W dniach 18-20 października 2022 roku członkowie Laboratorium Biologii RNA - Grupa ERA Chairs pod kierownictwem prof. Andrzeja Dziembowskiego (koordynatora projektu) spotkali się z partnerami: Centrum Nowych Technologii, Uniwersytet Warszawski (Polska) oraz Uniwersytet w Bergen (Norwegia).

Podczas spotkania omówiono postępy i przyszłe kierunki badań finansowanych z grantu GRIEG.

On October 18-20, 2022 members of the Laboratory of RNA Biology - ERA Chairs Group headed by Prof. Andrzej Dziembowski (coordinator of the project) met with partners: the Centre of New Technologies, University of Warsaw, Poland and University of Bergen, Norway.

During the meeting progress and future directions of research funded by the GRIEG grant was discussed.

Drug development projects

We cooperated with pharmaceutical companies in multiple drug development projects by performing structural studies of complexes of target proteins with inhibitors. Until the end of 2019 these projects were performed by a specialized laboratory – IIMCB Structural Biology Center with Marcin Nowotny as a CSO.

A published example of such cooperation concerns OATD-01 compound developed by OncoArendi Therapeutics company. OATD-01 is an inhibitor chitotriosidase/acidic mammalian chitinase which can be used in the treatment of idiopathic pulmonary fibrosis. The compound recently completed phase I clinical studies. We contributed the structural characterization of the complex between chitinase and the inhibitor.

Robert Koralewski, Barbara Dymek, Marzena Mazur, Piotr Sklepkiewicz, Sylwia Olejniczak, Wojciech Czestkowski, Krzysztof Matyszewski, Gleb Andryianau, Piotr Niedziejko, Michal Kowalski, Mariusz Gruza, Bartłomiej Borek, Karol Jedrzejczak, Agnieszka Bartoszewicz, Elżbieta Pluta, Aleksandra Rymaszewska, Magdalena Kania, Tomasz Rejczak, Sylwia Piasecka, Michal Mlacki, Marcin Mazurkiewicz, Michał Piotrowicz, Magdalena Salamon, Agnieszka Zagozdzon, Agnieszka Napiorkowska-Gromadzka, Aneta Bartlomiejczak, Witold Mozga, Paweł Dobrzański, Karolina Dzwonek, Jakub Golab, Marcin Nowotny, Jacek Olczak, and Adam Golebiowski, Discovery of OATD-01, a First-in-Class Chitinase Inhibitor as Potential New Therapeutics for Idiopathic Pulmonary Fibrosis. J Med Chem 2020 Dec 24;63(24):15527-15540. Published October 20, 2020, https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01179#

Close-up view of active site of hCHIT1 (represented as surface colored according to electrostatic potential) in complex with OATD-01 (blue sticks).

Nucleic acid-based therapeutics

Antisense technology is an emerging therapeutic approach for inhibiting gene expression through recognition and RNase H1-dependent degradation of cellular mRNAs. In this technology, short, synthetic, single-stranded oligonucleotides (ASOs) are designed that are complementary to the target mRNA.

ASOs are frequently chemically modified for enhancing the therapeutic properties of these molecules. The most widely used modifications include phosphorothioate (PS) backbone and modifications of the sugar moieties at 2’ site. The molecular mechanism of toxicity of chemically modified antisense oligonucleotides are not fully understood.

Hyjek-Składanowska M, Vickers T, Napiórkowska A, Anderson BA, Tanowitz M, Crooke ST, Liang X, Seth PP^&, Nowotny M^&. Origins of the increased affinity of phosphorothioate-modified therapeutic nucleic acids for proteins. J. Am. Chem. Soc., 2020, 142 (16): 7456-7468;
^& - corresponding authors

The first crystal structure of a complex between a protein and fully PS nucleic acid (DNA-binding domain of a model ASO-binding protein PC4 in complex with full PS 2′-OMe DNA gapmer ASO).
The structure reveals a possible mechanism of ASO-induced toxic protein aggregation: ASO is bound in hairpin-like conformation and its exposed gapmer part promotes the formation of a dimer of dimers of PC4 through base pairing.
The protein interacts with the PS-nucleic acid through a network of electrostatic and hydrophobic interactions. Importantly, the backbone of the PS ASO is able to form new and more extensive hydrophobic interactions than a natural phosphodiester backbone which provides insights into the origins for the enhanced affinity of PS for proteins.

The studies of PC4-ASO complex have been performed in cooperation with Ionis Pharmaceuticals (Carlsbad, California, USA).

Structure of PC4 in complex with PS 2′-OMe DNA gapmer ASO. The content of the asymmetric unit of the PTEN ASO complex crystal. PC4 protomers are color-coded and shown in surface representation, DNA is shown in cartoon (PS backbone is shown in yellow, 2′-OMe PS nucleotides are shown in aquamarine, DNA gapmer nucleotides are shown in dark gray). The base pairing between the nucleotides is shown as black dotted lines.

Hyjek-Składanowska M, Anderson BA, Mykhaylyk V, Orr C, Wagner A, Poznański JT, Skowronek K, Seth PP, Nowotny M. Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding. Nucleic Acids Research, 2022;, gkac774, https://doi.org/10.1093/nar/gkac774

The first crystal structure of a complex between a PS oligonucleotide and non-specific nucleic acids binding protein (core domain of annexin A2).
Interactions between the sulfur atom of the PS linkage and the protein surface are mainly of hydrophobic character - the hydrophobic nature of sulfur contributes to the association of PS ASOs with proteins.
Measurements of anomalous diffraction of sulfur confirmed that van der Waals contacts between the sulfur atom and hydrophobic parts of arginine and lysine side chains drive the enhanced interaction of PS ASOs with proteins.
Stereoisomer preference at given phosphorothioate in the DNA oligonucleotide is determined by the environment around the PS linkage coming from the protein and other adjacent structural features such as 5-Me groups on cytosine nucleobases.

The studies of AnxA2-ASO complex have been performed in cooperation with Ionis Pharmaceuticals (Carlsbad, California, USA) and Diamond synchrotron (Didcot, Oxfordshire, UK).

(A) Crystal structure of AnxA2 in complex with PS ASO duplex. Calcium-binding annexing domain I-IV are coloured light pink, violet, purple, and purple blue, respectively. Calcium ions are shown as orange spheres. DNA is shown in cartoon (PS backbone is shown in yellow, 2’-MOE PS nucleotides are shown in green, DNA gapmer nucleotides are shown in dark gray). (B) Close-up view on the phosphorothioate-binding surface in AnxA2. Polar interactions are shown as red dotted lines. Van der Waals interactions are shown as yellow dotted lines. (C) Difference Fourier anomalous map calculated based on long-wavelength X-ray diffraction data (λ = 2.7552 Ȧ), shown as teal mesh. The preferred occupancy of Rp PS stereoisomer is facilitated by the hydrophobic interactions with surrounding amino acids.

RNA processing

Human cap 2’-OH methyltransferase

mRNA contains a cap structure on its 5' terminus. In higher eukaryotes the first and the second ribonucleotide of the body of the mRNA are methylated on the 2' oxygen. This is thought to serve as a tag to distinguish self mRNA from the mRNA of invading viruses. To circumvent the mechanism, some viruses such as West Nile and yellow fever viruses encode their own 2'-OH methyltransferases.

Śmietanski M^*, Werner M^*, Purta E, Kamińska KH, Stepiński J, Darżynkiewicz E, Nowotny M^&, Bujnicki JM^&. Structural analysis of human 2’-O-ribose methyltransferases involved in mRNA cap structure formation. Nat. Commun., 2014 Jan 9;5:3004; ^* - equally contributing, ^& - corresponding authors

The first crystal structure of a cellular 2'-OH cap methyltransferase.
The mode of cap recognition markedly different from viral counterparts providing hints for antiviral drug development.

The studies of cap 2'-OH methyltransferase have been performed in collaboration with Prof. Janusz M.Bujnicki (IIMCB).

Crystal structure of human cap 2'-OH cap methyl transferase 1 (hMTR1). A fragment of capped mRNA is shown in red and the S-adenosylmethionine (methyl group donor) in green.

Mitochondrial exoribonuclease complex mtEXO

RNA degradation pathways play crucial roles in processing of various types of RNA, regulation of gene expression, and efficient removal of defective RNAs. The main executor of RNA turnover and surveillance activity in yeast mitochondria is the mtEXO complex, composed of Dss1 3ʹ-to-5ʹ exoribonuclease and Suv3 helicase.

Rażew M, Warkocki Z, Taube M, Kolondra A, Czarnocki-Cieciura M, Nowak E, Łabędzka-Dmoch K, Kawińska A, Piątkowski J, Golik P, Kozak M, Dziembowski A, Nowotny M. Structural analysis of mtEXO mitochondrial RNA degradosome reveals tight coupling of nuclease and helicase components. Nat Commun., 2018 Jan 8;9(1):97.

Crystal structure of Dss1 exoribonuclease from Candida glabrata reveals it is a unique member of the RNase II family with specialized domains responsible for interactions with Suv3 helicase.
Crystal structure of the mtEXO complex reveals the arrangement of both subunits in which the helicase motor feeds the 3' end of the RNA into the catalytic channel of Dss1 for its efficient degradation.
Co-operation of both helicase and nuclease activities within the complex is particularly important for degeneration of structured RNAs which cannot be handled by Dss1 on its own and for which the unwinding activity of Suv3 is required.

Crystal structure of Candida glabrata mtEXO complex shows the arrangement of the Suv3 helicase on top of Dss1 exoribonucleases and its accessory domains decorating the catalytic RNB domain (shown in blue) with the RNA molecule trapped inside (shown in black).

CutA terminal ribonucleotide transferase

Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3′-end of RNA molecules regulating their fate. A subgroup TENTs are 3′ CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines.

Malik D, Kobyłecki K, Krawczyk P, Poznański J, Jakielaszek A, Napiórkowska A, Dziembowski A, Tomecki R^&, Nowotny M^&, Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine, Nucleic Acids Res., 2020, 48(16):9387-9405; ^&- corresponding authors

The first structural characterization of a TENT adding C/U tails - structures solved for CutA in complex with incoming CTP analog and RNA with three adenosines.
The binding of GTP or a primer with a terminal guanosine is predicted to lead to clashes between NH2 of the guanine and the protein, explaining why CutA is unable to use these ligands as substrates.
Processive adenosine incorporation likely results from tighter binding of the primer with 3′-terminal adenosine and efficient stacking between adenosine bases of the primer and incoming ATP.
A dynamic process of NTP recognition proposed.

Overall structures of CutA complexes. (A) Structure of CutA–CMPCPP complex. The protein is shown as an orange cartoon, with β-strands in a darker shade of orange. Incoming nucleotide is shown as red sticks and active site residues are shown as orange sticks. (B) Structure of CutA–A3 complex. The protein is shown as pink cartoon, with β-strands in purple. The RNA is shown as red sticks and active site residues are shown as pink sticks.

Reverse transcriptases

XMRV reverse transcriptase

Reverse transcriptases use two enzymatic activities – DNA polymerase and RNase H to catalyze the conversion of single-stranded RNA to double-stranded DNA, a process essential for proliferation of retroviruses such as HIV and retrotransposons. Retroviral RTs are divided into two classes – dimeric (i. e. HIV) or monomeric (i. e. gammaretroviral enzyme from mouse Moloney leukemia virus and closely related XMRV).

Nowak E, Potrzebowski W, Konarev PV, Rausch JW, Bona MK, Svergun DI, Bujnicki JM, Le Grice SF, Nowotny M. Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid. Nucleic Acids Res., 2013 Apr 1;41(6):3874-87

The first crystal structure of a monomeric RTs in complex with RNA/DNA hybrid visualizing the polymerase-connection fragment of the enzyme.
Full-length protein modelled based on small-angle X-ray scattering data.
SAXS data demonstrated that the RNase H domain is mobile and only occasionally interacts with the substrate to cleave RNA. This is the mechanism to fine tune RNase H activity.
This mechanism of RNase H fine-tuning is different from dimeric RTs which use substrate deformations for that purpose.

Model of the full length XMRV RT based on SAXS data, a crystal structure of the polymerase-connection fragment in complex with RNA/DNA hybrid (blue-fingers, red-palm, green-thumb, yellow-connection) and the structure of isolated XMRV RNase H domain (orange, Zhou D, J Struct Biol. 2012). RNA template stand is in purple and DNA primer strand in blue.

Foamy viral protease-reverse transcriptase

One of the distinguishing features of foamy viruses (FV) is the different way of replication. Additionally they synthesize a separate mRNA from which the Pol protein is expressed as a precursor of PR-RT-IN, which is later cleaved into two proteins: PR-RT and IN. Thus, FV RT is the only protein of its type to contain an additional PR domain located at the N-terminus of the protein.

Nowacka M^*, Nowak E^*&, Czarnocki-Cieciura M, Jackiewicz J, Skowronek K, Szczepanowski RH, Wöhrl BM, Nowotny M^&. Structures of substrate complexes of foamy viral protease-reverse transcriptase. J Virol., 2021; 95(18): e0084821. ^*- equally contributing, ^& - corresponding authors

First structures of substrate complexes of a foamy viral protease-reverse transcriptase.
Novel mechanism of switching between protein monomer on RNA/DNA substrate and dimer on dsDNA.
Mobile RNase H domain of foamy viral PR-RT transiently interacts with the RNA/DNA substrate 18-21 nt from the polymerase active site.

Structures of nucleic acid complexes of MFV PR-RT. (A) Crystal structure of MFV PR-RT in complex with an RNA/DNA hybrid substrate. (B) Cryo-EM structure of MFV PR-RT ΔRH in complex with dsDNA. Lighter shades of colors are used for domains/subdomains of subunit B.

Ty3 reverse transcriptase

Retrotransposons are mobile genetic elements that replicate with an RNA intermediate. Reverse activity of element-encoded RT is essential for this process. Retroelements are one of the most potent forces shaping eukaryotic genomes – more than 40% of human genome derives from those elements. Ty3 is a yeast retrolement from long-terminal class termed Ty3/Copia. It is thought that retroviruses evolved from this class of retrotransposons.

Nowak E, Miller JT, Bona MK, Studnicka J, Szczepanowski RH, Jurkowski J, Le Grice SFJ^&, Nowotny M^&. Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry. Nature Struct. Mol. Biol., 2014; 21(4):389-96;
^& - corresponding authors

The first crystal structure of a retrotransposon RT.
Ty3 RT forms an asymmetric homodimer in which one subunit has the polymerase competent configuration and the other has an altered conformation and harbors the RNase H activity.
RNase H is postulated to undergo a conformational change to reach the position required for RNA/DNA cleavage, which regulates this activity.
Ty3 and HIV RT architecture differs: HIV enzyme is a constitutive heterodimer with both polymerase and RNase H activities residing in the larger subunit and Ty3 is a substrate-induced homodimer in which the two activities are located in different subunits.
The studies of XMRV and Ty3 reverse transcriptases have been performed in collaboration with Dr. Stuart Le Grice (National Institutes of Health, USA).

Crystal structure of Ty3 reverse transcriptase. The subunit with polymerase-competent configuration is shown in darker color (blue-fingers, red-palm, green-thumb, yellow-RNase H) and the subunit with altered conformation in lighter shades of the same colors. RNA template stand is in purple and DNA primer strand in blue. Active site residues for polymerase and RNase H domain are shown as sticks.

Cauliflower mosaic virus reverse transcriptase (CaMV RT)

CaMV is a prominent plant virus, important in both plant pathology and agricultural biotechnology. Even though the genome of CaMV is a double-stranded DNA, it replicates through reverse transcription. Interestingly, the structural features of CaMV RT make it similar to Ty3 RT, but its mode of action and enzymatic properties differ from other reverse transcriptases.

Chandrasekaran Prabaharan, Małgorzata Figiel, Roman H. Szczepanowski, Krzysztof Skowronek, Weronika Zajko, Vinuchakkaravarthy Thangaraj, Sebastian Chamera, Elzbieta Nowak, and Marcin Nowotny, Structural and biochemical characterization of cauliflower mosaic virus reverse transcriptase, J. Biol. Chem. (2024) 300(8), 107555.

The first crystal structure of a plant viral RT in complex with an RNA/DNA hybrid substrate.
CaMV RT forms a monomeric complex with the hybrid substrate and adopts a polymerase configuration. A single molecule of CaMV RT performs DNA synthesis.
Novel mechanism of RNase H activity that requires two copies of the enzyme for RNA hydrolysis. One molecule is involved in structural stabilisation and another forms a transient association with the monomeric complex for RNA cleavage.

Crystal structure ofCaMV RT-RNA/DNA hybrid complex. Domains of CaMV RT are labelled and RNA and DNA strands are shown in magenta and cyan, respectivel

HIV-1 reverse transriptase

HIV-1 RT is an important drug target in therapy of HIV-1 infection. The RT uses its polymerase and RNase H activities to catalyze the process of reverse transcription, in which the single-stranded RNA of the virus is converted into double-stranded DNA that can be integrated into the host cell genome.

Figiel M, Krepl M, Poznański J, Gołąb A, Šponer J, Nowotny M. Coordination between the polymerase and RNase H activity of HIV-1 reverse transcriptase. Nucleic Acids Res., 2017; 45(6):3341-3352.

Dynamics of HIV-1 RT-RNA/DNA complex were studied using a combination of chemical cross-linking and molecular dynamics simulations.
The RNA/DNA substrate can simultaneously interact with the polymerase and RNase H active sites.
Untwisting of the RNA/DNA substrate double helix is required for its productive interaction with the RNase H active site.
This allows HIV-1 RT to regulate the amount of the RNase H activity.

Molecular dynamics simulation of HIV-1 RT RNA/DNA complex (two views). Superposition of the starting model (light colors) and the final model in MD simulations (dark colors). Domains of HIV-1 RT are labeled. RNA and DNA strands of the substrate are shown in red and blue, respectively.

In order to be integrated into the host cell genome, the single-stranded RNA of HIV-1 is converted into double-stranded DNA. The synthesis of the (+)-strand DNA starts from the polypurine tract (PPT) primer. The PPT primer is generated by the RNase H domain of HIV-1 RT which cuts specifically at its termini, but leaves the body of the PPT intact.

Figiel M, Krepl M, Park S, Poznański J, Skowronek K, Gołąb A, Ha T, Šponer J, Nowotny M. Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase. J Biol. Chem., 2018; 293(1):191-202.

Factors involved in recognition of the PPT sequence by HIV-1 RT-RNA/DNA were studied using a combination of chemical cross-linking, molecular dynamics simulations, and single-molecule assays.
The PPT is specifically recognized after the complex with HIV-1 RT is formed and not at the stage of binding.
Recognition of the PPT is based on two elements: agreement with the sequence preference of RNase H and the indirect readout of the poly-rA/dT stretch. The rigid but brittle poly-rA/dT is not compatible with the catalytically relevant substrate geometry and is prone to undergo sequence slippage upon deformation.
The poor match with the RNase H sequence preference and the dynamic properties of the PPT explain the protection of its body from cleavage.

Studies of HIV-1 RT have been done in collaboration with Jiří Šponer (Institute of Biophysics, CAS) and Taekjip Ha (Johns Hopkins Univ.).

Model of PPT recognition by HIV-1 RT. Cleavage at the expected site involves both preferred residues at the cleavage consensus position and the ability of PPT sequence to undergo the appropriate conformational change without distortion (left panel). Cleavage in the middle of the PPT body (A-tract) is inhibited by three elements: non-preferred residues in the consensus positions, rigidity of the poly(rA-dT) sequence, and misalignment of the substrate at the RNAse H active site due to poly(rA-dT) sequence slippage

Abi polymerases

Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases and combine the ability to synthesize DNA in a template-independent manner with protein priming. The first reverse transcriptase-related proteins that were proposed to utilize the abortive infection antiphage strategy were AbiK, Abi-P2 and AbiA. Within this set, AbiA is the only protein to contain an additional component – a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain which is also found in several other systems of antiviral defense.

Figiel M^*&, Gapińska M^&, Czarnocki-Cieciura M, Zajko W, Sroka M, Skowronek K, Nowotny M*. Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases. Nucleic Acids Res., 2022; 50(17):10026-10040.

^&- equally contributing; * - corresponding authors

Cryo-EM and crystal structure of AbiK-DNA adduct, cryo-EM structure of protein-priming deficient AbiK variant, crystal structure of Abi-P2
AbiK-DNA structure is the first one showing a protein-DNA adduct resulting from protein-primed DNA synthesis
AbiK and Abi-P2 are the first examples of RT-related enzymes that adopt hexameric or trimeric architecture; the oligomerization is required for enzymatic activity
AbiK and Abi-P2 adopt a bilobal structure with an RT-like domain that comprises palm and fingers subdomains and a unique helical domain
Protein priming by Ll-AbiK involves a conformational change of a mobile loop harboring the priming tyrosine residue

Crystal structures of AbiK-DNA adduct (left) and Abi-P2 (right) polymerases shown in surface representation. Individual subunits are shown in different colors, DNA strands are shown as black cartoon.

Gapińska M, Zajko W, Skowronek K, Figiel M, Krawczyk PS, Egorov AA, Dziembowski A, Johansson MJO*, Nowotny M*. Structure-functional characterization of Lactococcus AbiA phage defense system. Nucleic Acids Res., 2024, 52(8):4723-4738.
* - corresponding authors

a crystal structure of AbiA-DNA complex
AbiA forms dimers and HEPN domain is essential for dimerization
AbiA exhibits template-independent DNA polymerase activity with nucleotide preference for adenosines and cytosines
HEPN domain is enzymatically inactive, yet its presence is required for DNA polymerase activity of AbiAProtein priming by Ll-AbiK involves a conformational change of a mobile loop harboring the priming tyrosine residue
DNA polymerase activity of AbiA is essential for protection against invading phages as evidenced by an infection assay in E. coli surrogate host
contrary to previous assumptions, the antiphage defense mediated by AbiA and AbiK appears not to rely on induced cell death

Studies of AbiA were performed in collaboration with Marcus Johansson (Lund University, Sweden) and Andrzej Dziembowski (IIMCB).

(left) Crystal structure of L. lactis AbiA protein dimer. Domains of one of the subunits are colored and labeled, DNA product of the enzyme is shown in green.
(right) Results of the infection assay, in which E. coli cells harboring wild-type and mutated versions of AbiA were challenged by different phages. Formation of plaques indicates successful infection.

DNA transposition

Tn7 Transposase TnsB

Bacterial Tn7 elements are among the best-studied and most widespread DNA transposons and are detected in 10%–20% of sequenced bacterial genomes. Tn7 mobility is tightly controlled and mediated by five element-encoded proteins. Translocation of the Tn7 is executed by a heteromeric transposase TnsA+TnsB. The TnsB transposase recognizes the left and right ends of the element, catalyzes cleavage at the 3′ ends of the transposon, and performs the strand-transfer reaction that leads to the insertion of the element into a target DNA.

Kaczmarska Z*, Czarnocki-Cieciura M*, Górecka-Minakowska KM*, Wingo RJ, Jackiewicz J, Zajko W, Poznański JT, Rawski M, Grant T, Peters JE&, Nowotny M.& Structural basis of transposon end recognition explains central features of Tn7 transposition systems. Mol Cell, 2022 Jul 21;82(14):2618-2632.e7

* - equally contributing, & - corresponding authors

Cryo-EM structure of Tn7 transposase TnsB in complex with transposon end DNA
TnsB chains interact with its binding sites in DNA in a tiled and intertwined fashion
TnsB exhibits a DNA sequence preference rather than strict specificity
Overlap of TnsB-binding sites in the right Tn7 end is key for its functionality

Beads-on-a-string organization of TnsB bound to the fragment of the right transposon end. The TnsB chains (labeled with capital letters) and DNA sites are colored as follows: olive for chain A, red for chain B, orange for chain C, yellow for chain D, burgundy for chain E, light cyan/cyan (top/bottom strands) for site 1, blue/dark blue for site 2, pink/purple for site 3, and black for transposon terminus. The axis of the DNA helix is shown as a dark blue tube.

DNA repair

Bacterial Nucleotide Excision Repair

Nucleotide excision repair (NER) is one of the major pathways of DNA repair and its main feature is its ability to recognize a wide spectrum of DNA lesions of various sizes and structures. In bacterial NER UvrA, a dimeric ATPase, plays the role of a DNA damage sensor. Damage verification is performed by UvrB helicase and UvrC double nuclease excises the damaged DNA fragment.

Jaciuk M, Nowak E, Skowronek K, Tanska A, Nowotny M. Structure of UvrA nucleotide excision repair protein in complex with modified DNA. Nature Struct. Mol. Biol., 2011; 18:191-197

The first crystal structure of UvrA interacting with damaged dsDNA obtained using the protein from T. maritima.
UvrA does not interact with the damage site directly but senses the deformed conformation of the DNA induced by the presence of the lesion – bending, stretching and unwinding.

Crystal structure of UvrA dimer (one monomer in color, the other in gray). One ATPase module is shown in cyan and red and the other in pink and blue. The DNA-binding domain is in green and UvrB-binding domain in yellow. Structural zinc ions are shown as orange spheres.

Jaciuk M^*, Swuec P^*, Gaur V^*, Kasprzak JM, Renault L, Dobrychłop M, Nirwal S, Bujnicki JM^& , Costa A^&, Nowotny M^&. A combined structural and biochemical approach reveals translocation and stalling of UvrB on the DNA lesion as a mechanism of damage verification in bacterial nucleotide excision repair. DNA Repair, 2019; 85, 102746; ^* - equally contributing, ^& - corresponding authors

Computational structural model of UvrA—UvrB—DNA complex involved in DNA damage verification in bacterial DNA repair was prepared and corroborated experimentally by electron microscopy.
UvrB uses a β-hairpin element to clamp one DNA strand, each one of the two UvrB molecules in the complex clamps a different DNA strand.
UvrB translocates in 3′ direction toward the DNA lesion where the UvrB molecule clamping the damaged strand stalls and recruits UvrC nuclease.
This mechanism explains how the initial imprecise localization of the damage by UvrA is converted to precise and strand-specific localization to promote accurate incisions by UvrC.

The studies of damage verification in bacterial NER have been performed in collaboration with Janusz M. Bujnicki (IIMCB) and Alessandro Costa (The Crick Institute).

Model of damage verification in bacterial NER. (A) Model of UvrA2—UvrB2—DNA complex. UvrA dimer shown in two shades of blue. UvrB shown in orange. DNA shown in black. (B) Proposed mechanism. The UvrA dimer bound at the site of DNA modification recruits two UvrB molecules. Each UvrB molecule clamps a different DNA strand under the B-hairpin element (upper panel). Both UvrB molecules then translocate toward the lesion with 5′ to 3′ polarity on the strand under the hairpin. The UvrB molecule that clamps the modified strand will stall at the lesion (green star indicates the site of DNA modification) and the other UvrB molecule (light orange) will dissociate (middle panel). The stalled UvrB recruits UvrC double nuclease (shown in yellow), which makes two incisions indicated with triangles.

Rad2

Eukaryotic nucleotide excision repair (NER) is one of the major DNA repair pathways. It involves the excision of the DNA fragment containing the damage. This is achieved through the action of two nucleases – XPF-ERCC1 complex and XPG (Rad2 in yeast). Rad2/XPG belongs to flap endonuclease metal ion-dependent enzymes along with FLAP1 and EXO1. Its unique feature within this family is the ability to cleave DNA bubbles – substrates with melted single stranded region flanked with double-stranded stretches of DNA.

Miętus M, Nowak E, Jaciuk M, Kustosz P, Studnicka J, Nowotny M. Crystal structure of the catalytic core of Rad2: insights into the mechanism of substrate binding. Nucleic Acids Res., 2014; 42(16):10762-75.

The first crystal structure of the catalytic core of Rad2 using a truncated version of the S. cerevisiae enzyme.
The main substrate specificity determinant of Rad2 is the interaction of the last exposed base pair of the double-stranded region with the so-called hydrophobic wedge of the enzyme. No interactions with the single stranded portion of the substrate are observed.
The main DNA-binding element is potassium-coordinating helix-two-turn-helix (H2TH) motif. It contains an additional charged helix binding the DNA, which is a unique feature of Rad2.
The likely explanation for the unique ability of Rad2 to clave DNA bubbles (substrate without a free 5’ DNA end) is the altered structure of the so-called helical arch. In FEN1 and EXO1 this element blocks the exit from the active site preventing cleavage of substrates without a free 5’ end. In Rad2 the helical arch has a different structure forming an exit route from the active site.

Crystal structure of Rad2-DNA complex. The complex contains two independent protein molecules – one is shown in color: cyan for H2TH motif, green for hydrophobic wedge and orange for helical arch. The DNA is shown in cyan and blue, potassium ion as a purple sphere and the calcium ion at the active site as a green sphere.

RecFOR

RecFOR pathway is one of the homologous recombination-based DNA repair pathways in bacteria. It involves RecF, RecO and RecR proteins, that bind at the junction of single-stranded (ss) and double-stranded (ds) DNA and then facilitate the replacement of the SSB protein, which initially covers ssDNA, with RecA which promotes the search for the homologous sequence.

Nirwal S, Czarnocki-Cieciura M, Chaudhary A, Zajko W, Skowronek K, Chamera S, Figiel M, Nowotny M. Mechanism of RecF–RecO–RecR cooperation in bacterial homologous recombination. Nature Structural & Molecular Biology, 2023; doi: 10.1038/s41594-023-00967-z.

The structure of RecF-DNA complex at 3.1 Å resolution, shows that RecF dimer uses its helical protrusions to clamp the dsDNA.
The structure of RecF-RecR-DNA subcomplex (3.1 Å resolution), shows that one protomer of RecF dimer binds two different regions of the tetrameric RecR ring.
The structures of RecR-RecO-DNA complex (6.1 Å resolution) and RecF-RecO-RecR-DNA assembly, explains how RecO is positioned to interact with ssDNA and SSB, which is proposed to lock the complex at a ss-dsDNA junction.

The cryo-EM reconstructions for the RecFOR-DNA assembly and the RecFR-DNA and RecOR-DNA subcomplexes of the assembly. RecF dimer is shown in yellow and sand; RecR tetramer in cyan, pink, purple and blue; RecO in red and DNA in white.

RuvC

RuvC is a canonical bacterial Holliday junction (HJ) resolvase, which functions as a dimer. HJ are four-way DNA structures formed by the exchange of strands between two helices. They are intermediates in homologous recombination, a process which is used to repair dangerous DNA lesions such as double-strand breaks.

Gorecka, KM, Komorowska W and Nowotny M. Crystal structure of RuvC resolvase in complex with Holliday junction substrate. Nucleic Acids Res., 2013; 41(21):9945-55

The first crystal structure of RuvC in complex with a DNA substrate and the first substrate complex structure of a cellular resolvase, solved at 3.8 Å resolution.
HJ in a novel tetrahedral conformation with two phosphate groups symmetrically located 1 nt from the HJ exchange point interacting with two active sites of RuvC dimer.
Novel mode of HJ recognition relative to phage enzymes for which complex crystal structures had been available.

Crystal structure of RuvC in complex with Holliday junction. The two protomers are shown in pink and orange. The DNA is in blue with cleaved phosphates indicated with spheres.

Górecka KM^*, Krepl M^*&, Szlachcic A, Poznański J, Šponer J, Nowotny M^&. RuvC uses dynamic probing of the Holliday junction substrate to achieve sequence specificity and efficient resolution. Nature Commun. 2019; 10(1):4102; ^*- equally contributing,
^& - corresponding authors

Mechanism of sequence specificity and cut coordination revealed by a combination of structural biology, biochemistry, and a computational approach.
Correct positioning of the substrate for cleavage requires conformational changes within the bound DNA, which are possible only for the cognate sequence.
The conformational changes and the relieving of protein-induced structural tension of the DNA facilitates coordination between the two cuts.
The unique DNA cleavage mechanism of RuvC demonstrates the importance of high-energy conformational states in nucleic acid readout.

Studies of RuvC mechanism have been done in collaboration with Jiří Šponer (Institute of Biophysics, CAS)

Cartoon representation of the mechanism of HJ resolution by RuvC. (a) Holliday junction. Cleaved and non-cleaved DNA strands are shown in purple and blue ladder-like representations, respectively. (b) Binding of the HJ DNA. The subunits of the dimer are shown as yellow-green and pink ovals. The scissile phosphate is marked as a purple circle. Cyan circles show active sites in an inactive configuration. (c) Flipping of the adenine (red) opposite the scissile base. The active site in the catalytic configuration is shown as a red circle. (d) The second cut. (e) Resolution products

Slx1-Slx4

Slx1 is a nuclease which cleaves various DNA structures during DNA repair and recombination. It associates with Slx4 platform protein which coordinates the action of multiple proteins. Slx1 together with Mus8-Eme1 nuclease constitute one of the major Holliday junction pathways in eukaryotes.

Gaur V, Wyatt HD, Komorowska W, Szczepanowski RH, de Sanctis D, Gorecka KM, West SC, Nowotny M. Structural and Mechanistic Analysis of the Slx1-Slx4 Endonuclease. Cell Rep., 2015; S2211-1247(15)00165-5

First structural information for Slx1 and Slx4CCD.
Fungal Slx1 forms a homodimer in which the active site is blocked, explaining why Slx1 alone is inactive.
Slx4CCD domain binding is mutually exclusive with homodimerization.
Slx4 binding exposes the active site of Slx1 and activates the nuclease. This mechanism ensures that the promiscuous and potentially dangerous Slx1 nuclease is only active when bound to Slx4 platform which regulates its activity and coordinates it with other proteins.

Crystal structures of Slx1 homodimer and Slx1 in complex with Slx4CCD domain (orange). Slx1 comprises GIY-IYG nuclease domain (yellow) and RING finger zinc-binding domain (green). Upon Slx4 binding the active site of the nuclease domain is exposed and the enzyme is activated.

Gaur V, Zajko W, Nirwal S, Szlachcic A, Gapińska M, Nowotny M. Recognition and processing of branched DNA substrates by Slx1-Slx4 nuclease. Nucleic Acids Res., 2019; 47:11681-11690

Based on a crystal structure, modeling, and biochemical studies, a mechanism was proposed to explain the specificity of Slx1 towards a wide range of branched DNA substrates.
Slx1 bends the DNA and identifies the branch point as a flexible discontinuity.
Cuts are introduced on the 3ʹ side of the branch point.

Models of Slx1–Slx4CCD3 interactions with branched DNA. (A) Schematic of the DNA flap substrates with terminology of the strands. (B) Model of Slx1–Slx4CCD3 bound to a 3′-flap substrate in configuration, which is conducive to incision in the continuous strand. (Left) Slx1 GIY-YIG domain is shown in yellow with -strands in orange and RING domain shown in blue. Slx4CCD is shown in green. The modeled DNA is based on the structure of R.Eco29kI restrictase (PDB ID: 3NIC) and is shown in black with the scissile phosphate shown as a sphere. A fragment of the DNA that is observed in the Slx1–Slx4CCD-DNA structure is shown in cyan. The possible link between the two DNA double helices is shown as a dashed cyan line. Residues of the active site are shown as red sticks. The same model with protein in surface representation, colored according to the surface potential (±3 Kt/e). (C) Model of Slx1–Slx4CCD3 bound to a 5′-flap substrate, in configuration which is conducive to incision of the flap strand. The representations are the same as in (B).

RNases H

Bacterial RNase H2

RNase H2 cleaves the 5′ phosphate of ribonucleotides in RNA-DNA junctions. It is the only known enzyme that can initiate the process of mutation-free removal of single ribonucleotides from genomic DNA. Such ribonucleotides are very often misincorporated by replicative polymerases and lead to genomic instability.

Rychlik MP, Chon H, Cerritelli SM, Klimek P, Crouch RJ, Nowotny M. Crystal Structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage. Mol. Cell, 2010; 40:658-670

First crystal structure of substrate complex of RNase H2 obtained using T. maritima protein.
Unique catalytic mechanism in which the substrate is deformed, so that the phosphate group of the RNA-DNA junction participates in metal ion coordination at the active site. This metal ion positions and activates the nucleophilic water molecule. Hence, the substrate is used to assemble the active site to promote its own cleavage.

The studies of RNases H2 were performed in collaboration with Dr. Robert Crouch (National Institutes of Health, USA).

Crystal structure of T. maritima RNase H2 in complex with the nucleic acid substrate. The catalytic domain is in orange and purple and the helical C-terminal extension in yellow. DNA is shown in blue and single ribonucleotide in red. Metal ions at the active site are shown as green spheres.

Human RNase H2

Human RNase H2 comprises three subunits – the catalytic subunit is very similar to monomeric bacterial and archaeal enzymes while the remaining two subunits do not share sequence similarity with any other known proteins. Mutations in human RNase H2 lead to a severe genetic autoimmune disease - Aicardi-Goutières syndrome.

Figiel M, Chon H, Cerritelli SM, Cybulska M, Crouch RJ, Nowotny M. The structural and biochemical characterization of human RNase H2 complex reveals the molecular basis for substrate recognition and Aicardi-Goutieres syndrome defects. J. Biol. Chem., 2011; 286:10540-50

Crystal structure of the trimeric human RNase H2.
All mutations from AGS patients mapped and their effect on the structure and mechanism of the enzyme proposed.

Crystal structure of human RNase H2. The catalytic subunit is shown in yellow and the auxiliary ones in purple in blue.

RNase H3

RNases H3 are present in certain bacterial and archaeal species. They share high sequence and structure similarity with RNases H2, however in terms of biochemical properties they are similar to RNases H1 – they prefer to cleave RNA/DNA hybrids in the middle of the RNA sequence. RNase H3 contains a unique N-terminal domain related to TATA-binding protein.

Figiel M, Nowotny M. Crystal structure of RNase H3-substrate complex reveals parallel evolution of RNA/DNA hybrid recognition. Nucleic Acids Res., 2014; 42(14):9285-94

The first crystal structure of RNase H3 in complex with the RNA/DNA substrate.
The RNA strand is recognized by contacts between 2’-OH groups of four consecutive ribonucleotides.
The DNA strand is recognized by deformation to B-form sugar puckers only allowed for DNA.
The N-domain specifically binds RNA/DNA hybrid by recognizing 2’-OH of RNA and forming stacking interactions with the ribose rings of the DNA.
The mechanism of N-domain is very similar to the structurally unrelated hybrid-binding domain present in the N-terminus of RNases H1. This is a likely case of convergent evolution of RNA/DNA recognition.

Crystal structure of RNase H3 (catalytic domain in yellow and N-domain in green) interacting with an RNA/DNA hybrid (RNA in red and DNA in blue). The cleaved phosphate is shown as a red sphere and the phosphate group of the deformed DNA residue as a blue sphere.

1.

May 9, 2021 - Recruitment announcement published
May 24, 2021 - Start of the recruitment
June 6, 2021 - Deadline for documents submission

Research projects for admissions 2021/2022-4:

Poly(A) tails - central hubs of mRNA stability control

Supervisor: Professor Andrzej Dziembowski

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of RNA Biology

Project description:

Gene expression is regulated at multiple levels. Our lab is interested in the regulation of mRNA stability, especially through the modifications of poly(A) tails. Recently, we have shown that the addition of untemplated uridines to the 3’ end of LINE1 retrotransposons precludes their propagation (Warkocki et al. Cell 2018). Moreover, we have identified a family of poly(A) polymerases TENT5, which reside in the cytoplasm and enhance the expression of mRNAs encoding secreted proteins (Moczek et al. Nature com. 2017; Bilska et al. Nature com. 2020; Gewartowska et al. Cell reports 2021). Those enzymes are differentially expressed in tissues and organs, affecting several aspects of animal physiology. TENT5C is an onco-suppressor in multiple myeloma and control immunoglobulin expression in B cells. TENT5A is essential for collagen secretion, and its mutations lead to congenital bone disease

In the future, we will continue to study the role and mechanism of action of TENT5 poly(A) polymerases, analyze global control of poly(A) tail lengths and develop bioinformatics tools for Direct RNA sequencing. Finally, we are planning to translate our knowledge out poly(A) tails for the design of mRNAs, which are more stable and better translated, which will be very valuable for mRNA-based therapeutics such as mRNA vaccines.

Aim:

The exact nature of the project will depend on the skills, predispositions, and interests of the selected PhD student. It may focus on:

functional analysis of the TENT5A poly(A) polymerases in transgenic mouse models generated in-house using CRISPR/Cas9 methodology
mastering of the DRS methodology in either the experimental part or bioinformatic analysis
analysis of principles of mRNA stability control and design of more efficient mRNA based therapeutics

Requirements:

Master's degree in biology, biochemistry or related field
Eligibility for PhD studies in Poland
Highly talented individuals who are passionate about research and are full of scientific curiosity
Experience in either: molecular biology/transcriptomics, animal models, bioinformatic analysis of transcriptomic data, will be a clear benefit
Written and spoken fluency in English
Willingness to learn and take new challenges, ability to work independently, analytical thinking
Good interpersonal skills and a collaborative attitude.

Number of positions available: 2

Contact: Andrzej Dziembowski

The role of mTOR-Brg1 interaction in normal and aberrant neuronal activity (NCN/MAESTRO)

Supervisor: Professor Jacek Jaworski

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of Molecular and Cellular Neurobiology

Project description:

Gene expression is key for brain development and function and is regulated by a complex protein apparatus, which, among other things, is responsible for changes in the spatial packing of DNA in the cellular nucleus. mTOR kinase is one of the basic regulators of metabolism Mutations in the mTOR regulating genes, i.e. TSC1 or TSC2 lead to multi-organ diseases with serious neurological and neuropsychological symptoms. One of such diseases is tuberous sclerosis complex characterized by the occurrence of epilepsy, mental retardation and autism spectrum disorders. mTOR acts on many proteins changing their function, but occurs mainly in cytoplasm. However, the results of our previous research and the preliminary data which form the basis of this research proposal indicate that neuronal activity causes mTOR to move to the nucleus of the cell, where it interacts with Brg1. Using advanced molecular, cellular biology and microscopy methods we plan to study how mTOR-Brg1 interaction affects neuronal activity, epileptogenesis and social interactions. The research will use in vitro cultured neurons (rat and human) and Danio rerio. The results will contribute to a better understanding of the role of mTOR in physiology and brain diseases.

Aim:

On the basis of our previous research, we hypothesize that neuronal activity causes mTOR to move to the nucleus, where it regulates the chromatin-modelling complexes and gene expression. At the same time, we hypothesize that this sequence of events is disturbed in tuberous sclerosis complex leading to epilepsy as well as disturbances in social interactions characteristic of autism spectrum diseases. The aim of the project is to verify these hypotheses.

Requirements:

Master's degree in biology, biochemistry or related field
Eligibility for PhD studies in Poland
Good knowledge of basics of molecular and cell biology and/or neurobiology
Basic hands-on experience in one of the fields: molecular & cell biology, genetic engineering, fluorescent microscopy, danio rerio animal model
Basic programing skills (e.g., R)
Written and spoken fluency in English
Willingness to learn and take new challenges, ability to work independently, analytical thinking
Good interpersonal skills and a collaborative attitude

Number of positions available: 1

Contact: Jacek Jaworski

Signaling of AXL receptor in cancer cells

Supervisor: Professor Marta Miączyńska, auxiliary supervisor Daria Zdżalik-Bielecka, PhD

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of Cell Biology

Project description:

AXL is a tyrosine kinase receptor that upon activation by its GAS6 ligand regulates cell growth, survival, migration and invasion. Overexpression of AXL occurs in a wide variety of cancer types
and is associated with increased invasiveness and metastasis, as well as resistance of cancer cells to anticancer therapies. In our previous studies, we identified the AXL receptor interactome (a set of proteins with which the receptor interacts) and discovered that AXL binds numerous proteins that regulate actin cytoskeleton dynamics. Through such interactions, activation of AXL receptor stimulates the formation of peripheral and circular membrane ruffles that promote macropinocytosis and ultimately lead to cell invasion (see our manuscript at https://bit.ly/2QfEVpF for more details). Macropinocytosis is a form of endocytosis that allows cells to take up large volumes of extracellular fluid and the compounds dissolved within it. The macromolecules taken up through this pathway can be an important source of nutrients that fuel cancer cell growth. It has been recently proposed that macropinocytosis may also contribute to resistance of cancer cells to chemotherapeutics. We found that proteins in the AXL interactome may be involved in nutrient uptake by macropinocytosis. Elucidating their mechanisms of action in AXL receptor signaling will be the goal of the proposed project.

Aim:

The goal of this project is to investigate the molecular mechanisms of AXL receptor signaling that regulates cancer cell growth. Specifically, we will investigate how the AXL receptor and its ligand GAS6 stimulate uptake of nutrients from the extracellular environment via macropinocytosis.

We will also determine whether and how this phenomenon contributes to drug resistance of cancer cells and, consequently, to the growth of tumors despite the applied therapy.

Requirements:

Master's degree in biology, biochemistry or related field
Eligibility for PhD studies in Poland
Solid understanding of the principles of molecular and cell biology
Previous experience in laboratory work and familiarity with basic molecular biology techniques
Written and spoken fluency in English
Good interpersonal skills and a collaborative attitude

Number of positions available: 1

Contact: This email address is being protected from spambots. You need JavaScript enabled to view it., Daria Zdżalik-Bielecka

RNA-Protein Interactions in Human Health and Disease (NCN/DIOSUCRI)

Supervisor: Gracjan Michlewski, PhD, Professor

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of RNA-Protein Interactions

Project description:

RNA-binding proteins (RBPs) are key molecules that control gene expression through RNA-protein interactions. Consequently, they contribute to cellular homeostasis, normal development and majority of human diseases. Importantly, new RBPs are being discovered by high-throughput proteomics, but we still have a limited understanding of their function.

RNA viruses have caused several epidemics in the 21st century. Taking influenza A virus (IAV) infection as an exemplar, it kills 250,000 to 500,000 people annually and generates a significant global socioeconomic burden. Importantly the emergence of COVID-19 pandemic caused by an RNA virus SARS-CoV-2 continue to have catastrophic consequences on public health and world economy. Thus, a detailed molecular understanding of host-virus interactions is imperative in order to know how best to inactivate these viruses and prevent major disruptions in the future.

We have recently discovered and started characterising novel RNA binding protein – E3 ubiquitin ligase TRIM25 (Choudhury et al. 2014; Choudhury et al. 2017). TRIM25 belongs to a large family of tripartite motif-containing proteins (more than 80), most of which have E3 ubiquitin ligase activity. Many of TIRIMs are positive or negative regulators of innate immune response pathways. Importantly, TRIM25 is emerging as a key factor in the innate immune response to RNA viruses (including IAV, CoV, dengue virus and many others). Despite the essential involvement of TRIM25 in viral RNA-induced innate immunity, its RNA-binding functions are still poorly understood.

Aim:

With this project, we aim to take advantage of an assembled multi-disciplinary team to uncover the roles of the novel RNA-protein interactions in the antiviral response to selected RNA virus infections. We hypothesise that TRIM25 binds directly to viral RNAs to restrict virus propagation. We also hypothesise that other members from TRIM family bind RNA. Finally, we hypothesise that specific host RBPs bind to virus derived RNAs and inhibit or augment innate immune response. In summary, this project has the potential to make crucial contributions to understanding the innate immune response to RNA viruses and provide a platform for the development of novel, RNA-based antiviral therapeutics.

Requirements:

Master's degree in biology, biochemistry or related field
Solid knowledge of the principles of cell and molecular biology, virology or biochemistry
Hands-on experience in laboratory work and is familiar with basic cell and molecular biology techniques
Prior experience in virus handling and analysis, cell culture, mass spectrometry or bioinformatics will be an advantage
Proficiency in written and spoken English
Excellent interpersonal skills, initiative and ability to work independently and in a high-performance team

Number of positions available: 1

Contact: Gracjan Michlewski

Identifying unique adaptive responses of red pulp macrophages to iron deficiency (NCN/SONATA)

Supervisor: Wojciech Pokrzywa, PhD DSc, auxiliary supervisor, PI: Katarzyna Mleczko-Sanecka, PhD

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of Iron Homeostasis

Project description:

Iron deficiency is a global health burden with profound socio-medical impacts, but little is known about how functions of specialized cells are affected by low systemic iron levels. Red pulp macrophages (RPMs) residing in the spleen are responsible for removing aged erythrocytes from the bloodstream. Following erythrocyte lysis, RPMs release iron to the circulation to replenish the pool of serum iron necessary for sustaining erythropoiesis. RPMs are thus critical for maintaining blood and iron homeostasis in the body. Interestingly, it was largely unknown if and how key cellular functions of RPMs are affected by low body iron status. Using a mouse model of nutritional iron deficiency, we uncovered that iron deficiency triggers specific but still elusive signaling mechanisms that modulate RPMs’ phagocytic and metabolic functions. We expect that these newly identified responses likely contribute to the adaptation of the whole organism to limited iron supplies. Within our project, we apply both in vivo and in vitro approaches to decipher the molecular mechanism responsible for the adaptive functional ‘rewiring’ of RPMs in iron deficiency and determine their physiological role for the whole organism. The new knowledge generated by this research is expected to significantly advance our understanding of an organism’s adaptation to iron deficiency.

Aim:

We aim to comprehensively characterize the functional adaptation of RPMs to iron deficiency and identify its molecular triggers. We also plan to determine how this ‘rewiring’ affects RPMs’ inflammatory status. We will explore how the abrogation of this ‘adaptation program’ affects the organism, including iron and blood homeostasis. To this end, we will create and characterize new conditional knock-out mice characterized by specific suppression of the RPMs’ adaptation to low iron conditions.

Requirements:

Master's degree in biology, biochemistry or related field
Eligibility for PhD studies in Poland
Interests in molecular aspects of physiology, motivation for experimental work, passion for science, hands-on experience in laboratory work
Experience in mouse/rat-based studies or willingness to work with animals
Written and spoken fluency in English
Willingness to learn and take new challenges, ability to work independently, analytical thinking
Good interpersonal skills and a collaborative attitude.
Research achievements (eg, publications or manuscripts in preparation) and experience abroad will be of advantage

Number of positions available: 2

Contact: This email address is being protected from spambots. You need JavaScript enabled to view it.

2.

August 4, 2021 - Start of the recruitment
August 18, 2021 - Deadline for documents submission

Research projects for admissions 2021/2022-5:

Genomics and Epigenomics of acute myelogenous leukemia (AML)

Supervisor: Professor Matthias Bochtler

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of Structural Biology

Project description

Epigenomic changes play a prominent role in acute myelogenous leukemia (AML). Mutations in the methyltransferase DNMT3A and the dioxygenase TET2 are among the most frequent alterations in this type of malignancy. It has been proposed that defects in epigenomics entail DNA repair defects, which in turn lead to karyotype degradation. This contrasts with information from the Cancer Genome Atlas and the COSMIC database, which both identify AML as a typical M-type malignancy, i.e. a malignancy that is driven by mutations, rather than by copy-number variation. However, clinical observation suggests that a considerable fraction of AML patients have karyotype aberrations. In some cases, the chromosomal changes can be drastic and resemble the chromothripsis seen in other malignancies. Highly karyotype aberrant AMLs are poorly characterized. It is not clear whether the spectrum of exome mutations is similar in these leukemias and in M-type leukemias and what drives the karyotype degradation. It is also unclear whether changes in the epigenomic machinery and their possible effects on DNA signaling play a role in this process. We hope to clarify these issues, primarily by sequencing approaches, in collaboration with clinicians in Heidelberg and Dresden (Germany).

Aim

The aim of the project is to obtain deep sequencing data for AML patients (bulk and single cell). We plan to compare the spectrum of mutations and copy number variation in highly aberrant and normal karyotype AMLs. We plan to test if the spectrum of mutations in the coding genome and tumor clonal histories are similar in the two types of AML. We want to learn if mutations in the epigenomic enzymes cause DNA repair phenotypes, which could ultimately lead to karyotype degradation.

Requirements:

Master's degree in biology, biochemistry or a related field
Eligibility for PhD studies in Poland
Theoretical knowledge of genetics and epigenetics.
Practical experience with cellular fractionation by FACS.
Experience with preparation of libraries for DNA sequencing using Nanopore and Illumina technologies.
Experience with genotyping (high resolution melting analysis).
Experience with or at least interest in bioinformatic analysis of deep sequencing data.
Written and spoken fluency in English
Willingness to learn and take new challenges, ability to work independently, analytical thinking
Good interpersonal skills and a collaborative attitude

Number of positions available: 1

Contact: This email address is being protected from spambots. You need JavaScript enabled to view it., This email address is being protected from spambots. You need JavaScript enabled to view it.

Poly(A) tails - central hubs of mRNA stability control

Supervisor: Professor Andrzej Dziembowski

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of RNA Biology

Project description:

Gene expression is regulated at multiple levels. Our lab is interested in the regulation of mRNA stability, especially through the modifications of poly(A) tails.
Recently, we have shown that the addition of untemplated uridines to the 3’ end of LINE1 retrotransposons precludes their propagation (Warkocki et al. Cell 2018). Moreover, we have identified a family of poly(A) polymerases TENT5, which reside in the cytoplasm and enhance the expression of mRNAs encoding secreted proteins (Moczek et al. Nature com. 2017; Bilska et al. Nature com. 2020; Gewartowska et al. Cell reports 2021). Those enzymes are differentially expressed in tissues and organs, affecting several aspects of animal physiology. TENT5C is an onco-suppressor in multiple myeloma and control immunoglobulin expression in B cells. TENT5A is essential for collagen secretion, and its mutations lead to congenital bone disease.

To study the dynamics of poly(A) tails genome-wide, we have implemented a Direct RNA sequencing Nanopore methodology. It is now widely used in our projects, and we also collaborate with other laboratories interested in post-transcriptional gene expression regulation (for instance, Scheer et al. Nature com. 2021). Moreover, we use Direct RNA sequencing to look globally at the regulation of poly(A) tails (Tudek et al. Nature com., under revision).
In the future, we will continue to study the role and mechanism of action of TENT5 poly(A) polymerases, analyze global control of poly(A) tail lengths and develop bioinformatics tools for Direct

RNA sequencing. Finally, we are planning to translate our knowledge out poly(A) tails for the design of mRNAs, which are more stable and better translated, which will be very valuable for mRNA-based therapeutics such as mRNA vaccines.

Aim:

The exact nature of the project will depend on the skills, predispositions, and interests of the selected PhD student. It may focus on:

functional analysis of the TENT5A poly(A) polymerases in transgenic mouse models generated in-house using CRISPR/Cas9 methodology.
mastering of the DRS methodology in either the experimental part or bioinformatic analysis.
analysis of principles of mRNA stability control and design of more efficient mRNA based therapeutics

Requirements:

Master's degree in biology, biochemistry or related field
Eligibility for PhD studies in Poland
Highly talented individuals who are passionate about research and are full of scientific curiosity
Experience in either: molecular biology/transcriptomics, animal models, bioinformatic analysis of transcriptomic data, will be a clear benefit
Written and spoken fluency in English
Willingness to learn and take new challenges, ability to work independently, analytical thinking
Good interpersonal skills and a collaborative attitude

Number of positions available: 2

Contact: This email address is being protected from spambots. You need JavaScript enabled to view it.

Experimental analysis of molecular determinants involved in epilepsy (NCN/OPUS)

Supervisor: Professor Jacek Kuźnicki, auxiliary supervisor/PI: Vladimir Korzh, PhD

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of Neurodegeneration

Project description:

The effects of KCNB1 mutations that cause epileptic encephalopathy were analyzed mechanistically mainly using electrophysiology in heterologous cells in vitro. The developmental analysis was limited by the availability of single mutants in mice and zebrafish and did not explore the whole range of effects caused by KCNB1 mutations (Shen et al., 2016). The KCNB1 loss of function (LOF) or gain of function (GOF) cause specific morphological changes in the brain ventricles (Shen et al., 2016) and inner ear (Jedrychowska et al., 2020) in developing zebrafish embryos and larvae. The zebrafish transgenics express fluorescent markers in specific manner. The high-resolution microscopy of transgenic embryos and larvae in vivo provides information about developmental mechanisms as well as changes in activity of specific signaling pathways. These tools satisfy conditions necessary to study the developmental effect of different KCNB1 mutations in real time. This rationale was confirmed in preliminary experiments when analyzing the effect of overexpression of human mutated KCNB1 mRNA. Kcnb1 GOF causes cell delamination in brain ventricles and their expansion (hydrocephalus), and enlargement of the inner ear and otoliths. These features recapitulate the phenotype of the kcnb1 GOF zebrafish mutant and constitute the rationale for the "brain and ear" in vivo test to be used when defining an effect of human mutations.

Aim:

Using the site-specific CRISPR-Cas9 mutagenesis in zebrafish, the representative Kcnb1 mutations that mimic the known human mutations of KCNB1 will be generated and analyzed by a combination of bioimaging, single-cell transcriptomics, electrophysiology and behavioral analysis. This will provide rationale for subfunctionalization of human KCNB1 mutations.

Requirements:

Master's degree in biology, biochemistry or related field
Eligibility for PhD studies in Poland
Prior experience in molecular developmental biology and zebrafish studies is a bonus during selection of candidates, but necessary training will be provided
Written and spoken fluency in English
Willingness to learn and take new challenges, ability to work independently, analytical thinking
Good interpersonal skills and a collaborative attitude

Number of positions available: 2

Contact: Vladimir Korzh

Identifying unique adaptive responses of red pulp macrophages to iron deficiency (NCN/SONATA)

Supervisor: Wojciech Pokrzywa, PhD DSc, auxiliary supervisor, PI: Katarzyna Mleczko-Sanecka, PhD

Institute: International Institute of Molecular and Cell Biology in Warsaw

Laboratory: Laboratory of Iron Homeostasis

Project description:

Aim:

We aim to comprehensively characterize the functional adaptation of RPMs to iron deficiency and identify its molecular triggers. We will explore how the abrogation of this ‘adaptation program’ affects the organism, including iron and blood homeostasis. To this end, we will create and characterize new conditional knock-out mice characterized by specific suppression of the RPMs’ adaptation to low iron conditions.

Requirements:

Master's degree in biology, biochemistry or related field
Eligibility for PhD studies in Poland
Interests in molecular aspects of physiology, motivation for experimental work, passion for science, hands-on experience in laboratory work
Experience in mouse/rat-based studies or willingness to work with animals
Written and spoken fluency in English
Willingness to learn and take new challenges, ability to work independently, analytical thinking
Good interpersonal skills and a collaborative attitude.
Research achievements (eg, publications or manuscripts in preparation) and experience abroad will be of advantage

Number of positions available: 1

Contact: Katarzyna Mleczko-Sanecka

3.

Research projects for admissions 2021/2022-6:

9.1 Poly(A) tails - central hubs of mRNA stability control (prof. Andrzej Dziembowski)9.1 Poly(A) tails - central hubs of mRNA stability control (prof. Andrzej Dziembowski) - 2 places available

9.2 Mechanistic and structural studies of th replication of (+)ssRNA viruses (NCN/OPUS) (prof. Marcin Nowotny)

9.3 Ten eleven translocation 2 (TET2) in acute myeloid leukemia (prof. Matthias Bochtler)

9.4 Experimental analysis of molecular determinants involved in epilepsy (NCN/OPUS) (prof. Jacek Kuźnicki / Vladimir Korzh, PhD)

Results of the recruitment to the Warsaw Doctoral School in Natural and Biomedical Sciences for IIMCB research projects

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Drug development projects

Nucleic acid-based therapeutics

Antisense technology is an emerging therapeutic approach for inhibiting gene expression through recognition and RNase H1-dependent degradation of cellular mRNAs. In this technology, short, synthetic, single-stranded oligonucleotides (ASOs) are designed that are complementary to the target mRNA.

RNA processing

Human cap 2’-OH methyltransferase

Mitochondrial exoribonuclease complex mtEXO

CutA terminal ribonucleotide transferase

Reverse transcriptases

XMRV reverse transcriptase

Foamy viral protease-reverse transcriptase

Ty3 reverse transcriptase

Cauliflower mosaic virus reverse transcriptase (CaMV RT)

HIV-1 reverse transriptase

Abi polymerases

DNA transposition

Tn7 Transposase TnsB

DNA repair

Bacterial Nucleotide Excision Repair

Rad2

RecFOR

RuvC

Slx1-Slx4

Slx1 is a nuclease which cleaves various DNA structures during DNA repair and recombination. It associates with Slx4 platform protein which coordinates the action of multiple proteins. Slx1 together with Mus8-Eme1 nuclease constitute one of the major Holliday junction pathways in eukaryotes.

RNases H

Bacterial RNase H2

Human RNase H2

RNase H3

1.

Research projects for admissions 2021/2022-4:

Poly(A) tails - central hubs of mRNA stability control

The role of mTOR-Brg1 interaction in normal and aberrant neuronal activity (NCN/MAESTRO)

Signaling of AXL receptor in cancer cells

RNA-Protein Interactions in Human Health and Disease (NCN/DIOSUCRI)

Identifying unique adaptive responses of red pulp macrophages to iron deficiency (NCN/SONATA)

2.

Research projects for admissions 2021/2022-5:

Genomics and Epigenomics of acute myelogenous leukemia (AML)

Poly(A) tails - central hubs of mRNA stability control

Experimental analysis of molecular determinants involved in epilepsy (NCN/OPUS)

Identifying unique adaptive responses of red pulp macrophages to iron deficiency (NCN/SONATA)

3.

Research projects for admissions 2021/2022-6:

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