Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• Our work demonstrated, for the first time in any eukaryote, that a TFIIS homolog is an essential factor for the production of regulatory non-coding zygotic transcripts.
• We provided the first description of zygotic long ncRNAs that participate in the control of developmentally programmed DNA elimination.
• We found that the developmentally upregulated Spt5 homolog, in cooperation with Spt4 factors, is necessary for the sexual reproduction and production of meiosis-specific short non-coding scnRNAs (similar to piRNAs in animals).
• We discovered the role of the PIWI-interacting protein Gtsf1 in the selective degradation of small RNAs.

Opis badań

Nearly all kinds of genomic regions in eukaryotes have been reported to have some transcriptional activity, and give rise to a variety of non-coding transcripts (ncRNAs). The important question in the field is whether ncRNA synthesis requires a particular composition of the transcriptional machinery. To answer this, we use the ciliate Paramecium tetraurelia, a model unicellular organism in which different types of ncRNAs are implicated in the regulation of developmental genome rearrangements. At each sexual cycle, during the development of the somatic nucleus (macronucleus; MAC) from the germline nucleus (micronucleus; MIC), the Paramecium genome is massively rearranged through the reproducible elimination of germline-specific sequences, including multi-copy transposons and thousands of short, single-copy, non-coding internal eliminated sequences (IESs). According to the current model proposed for P. tetraurelia, these programmed genome rearrangements involve a specialized, RNA interference-related machinery and is mediated by different types of non-coding RNA molecules: a special class of developmentally regulated small RNAs from the MIC (scnRNAs) and longer, non-coding transcripts produced by the maternal MAC as well as by the new MAC. 25-nt scnRNAs are produced from the entire germline genome, bound by PIWI proteins and transported to the maternal somatic nucleus, where selection of scnRNAs corresponding to germline-specific sequences is thought to take place. Selected scnRNAs are then translocated again into the developing somatic nucleus guide the elimination of transposable elements and IES sequences. Epigenetic control of genome rearrangements in P. tetraurelia by RNA-mediated, homology-dependent mechanisms provides evolutionary insight into the diversity of non-coding RNA pathways involved in genome plasticity.

Our research involves in-depth studies of factors specifically responsible for epigenetic control of developmentally programmed DNA elimination mediated by non-coding transcripts in Paramecium. We focus our study on the characterization of proteins influencing transcription, including families of the TFIIS, Spt5 and Spt4 elongation factors as well as other components of the RNA polymerase II complex. We are testing the hypothesis concerning the possible role of these proteins in initialization of genome-wide non-coding transcription. Our research interest also includes other proteins involved in RNA metabolism – PIWI proteins and its associated factors responsible for RNA-RNA and protein-chromatin interactions as well as selective degradation of scnRNAs.

Sequencing and annotation of the largest somatic chromosome and establishment of the whole genome sequence of Paramecium tetraurelia
The DNA Sequencing and Oligonucleotide Synthesis Laboratory from IBB PAS took part in a pilot project for the random sequencing of the genome of Paramecium (1999-2000) and sequencing of the entire chromosome of the macronucleus (2001-2003). The largest Paramecium chromosome we sequenced, one-million base pair in length, was the longest DNA sequence established in Poland at that time and the first ciliate chromosome ever sequenced. The data obtained in these projects were instrumental for the macronuclear genome sequencing (2003-2006).

Functional study of genes essential for sexual processes in Paramecium
In a subsequent project (2004-2010), we applied a global strategy to monitor gene expression profiles of the genes carried by the largest somatic chromosome. Transcriptome analysis involving dedicated DNA arrays revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes.

Discovery and characterization of TFIIS4 elongation factor involved in synthesis of long non-coding RNA
In the course of our studies (2009-2014) we identified an autogamy-specific essential function of the TFIIS4 putative elongation factor that is involved in regulation of genome rearrangements and transcription in the new MAC. We obtained the first evidence for the existence of germline-specific non-coding transcripts at an early stage of MAC development and proved that TFIIS4 plays an important role in their production. We postulated that TFIIS4 may be involved in transcription of an entire genome, providing an interaction RNA scaffold for small RNAs. Our work demonstrated, for the first time in any eukaryote, that a TFIIS homolog is an essential factor for the production of regulatory non-coding zygotic transcripts.

Discovery of a role of Spt5-Spt4 complex in production of development-specific small ncRNAs
Spt5-Spt4 complex is a component of the RNAPII elongation complex. We found that the developmentally up-regulated Spt5 homolog, in cooperation with Spt4 factors, is necessary for sexual reproduction and production of meiosis-specific short non-coding RNAs – scnRNAs. Spt5m and Spt4 proteins are also responsible for correct genome rearrangements in the new MAC. Mass spectrometry analysis following affinity purification of FLAG-tagged proteins confirmed interaction between Spt5m and Spt4mA/Spt5mB/Spt4vA as well as between Spt4mA and Spt5m/Spt5v, leading to the conclusion that different configurations of DSIF complex may exist in Paramecium. These studies established a novel connection between non-coding transcription and the control of genome plasticity.

Discovery of the role of the PIWI-interacting protein Gtsf1 in the selective degradation of small RNAs
We provided important mechanistic insights into the scnRNA selection pathway by identifying a Paramecium homolog of Gtsf1 as essential for the selective degradation of scnRNAs corresponding to retained somatic sequences. We also showed that Gtsf1 is localized in the maternal somatic nucleus where it associates with the scnRNA-binding protein Ptiwi09. Furthermore, we demonstrated that the scnRNA selection process is critical for genome elimination. We proposed that Gtsf1 is required for the coordinated degradation of Ptiwi09-scnRNA complexes that pair with target RNA via the ubiquitin pathway.

Metodologia

The experimental approaches that are used in our laboratory range from molecular genetics to genomics, proteomics, and cell imaging techniques. Our model organism, Paramecium tetraurelia, is a free-living unicellular eukaryote that feeds on bacteria and can easily be cultured. It is well suited for genetic studies because it has two alternative modes of sexual reproduction, conjugation and autogamy, that can be induced under laboratory conditions. We routinely use the “RNAi by feeding” technique to silence the expression of one, two, or even three genes in parallel. We perform genome-wide analyses of genome rearrangement using the high-throughput sequencing of DNA from purified developing new MACs. The deep sequencing of short RNA libraries enables evaluations of the synthesis of scnRNAs and iesRNAs, two classes of ncRNA that are implicated in the regulation of genome rearrangements. We successfully use tagged proteins for the purification of protein complexes that are analyzed using mass spectrometry approaches. The relatively large size of the P. tetraurelia cell (120 μm) and nuclear dimorphism allow microscopic (fluorescence and confocal) studies of dynamic changes in the localization of fluorescently labeled proteins that are active in different nuclear compartments during the course of the sexual cycle.

Wybrane publikacje

• The PIWI-interacting protein Gtsf1 controls the selective degradation of small RNAs in Paramecium. Charmant O.*, Gruchota J.*, Arnaiz O., Nowak K.P., Moisan N., Zangarelli C., Bétermier M., Anielska-Mazur A., Legros V., Chevreux G., Nowak J.K., Duharcourt S. Nucleic Acids Research. 2025. doi: 110.1093/nar/gkae1055.
• The transient Spt4-Spt5 complex as an upstream regulator of non-coding RNAs during development. Owsian, D., Gruchota, J., Arnaiz, O., Nowak, J.K. Nucleic Acids Research 2022. doi: 10.1093/nar/gkac106.
• A meiosis-specific Spt5 homolog involved in non-coding transcription. Gruchota, J., Denby Wilkes, C., Arnaiz, O., Sperling, L., and Nowak, J.K. Nucleic Acids Res. 2017. doi: 10.1093/nar/gkw1318.
• TFIIS-dependent noncoding transcription regulates developmental genome rearrangements. Maliszewska-Olejniczak, K., Gruchota, J., Gromadka, R., Denby Wilkes, C., Arnaiz, O., Mathy, N., Duharcourt, S., Bétermier, M., and Nowak, J.K. PLOS Genet. 2015. doi: 10.1371/journal.pgen.1005383.
• Functional study of genes essential for autogamy and nuclear reorganization in Paramecium. Nowak JK, Gromadka R, Juszczuk M, Jerka-Dziadosz M, Maliszewska K, Mucchielli MH, Gout JF, Arnaiz O, Agier N, Tang T, Aggerbeck LP, Cohen J, Delacroix H, Sperling L, Herbert CJ, Zagulski M, Bétermier M. Eukaryot Cell. 2011. doi: 10.1128/EC.00258.

Współpraca

• Mireille Betermier, I2BC - Institute for Integrative Biology of the Cell, Gif-sur-Yvette, France
• Sandra Duharcourt, Institut Jacques Monod, Paris, France

Nagrody i wyróżnienia

• Kamila Maliszewska-Olejniczak. START Fellowship. 2016. Foundation for Polish Science, Poland.