ModeRNA: a tool for comparative modeling of RNA 3D structure

RNA is a large group of functionally important biomacromolecules. In striking analogy to proteins, the function of RNA depends on its structure and dynamics, which in turn is encoded in the linear sequence. However, while there are numerous methods for computational prediction of protein three-dimensional (3D) structure from sequence, with comparative modeling being the most reliable approach, there are very few such methods for RNA. Here, we present ModeRNA, a software tool for comparative modeling of RNA 3D structures. As an input, ModeRNA requires a 3D structure of a template RNA molecule, and a sequence alignment between the target to be modeled and the template. It must be emphasized that a good alignment is required for successful modeling, and for large and complex RNA molecules the development of a good alignment usually requires manual adjustments of the input data based on previous expertise of the respective RNA family. ModeRNA can model post-transcriptional modifications, a functionally important feature analogous to post-translational modifications in proteins. ModeRNA can also model DNA structures or use them as templates. It is equipped with many functions for merging fragments of different nucleic acid structures into a single model and analyzing their geometry. Windows and UNIX implementations of ModeRNA with comprehensive documentation and a tutorial are freely available

RNA and protein 3D structure modeling: similarities and differences

In analogy to proteins, the function of RNA depends on its structure and dynamics, which are encoded in the linear sequence. While there are numerous methods for computational prediction of protein 3D structure from sequence, there have been very few such methods for RNA. This review discusses template-based and template-free approaches for macromolecular structure prediction, with special emphasis on comparison between the already tried-and-tested methods for protein structure modeling and the very recently developed “protein-like” modeling methods for RNA. We highlight analogies between many successful methods for modeling of these two types of biological macromolecules and argue that RNA 3D structure can be modeled using “protein-like” methodology. We also highlight the areas where the differences between RNA and proteins require the development of RNA-specific solutions

Formation of the Immunoglobulin Repertoire in Precursor-B-cell Development

Precursor-B cells develop in bone marrow (BM) from hematopoietic stem cells (HSC) with the ultimate goal to generate mature B-cells with unique immunoglobulins (Ig), whereby all B cells together provide an enormous Ig repertoire diversity. Formation of the Ig occurs through somatic V(D)J recombination of the Ig heavy chain (IGH) and the Ig light chain (IGK or IGL) loci that contain multiple variable (V), diverse (D) and joining (J) coding elements. The Ig loci recombine in a ordered manner. The IGH locus rearranges before the Ig light chain loci and functional Ig rearrangements are generally restricted to one allele. Thus, the ordered accessibility of Ig loci for rearrangements is tightly controlled. Studies described in this thesis aimed to better understand the mechanisms underlying the stage-specific regulation of V(D) J recombination and the generation of a diverse Ig repertoire during precursor-B-cell development. The stepwise V(D)J recombination process is controlled by transcriptional and epigenetic mechanisms which must cooperate together to provide the developmental increase in Ig loci accessibility for rearrangement. The formation of the broad Ig repertoire is dependent on the B-cell developmental niche. The BM niche undergoes physiological changes during life and therefore provides different signals to developing B cells, resulting in distinct Ig gene repertoires with distinct reactivity. Future studies should reveal which environmental factors modify the Ig loci accessibility for the V(D)J recombinase and thereby control the Ig repertoire and reactivity. Such knowledge might be applicable for shaping the Ig repertoire in immune deficient patients

PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.España Ministerio de Ciencia e Innovacion SAF2016-80626-REspaña, Fundación Canaria Instituto de Investigación Sanitaria de Canarias (FIISC) [PIFUN16/18

Nuclear positioning rather than contraction controls ordered rearrangements of immunoglobulin loci

Progenitor-B cells recombine their immunoglobulin (Ig) loci to create unique antigen receptors. Despite a common recombination machinery, the Ig heavy and Ig light chain loci rearrange in a stepwise manner. We studied pre-pro-B cells and Rag-/- progenitor-B cells to determine whether Ig locus contraction or nuclear positioning is decisive for stepwise rearrangements. We found that both Ig loci were contracted in pro-B and pre-B cells. Igh relocated from the nuclear lamina to central domains only at the pro-B cell stage, whereas, Igê remained sequestered at the lamina, and only at the pre-B cell stage located to central nuclear domains. Finally, in vitro induced re-positioning of Ig alleles away from the nuclear periphery increased germline transcription of Ig loci in pre-pro-B cells. Thus, Ig locus contraction juxtaposes genomically distant elements to mediate efficient recombination, however, sequential positioning of Ig loci away from the nuclear periphery determines stage-specific accessibility of Ig loci

Pre-B Cell Receptor Signaling Induces Immunoglobulin κ Locus Accessibility by Functional Redistribution of Enhancer-Mediated Chromatin Interactions

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vκ transcription. To investigate whether pre-BCR signaling modulates Vκ accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb Vκ region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3′κ enhancer with Vκ genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vκ genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vκ genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the Vκ region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vκ region, whereby the two enhancers play distinct roles

Decreased IL7Rα and TdT expression underlie the skewed immunoglobulin repertoire of human B-cell precursors from fetal origin

Newborns are unable to mount antibody responses towards certain antigens. This has been related to the restricted repertoire of immunoglobulin (Ig) genes of their B cells. The mechanisms underlying the restricted fetal Ig gene repertoire are currently unresolved. We here addressed this with detailed molecular and cellular analysis of human precursor-B cells from fetal liver, fetal bone marrow (BM), and pediatric BM. In the absence of selection processes, fetal B-cell progenitors more frequently used proximal V, D and J genes in complete IGH gene rearrangements, despite normal Ig locus contraction. Fewer N-nucleotides were added in IGH gene rearrangements in the context of low TdT and XRCC4 expression. Moreover, fetal progenitor-B cells expressed lower levels of IL7Rα than their pediatric counterparts. Analysis of progenitor-B cells from IL7Rα-deficient patients revealed that TdT expression and N-nucleotides additions in Dh-Jh junctions were dependent on functional IL7Rα. Thus, IL7Rα affects TdT expression, and decreased expression of this receptor underlies at least in part the skewed Ig repertoire formation in fetal B-cell precursors. These new insights provide a better understanding of the formation of adaptive immunity in the developing fetus

Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome

ModeRNA: a tool for comparative modeling of RNA 3D structure

Wydział Biologii: Instytut Biologii Molekularnej i BiotechnologiiRNA pełni ważne role we wszystkich organizmach żywych. Pełne zrozumienie funkcji i mechanizmów działania cząsteczek RNA jest możliwe jedynie w kontekście ich struktury przestrzennej. Metody doświadczalne pozwalające określić położenie poszczególnych atomów w przestrzeni są jednak drogie i czasochłonne. Powoduje to olbrzymią dysproporcję pomiędzy liczbą znanych sekwencji i znanych struktur. Bazując na podejściu modelowania homologicznego w tej pracy powstał program komputerowy ModeRNA do przewidywania struktury przestrzennej RNA. Buduje on model na podstawie szablonu strukturalnego i przyrównania pomiędzy sekwencją celu i szablonu. Program pozwala ponadto na analizę oraz redagowanie struktury RNA. Jego unikatową cechą jest możliwość modelowania 115 modyfikowanych nukleotydów. Oprócz wersji instalacyjnej programu powstał serwer, który udostępnia operacje do modelowania oraz ułatwia odnalezienie szablonu i przygotowanie przyrównania. Program ModeRNA wspiera wszystkie kroki niezbędne do zbudowania modelu homologicznego cząsteczki RNA. Działanie programu zostało sprawdzone na zbiorze 99 tRNA, dla których zostały zbudowane i ocenione 9802 modele. Średnie RMSD wyniosło 5,6 Å i jest zbliżona do zmienności występującej w zbiorze struktur natywnych. Program ModeRNA posiada obszerną dokumentację, która wraz z kodem dostępna jest pod licencją wolnego i otwartego oprogramowania (GNU GPL).RNA carries out important roles in all living organisms. Complete understanding of its function and mechanisms of action is possible only in the context of 3D structure. Experimental methods that give knowledge about the spatial position of all atoms in a structure are expensive and time-consuming. This has led to a great disproportion between the number of known sequences and the number of known structures. In this thesis, the computer program ModeRNA for RNA structure prediction was developed based on the homology modeling approach. It builds models using a template structure and a pairwise alignment of target and template sequences. The software has also functions for RNA analysis and editing. A unique feature of ModeRNA is the ability to model 115 posttranscriptional modifications. In addition to the standalone software an internet server was implemented. It provides modeling operations and facilitates template search as well as alignment preparation. The ModeRNA software supports all steps required for building homology models. The software was tested on a set of 99 tRNA structures for which 9802 models were built and evaluated. The mean RMSD value for all models is 5.6 Å which is comparable to the variability occurring in native structures. The ModeRNA software has extensive documentation which is available as well as the source code under the GNU GPL open source license