Regulation of the MDM4 oncogene by the ribosomal protein L22 through alternative mRNA splicing
by Jennifer Jansen
Date of Examination:2024-09-03
Date of issue:2024-09-27
Advisor:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Markus Bohnsack
Persistent Address:
http://dx.doi.org/10.53846/goediss-10766
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Abstract
English
p53 is the most frequently mutated tumor suppressor in human cancer. By acting as a transcription factor for its target genes, it causes downstream effects such as cell cycle arrest or apoptosis. To avoid excessive p53 activity under unstressed conditions, negative regulation of p53 by its antagonists MDM2 and MDM4 is essential. Regulation of MDM2 and MDM4 in turn allows for p53 activation upon different stresses, including nucleolar stress resulting from an impairment of ribosome biogenesis. An important mechanism of how MDM4 is regulated consists in alternative splicing of the MDM4 pre-mRNA and particularly the differential inclusion of its exon 6. MDM4 exon 6 inclusion results in the formation of the MDM4-FL variant encoding full-length, functional MDM4 protein. In contrast, upon exon 6 skipping, the truncated, nonfunctional MDM4-S variant is formed. Correlation analyses of differential RNA splicing and gene mutations in large panels of cancer cell lines revealed a correlation of mutations of the ribosomal protein L22 (RPL22; eL22) and enhanced MDM4 exon 6 inclusion. Based on this, we asked whether L22 regulates MDM4 splicing, promoting exon 6 skipping. We found that L22 indeed regulates MDM4 splicing, promoting exon 6 skipping. This effect was particularly pronounced when impaired ribosome biogenesis caused nucleolar stress and redistribution of L22 to the nucleoplasm. L22 depletion not only led to the presence of functional MDM4 despite nucleolar stress, but it also reduced p53 activity. Previous work revealed that some RNAs bind L22, and that this is mediated by a specific L22-binding consensus motif within RNA. We identified three L22-binding consensus sequences in MDM4 intron 6, and these directly associated with L22. Deletion of these sequences abolished the regulation of MDM4 splicing by L22, reduced p53 activity, and caused resistance of cells to nucleolar stress-induced growth arrest. Overlapping sequences were required for the MDM4 splicing regulation by ZMAT3, but this involved a mechanism that was at least partially independent of L22. Finally, L22 regulated the splicing of additional genes besides MDM4, i.e. L22L1 (RPL22L1) and UBAP2L. In summary, we identified a new mechanism of how p53 is activated and cell proliferation is reduced upon disrupted ribosome biogenesis and nucleolar stress. In this context, L22 redistributes to the nucleoplasm of cells. This allows L22 to bind MDM4 pre-mRNA at its consensus sequence and promote the skipping of exon 6, thereby lowering functional MDM4 levels. This increases p53 activity and suppresses cell proliferation. Through this mechanism, L22 interconnects different layers of gene expression, i.e. ribosome biogenesis, RNA splicing, and transcription.
Keywords: RNA stem-loop; Ribosomal proteins L22, L22L1, S13; Nucleolus; RNA polymerase I; ZMAT3; UBAP2L; Pre-mRNA splicing; Exon skipping
