RNA modulation of structure and function of the Drosophila MSL complex in vitro

Kiss, Anna Elisabeth (2024): RNA modulation of structure and function of the Drosophila MSL complex in vitro. Dissertation, LMU München: Medizinische Fakultät

Abstract

Gene expression is highly regulated in eukaryotes by histone modifications and corresponding reader-writer proteins. In the fruit fly Drosophila melanogaster the gene expression from the single male X chromosome is doubled, matching the transcriptional output from the two female X chromosomes. Absence of this gene dosage compensation results in male fly lethality. The molecular machinery involved is called dosage compensation complex (DCC), which consists of five proteins and a long non-coding RNA. The proteins of the DCC are the histone acetyltransferase MOF, the male-specific lethal proteins (MSL1, MSL2, MSL3) and the helicase MLE. At least one of two long non-coding RNAs roX1 or roX2 are critical for male fly viability in vivo. How the lncRNA impacts the function of the DCC remains hitherto unknown. A hypothesis states that roX incorporation leads to structural changes of the DCC and potentially enhances one of its enzymatic activities, particularly its acetylation activity. To date, only limited information on the structure of the complete complex is available. Thus, I applied crosslinking mass spectrometry (XL-MS) to analyze structural interfaces within the DCC in absence or presence of roX RNA. Novel MSL-MSL protein interactions were found and previously reported contact sites were confirmed. Addition of roX2 RNA subtly changed XL patterns in MLE and the MSL1-MSL2 submodule of the MSL complex, which could hint towards a conformational change upon roX2 integration. Transcriptional upregulation of the male X chromosome is molecularly linked to H4K16 acetylation by the HAT MOF. Hypothetically the activity and the specificity of MOF could be regulated by roX2 RNA. To address this hypothesis, I reconstituted nucleosome arrays in vitro and used purified MSL complexes for acetylation assays. These assays were evaluated by mass spectrometry, which allows for accurate site-specific acetylation identification and quantification. In absence of RNA MOF within the MSL complex is active, however not selective for H4K16ac. At longer incubation times the complete H4 tail is acetylated, starting at H4K16 and progressing outwards to H4K12, K8 and K5. This zipper-like processive behavior is supported by mathematical modelling. Upon addition of roX2 RNA or unrelated long RNA the oligo-acetylation of the H4 tail is suppressed, even at prolonged incubation times. If this effect can be linked to roX RNA in vivo or can be ensured as well by heterogeneous nuclear RNA (hnRNA) remains to be elucidated. Finally, dTIP60, which is a HAT complex of the same enzymatic family, does not show the processive oligo-acetylation mechanism and is not impacted by RNA. In conclusion, the lncRNA roX2 induces subtle conformational changes in MLE and the MSL1-MSL2 submodule of the MSL complex. Moreover, it increases the specificity of the HAT MOF towards H4K16ac in nucleosome arrays. The possible connection between these roles via a shared allosteric mechanism awaits further investigation.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: RNA, Histone, Acetylation, Crosslinking, Mass spectrometry
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 27. September 2024
• 1. Bericht­erstatter:in: Becker, Peter