retention as well as specific splicing defects

While ecd loss is tolerated by terminally differentiated cells, actively replicating cells demand Ecd for their survival and function. Importantly, the expression of human Ecd rescues the cellular and molecular defects caused by loss of the Drosophila ortholog (Claudius et al., 2014). Our comparative transcriptomics revealed a trans-criptome-wide increase in intron retention and greater susceptibility of specific weak and non-canonical splice sites to missplicing in ecd-deficient mitotically active cells compared to the differ-entiated neurons and control tissues (Figure 2).

Ecd role in tissue homeostasis and tumorigenesis

Intriguingly, Ecd has been found upregulated in various carcinomas and correlated with poor patient prognosis. Moreover, missense and nonsense mutations have been detected in human intestinal and prostate tumors. To address the pathogenicity of the most frequently detected Ecd cancer-associated mutations, we expressed the human Ecd mutant variants either in the ecd-deficient or wild-type Drosophila imaginal clones using the eyFLP MARCM system. Similar to wild-type human Ecd, the cancer-associated mutant proteins were sufficient to rescue the cellular and molecular defects inflicted by the loss of endogenous ecd (Figure 3). However, neither of the mutant proteins was sufficient to promote growth when overexpressed in a wild-type background, indicating that they likely represent “bystander” rather than oncogenic driver mutations. To translate our findings from the fly to a mammalian systemand to address how altered spliceosome activity contributes to the breakdown of tissue homeostasis and tumorigenesis, we have generated the full body and the conditional knock-out mouse lines using the easy-CRISPR strategy.

Perspectives

Alterations of splicing factors in various human cancers prompted the development of spliceosome-targeting compounds, which showed marked anti-tumor effects and are now in clinical trials. Advancing our knowledge about individual splicing regulators in health and disease will yield additional therapeutic options with which splicing in tumor cells can be modulated or inhibited to block or reverse tumor development.

Cosolo, A., Jaiswal, J., Csordas, G., Grass, I., Uhlirova, M., and Classen, A.K. (2019). JNK-dependent cell cycle stalling in G2 promotes survival and senescence-like phenotypes in tissue stress. Elife 8.

Mundorf, J., Donohoe, C.D., McClure, C.D., Southall, T.D., and Uhlirova, M. (2019). Ets21c Governs Tissue Renewal, Stress Tolerance, and Aging in the Drosophila Intestine. Cell Rep 27, 3019-33 e5.

Donohoe, C.D., Csordas, G., Correia, A., Jindra, M., Klein, C., Habermann, B., and Uhlirova, M. (2018). Atf3 links loss of epithelial polarity to defects in cell differentiation and cytoarchitecture. PLoS Genet 14, e1007241.

Prof. Dr. Mirka Uhlirova CMMC Cologne
Prof. Dr. Mirka Uhlirova

Institute for Genetics and CECAD Cologne / RG location - CECAD Building

Principal Investigator A 12

+49 221 478 84334

+49 221 478 7789

Institute for Genetics and CECAD Cologne / RG location - CECAD Building

Joseph-Stelzmann-Str. 26

50931 Cologne

CMMC Profile Page

Curriculum Vitae (CV)

Publications - Mirka Uhlirova

Link to PubMed

Group Members

Gábor Csordás (PostDoc)
Steffen Erkelenz (PostDoc)
Dimitrije Stanković (PhD student)
Andreia Correia (PhD student)
Naziha Mroueh (PhD student)
Dominik Aschemeier (Bc student)
Eva Külshammer (Lab manager)
Tina Bresser (technician)
Nils Teuscher (technician)
Elke Mainz (secretary)

Figure 1

Figure 2

Figure 3