Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We uncovered an RNA surveillance pathway that is responsible for the degradation of G4-containing non-coding RNA in human mitochondria.
• We discovered that mitochondria are a source of double-stranded RNA that can be released into the cytoplasm and trigger the interferon response.
• We identified a protein (MTRES1), the upregulation of which counteracts mitochondrial transcription arrest.

Opis badań

• MITOCHONDRIAL RNA EXPRESSION AND SURVEILLANCE

Mitochondria are unique organelles in human cells because their function depends on crosstalk between two genomes: nuclear and mitochondrial. Human mitochondrial DNA (mtDNA) contains only a few genes, but all of them are essential. mtDNA exists in multiple copies per cell, and the expression of mitochondrial genes can be regulated by controlling the gene copy number.

Although the human mitochondrial genome was one of the first to be sequenced, the way it functions is still not entirely understood. Our studies focus on unraveling mitochondrial gene expression mechanisms. To date, we have been mainly interested in post-transcriptional mechanisms, but our ongoing and future studies concern the mechanisms that regulate mitochondrial gene copy number. We also intend to investigate the processing of individual mitochondrial transcripts and the ways in which mitochondrial nucleic acid metabolism responds to stresses (e.g., viral infection). We also plan to explore the recent discovery that mtDNA expression leads to the formation of double-stranded RNA.

• RNA decay and surveillance

The regulation of human mitochondrial gene expression at the initiation of mtDNA transcription appears to be limited. Thus, post-transcriptional processes, including RNA decay, are critical for shaping the mitochondrial transcriptome. We contributed significantly to the identification of an RNA-degrading complex in human mitochondria (1). This complex—the mitochondrial degradosome—consists of RNA helicase SUV3 and the ribonuclease PNPase. We showed that the degradosome is essential for the surveillance and decay of human mtRNAs, particularly antisense transcripts (1). Our recent studies revealed that short mtRNAs that are generated by mtRNA processing and decay machinery are removed by REXO2 oligoribonuclease, proving that REXO2 controls short mtRNAs (2).

• Mitochondrial RNA binding proteins

RNA molecules can form different structures, many of which involve non-canonical base pairing, such as the case of G-quadruplexes (G4s). Mitochondrial genomes of vertebrates exhibit extraordinary GC skews (i.e., high guanine content on one strand). Therefore, the transcription of human mtDNA results in the synthesis of G-rich RNAs that are prone to form G4s. Such mtRNAs, mostly antisense RNAs, are transcribed at high rates, but their steady-state levels are extremely low. We described a mechanism by which G4-containing antisense mtRNAs are efficiently degraded in humans (3, 4). We showed that the RNA-binding protein GRSF1 melts G4s in mtRNAs, facilitating degradosome-mediated decay. Based on phylogenetic analyses, we proposed that GRSF1 appears in mitochondria when genomes undergo a G4-poor to G4-rich transition. This evolutionary adaptation enabled the control of G4 mtRNA levels.

• Double-stranded RNA in mitochondrial biology

The importance of degradosome-dependent mtRNA surveillance was underscored by our studies that were performed in collaboration with the Proudfoot laboratory (Oxford University, UK) and others, showing that SUV3 and PNPase are major regulators of mitochondrial dsRNA (mt-dsRNA) (5). We revealed that mtDNA transcription is a significant source of dsRNA in humans. We showed that the depletion of SUV3 or PNPase leads to the accumulation of mt-dsRNA, and in the case of PNPase, to the release of these species into the cytosol. Remarkably, once in the cytosol, mt-dsRNA triggers an innate immune response through induction of the interferon pathway (5). Our work identified a new mechanism by which mitochondria contribute to cell fate and human health. Currently, we are investigating the role of other proteins in the regulation of mt-dsRNA levels.

• Mitochondrial genome expression in stress

The maintenance of mitochondrial gene expression is crucial for cell homeostasis. Stress conditions may lead to a temporary reduction of mtDNA copy number, raising the risk of the insufficient expression of mtDNA-encoded genes. We applied a quantitative proteomic screen to search for proteins that sustain mtDNA expression under stress conditions. We found that the novel mtRNA-binding protein MTRES1 is elevated in cells with perturbances in mtDNA expression. Our study showed that MTRES1 prevented mtRNA depletion during transcription arrest (6).

• NUCLEAR-ENCODED RNA SURVEILLANCE AND PROCESSING
• Regulation of double-stranded RNA in the nucleus

Transcription of the nuclear genome has the potential to produce long dsRNA species. However, under normal conditions, such RNAs are hardly detectable, suggesting their tight control. To identify proteins that are involved in this regulation, we performed a loss-of-function screen. This screen indicated a pathway, the inhibition of which led to the upregulation of nuclear dsRNA. The molecular mechanism that underlies dsRNA accumulation is the subject of our ongoing studies.

• Bibliography

1. Borowski et al. Nucleic Acids Res. 2013. doi: 10.1093/ar/gks1130
2. Szewczyk et al. Nucleic Acids Res. 2020. doi: 10.1093/nar/gkaa302
3. Pietras et al. Nat Commun. 2018. doi: 10.1038/s41467-018-05007-9
4. Pietras et al. Mol Cell Oncol. 2018. doi: 10.1080/23723556.2018.1516452
5. Dhir et al. Nature. 2018. doi: 10.1038/s41586-018-0363-0
6. Kotrys et al. Nucleic Acids Res. 2019. doi: 10.1093/nar/gkz542
7. Szczesny et al. PLoS One. 2018. doi: 10.1371/journal.pone.0194887

Metodologia

• We combine a range of molecular biology, biochemical, and cellular biology methods to achieve our research goals.
• We perform high-throughput genome-wide siRNA screens. We are equipped with necessary instruments and human siRNAs libraries (genome-wide and targeted).
• We conduct siRNA and DNA plasmid transfections, immunofluorescence staining, and other cell culture experiments in high-throughput compatible 384-well formats.
• We use our Attune NxT flow cytometer to apply a range of cell biology assays.
• We apply confocal and high-content fluorescent microscopy (thanks to a collaboration with IGiB UW).
• We create human cellular models for functional studies (gene silencing and overexpression). We co-developed a straightforward method for DNA cloning into more than 50 vectors that is dedicated to mammalian cell-based studies (7).
• We use various biochemical assays to study the activity of proteins that act on nucleic acids (e.g., degradation, binding, and polymerization).
• We use next-generation sequencing-based approaches, proteomics, and cryo-electron microscopy in collaboration with other laboratories.

Wybrane publikacje

• Dedicated surveillance mechanism controls G-quadruplex forming non-coding RNAs in human mitochondria. Pietras Z, Wojcik MA, Borowski LS, Szewczyk M, Kulinski TM, Cysewski D, Stepien PP, Dziembowski A*, Szczesny RJ*. Nat Commun. 2018. doi: 10.1038/s41467-018-05007-9.
• Mitochondrial double-stranded RNA triggers antiviral signalling in humans. Dhir A*, Dhir S, Borowski LS, Jimenez L, Teitell M, Rötig A, Crow YJ, Rice GI, Duffy D, Tamby C, Nojima T, Munnich A, Schiff M, de Almeida CR, Rehwinkel J, Dziembowski A, Szczesny RJ*, Proudfoot NJ*. Nature. 2018. doi: 10.1038/s41586-018-0363-0.
• Human REXO2 controls short mitochondrial RNAs generated by mtRNA processing and decay machinery to prevent accumulation of double-stranded RNA. Szewczyk M, Malik D, Borowski LS, Czarnomska SD, Kotrys AV, Klosowska-Kosicka K, Nowotny M, Szczesny RJ*. Nucleic Acids Res. 2020. doi: 10.1093/nar/gkaa302 .
• Quantitative proteomics revealed C6orf203/MTRES1 as a factor preventing stress-induced transcription deficiency in human mitochondria. Kotrys AV, Cysewski D, Czarnomska SD, Pietras Z, Borowski LS, Dziembowski A, Szczesny RJ*. Nucleic Acids Res. 2019. doi: 10.1093/nar/gkz542.

Współpraca

• Marcin Nowotny, Laboratory of Protein Structure, International Institute of Molecular and Cell Biology in Warsaw, Poland, www.iimcb.gov.pl/en/research/laboratories/6-laboratory-of-protein-structure-nowotny-laboratory.
• Michał Szymański, Structural Biology Laboratory, University of Gdańsk, Poland. https://mrslab.ug.edu.pl/

Nagrody i wyróżnienia

• Roman Szczęsny. Prime Minister Award for Habilitation. 2020. Prime Minister, Poland.
• Anna Kotrys. START Fellowship. 2020. Foundation for Polish Science, Poland.
• Roman Szczęsny. NCN Award. 2019. National Science Centre, Poland.
• Roman Szczęsny. Minister Award for Scientific Achievements. 2019. Minister of Science and Higher Education, Poland.
• Roman Szczęsny and others. Jan Karol Parnas Award for the best Polish biochemical publication. 2019. The Polish Biochemical Society, Poland.