Unraveling the Molecular Roles of Novel Proteins that Influence Excision of Genomic DNA in Paramecium

Abstract

A significant portion of many genomes comprises sequences such as transposable elements, which carry the potential risk of disrupting genes when they are expressed. While most organisms silence these regions to prevent their proliferation, ciliates tend to eliminate transposable elements and their remnants, Internal Eliminated Sequences (IESs), from the somatic genome. This drastic step of removing substantial portions of the genome necessitates reliable IES targeting, making it a highly orchestrated process. In Paramecium tetraurelia (henceforth Paramecium), IESs lack a conserved motif that provides sufficient specificity for sequence-based targeting alone. To address this challenge, small RNA-guided nuclear crosstalk has been proposed to identify sequences for excision by comparing the fully reorganized somatic genome with the non-reorganized germline genome. However, it is unclear how precise elimination is achieved for all IESs, as only a subset of IESs relies on the small RNA pathway for efficient excision. It is evident that additional, as yet unidentified factors play a role in facilitating the recruitment of the excision machinery to IESs. Therefore, the primary objective of this study is to characterize novel proteins that contribute to IES excision. First, ISWI1, a member of the highly conserved Imitation Switch (ISWI) family of ATP- dependent chromatin remodelers, is characterized. ISWI1 is the first protein to be reported that influences the precision of cutting the exact boundaries of IESs, presumably by nucleosome positioning. In other eukaryotes, ISWI always relies on complex partners for its full activity. After we identified two complex partners of ISWI1, ISWI1 Complex Protein (ICOP) 1 and ICOP2, we show that ISWI1 and the ICOPs localize to the maturing somatic nuclei, where IES excision occurs. The ICOPs interact with ISWI1 both in vitro and in vivo. In knockdown experiments, all three proteins show phenotypic similarities including IES retention in the reorganized somatic genome, imprecise excision at alternative IES boundaries and alterations in the nucleosome densities on IESs, suggesting shared functionality. Additionally, we screened for novel candidates among ISWI1-associated proteins identified by mass spectrometry and characterize two paralogous PHD finger proteins: development-specific PHD finger (DevPF) 1 and DevPF2. Despite their high similarity at the nucleotide and amino acid level, we show that these paralogs contribute differently to IES excision. The early-expressed DevPF1 localizes into some, though not all, of the meiotic germline nuclei, a localization pattern not yet reported for Paramecium proteins. In later stages, DevPF1 localizes to the maturing somatic nuclei, as does the late-expressed DevPF2. The knockdown of DevPF1 completely abolishes development-specific small RNA production, while DevPF2 knockdown mainly affects the late-produced small RNA population. We also demonstrate that DevPF1 knockdown exhibits no preference regarding IES length while in DevPF2 knockdown, preferably long IESs are retained. Taken together, I present work that characterizes five new players essential for maintaining genome integrity during nuclear maturation, adding to our picture of IES excision in Paramecium. These and contemporary studies suggest that additional proteins involved in Paramecium DNA elimination remain to be discovered – it just remains to be seen how many. Unravelling the molecular systems regulating DNA elimination has implications for anyone considering long-term introductions of unnatural genome editing components (such as CRISPR) into eukaryotes. Further knowledge of fundamental processes like genome reorganization are vital for our understanding of cell biology in general and can potentially be transferred to other species.