PfGBP2 is a novel G-quadruplex binding protein in Plasmodium falciparum.

Guanine-quadruplexes (G4s) are non-canonical DNA structures that can regulate key biological processes such as transcription, replication and telomere maintenance in several organisms including eukaryotes, prokaryotes and viruses. Recent reports have identified the presence of G4s within the AT-rich genome of Plasmodium falciparum, the protozoan parasite causing malaria. In Plasmodium, potential G4-forming sequences (G4FS) are enriched in the telomeric and sub-telomeric regions of the genome where they are associated with telomere maintenance and recombination events within virulence genes. However, there is a little understanding about the biological role of G4s and G4-binding proteins. Here, we provide the first snapshot of G4-interactome in P. falciparum using DNA pull-down assay followed by LC-MS/MS. Interestingly, we identified ~24 potential G4-binding proteins (G4-BP) that bind to a stable G4FS (AP2_G4). Furthermore, we characterised the role of G-strand binding protein 2 (PfGBP2), a putative telomere-binding protein in P. falciparum. We validated the interaction of PfGBP2 with G4 in vitro as well as in vivo. PfGBP2 is expressed throughout the intra-erythrocytic developmental cycle and is essential for the parasites in the presence of G4-stabilising ligand, pyridostatin. Gene knockout studies showed the role of PfGBP2 in the expression of var genes. Taken together, this study suggests that PfGBP2 is a bona fide G4-binding protein, which is likely to be involved in the regulation of G4-related functions in these malarial parasites. In addition, this study sheds light on this understudied G4 biology in P. falciparum.

Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage.

BACKGROUND: The main function of telomerase is at the telomeres but under adverse conditions telomerase can bind to internal regions causing deleterious effects as observed in cancer cells. RESULTS: By mapping the global occupancy of the catalytic subunit of telomerase (Est2) in the budding yeast Saccharomyces cerevisiae, we reveal that it binds to multiple guanine-rich genomic loci, which we termed "non-telomeric binding sites" (NTBS). We characterize Est2 binding to NTBS. Contrary to telomeres, Est2 binds to NTBS in G1 and G2 phase independently of Est1 and Est3. The absence of Est1 and Est3 renders telomerase inactive at NTBS. However, upon global DNA damage, Est1 and Est3 join Est2 at NTBS and telomere addition can be observed indicating that Est2 occupancy marks NTBS regions as particular risks for genome stability. CONCLUSIONS: Our results provide a novel model of telomerase regulation in the cell cycle using internal regions as "parking spots" of Est2 but marking them as hotspots for telomere addition.

Clinical and immunological effects of mRNA vaccines in malignant diseases.

In vitro-transcribed messenger RNA-based therapeutics represent a relatively novel and highly efficient class of drugs. Several recently published studies emphasize the potential efficacy of mRNA vaccines in treating different types of malignant and infectious diseases where conventional vaccine strategies and platforms fail to elicit protective immune responses. mRNA vaccines have lately raised high interest as potent vaccines against SARS-CoV2. Direct application of mRNA or its electroporation into dendritic cells was shown to induce polyclonal CD4+ and CD8+ mediated antigen-specific T cell responses as well as the production of protective antibodies with the ability to eliminate transformed or infected cells. More importantly, the vaccine composition may include two or more mRNAs coding for different proteins or long peptides. This enables the induction of polyclonal immune responses against a broad variety of epitopes within the encoded antigens that are presented on various MHC complexes, thus avoiding the restriction to a certain HLA molecule or possible immune escape due to antigen-loss. The development and design of mRNA therapies was recently boosted by several critical innovations including the development of technologies for the production and delivery of high quality and stable mRNA. Several technical obstacles such as stability, delivery and immunogenicity were addressed in the past and gradually solved in the recent years.This review will summarize the most recent technological developments and application of mRNA vaccines in clinical trials and discusses the results, challenges and future directions with a special focus on the induced innate and adaptive immune responses.

G-quadruplexes: a promising target for cancer therapy.

DNA and RNA can fold into a variety of alternative conformations. In recent years, a particular nucleic acid structure was discussed to play a role in malignant transformation and cancer development. This structure is called a G-quadruplex (G4). G4 structure formation can drive genome instability by creating mutations, deletions and stimulating recombination events. The importance of G4 structures in the characterization of malignant cells was currently demonstrated in breast cancer samples. In this analysis a correlation between G4 structure formation and an increased intratumor heterogeneity was identified. This suggests that G4 structures might allow breast cancer stratification and supports the identification of new personalized treatment options. Because of the stability of G4 structures and their presence within most human oncogenic promoters and at telomeres, G4 structures are currently tested as a therapeutic target to downregulate transcription or to block telomere elongation in cancer cells. To date, different chemical molecules (G4 ligands) have been developed that aim to target G4 structures. In this review we discuss and compare G4 function and relevance for therapeutic approaches and their impact on cancer development for three cancer entities, which differ significantly in their amount and type of mutations: pancreatic cancer, leukemia and malignant melanoma. G4 structures might present a promising new strategy to individually target tumor cells and could support personalized treatment approaches in the future.

The Relevance of G-Quadruplexes for DNA Repair.

DNA molecules can adopt a variety of alternative structures. Among these structures are G-quadruplex DNA structures (G4s), which support cellular function by affecting transcription, translation, and telomere maintenance. These structures can also induce genome instability by stalling replication, increasing DNA damage, and recombination events. G-quadruplex-driven genome instability is connected to tumorigenesis and other genetic disorders. In recent years, the connection between genome stability, DNA repair and G4 formation was further underlined by the identification of multiple DNA repair proteins and ligands which bind and stabilize said G4 structures to block specific DNA repair pathways. The relevance of G4s for different DNA repair pathways is complex and depends on the repair pathway itself. G4 structures can induce DNA damage and block efficient DNA repair, but they can also support the activity and function of certain repair pathways. In this review, we highlight the roles and consequences of G4 DNA structures for DNA repair initiation, processing, and the efficiency of various DNA repair pathways.

Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes.

We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6'U7' and 6'A7' on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step. We observe a stable alignment for the looped-out 6'N7' bulge base, which stacks on the unpaired first base of the guide strand, with the looped-out alignment facilitated by weakened Watson-Crick and reversed non-canonical flanking pairs. These structural studies are complemented by cleavage assays that independently monitor the impact of bulges on TtAgo-mediated cleavage reaction.

A Novel G-Quadruplex Binding Protein in Yeast-Slx9.

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.

Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus.

Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the 5' end. The exact structure of RNA activating RIG-I remains controversial. Here, we established a chemical approach for 5' triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5' triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double-strand conformation with base pairing of the nucleoside carrying the 5' triphosphate was required. RIG-I activation was impaired by a 3' overhang at the 5' triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative-strand RNA viruses that lack long double-stranded RNA but do contain blunt short double-stranded 5' triphosphate RNA in the panhandle region of their single-stranded genome.

LARP4B is an AU-rich sequence associated factor that promotes mRNA accumulation and translation.

mRNAs are key molecules in gene expression and subject to diverse regulatory events. Regulation is accomplished by distinct sets of trans-acting factors that interact with mRNAs and form defined mRNA-protein complexes (mRNPs). The resulting "mRNP code" determines the fate of any given mRNA and thus controlling gene expression at the post-transcriptional level. The La-related protein 4B (LARP4B) belongs to an evolutionarily conserved family of RNA-binding proteins characterized by the presence of a La-module implicated in direct RNA binding. Biochemical experiments have shown previously direct interactions of LARP4B with factors of the translation machinery. This finding along with the observation of an association with actively translating ribosomes suggested that LARP4B is a factor contributing to the mRNP code. To gain insight into the function of LARP4B in vivo we tested its mRNA association at the transcriptome level and its impact on the proteome. PAR-CLIP analyses allowed us to identify the in vivo RNA targets of LARP4B. We show that LARP4B binds to a distinct set of cellular mRNAs by contacting their 3' UTRs. Biocomputational analysis combined with in vitro binding assays identified the LARP4B-binding motif on mRNA targets. The reduction of cellular LARP4B levels leads to a marked destabilization of its mRNA targets and consequently their reduced translation. Our data identify LARP4B as a component of the mRNP code that influences the expression of its mRNA targets by affecting their stability.

Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes.

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg(2+)-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

A genome-wide view of the expression and processing patterns of Thermus thermophilus HB8 CRISPR RNAs.

The CRISPR-Cas system represents an RNA-based adaptive immune response system in prokaryotes and archaea. CRISPRs (clustered regularly interspaced short palindromic repeats) consist of arrays of short repeat-sequences interspaced by nonrepetitive short spacers, some of which show sequence similarity to foreign phage genetic elements. Their cistronic transcripts are processed to produce the mature CRISPR RNAs (crRNAs), the elements that confer immunity by base-pairing with exogenous nucleic acids. We characterized the expression and processing patterns of Thermus thermophilus HB8 CRISPRs by using differential deep-sequencing, which differentiates between 5' monophosphate and 5' non-monophosphate-containing RNAs and/or between 3' hydroxyl and 3' non-hydroxyl-containing RNAs. The genome of T. thermophilus HB8 encodes 11 CRISPRs, classified into three distinct repeat-sequence types, all of which were constitutively expressed without deliberately infecting the bacteria with phage. Analysis of the differential deep sequencing data suggested that crRNAs are generated by endonucleolytic cleavage, leaving fragments with 5' hydroxyl and 3' phosphate or 2',3'-cyclic phosphate termini. The 5' ends of all crRNAs are generated by site-specific cleavage 8 nucleotides upstream of the spacer first position; however, the 3' ends are generated by two alternative, repeat-sequence-type-dependent mechanisms. These observations are consistent with the operation of multiple crRNA processing systems within a bacterial strain.

Structure of the guide-strand-containing argonaute silencing complex.

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.

Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex.

Here we report on a 3.0 A crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10-11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson-Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of argonaute widens on formation of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2'-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.

UV-induced G4 DNA structures recruit ZRF1 which prevents UV-induced senescence.

Senescence has two roles in oncology: it is known as a potent tumor-suppressive mechanism, which also supports tissue regeneration and repair, it is also known to contribute to reduced patient resilience, which might lead to cancer recurrence and resistance after therapy. Senescence can be activated in a DNA damage-dependent and -independent manner. It is not clear which type of genomic lesions induces senescence, but it is known that UV irradiation can activate cellular senescence in photoaged skin. Proteins that support the repair of DNA damage are linked to senescence but how they contribute to senescence after UV irradiation is still unknown. Here, we unraveled a mechanism showing that upon UV irradiation multiple G-quadruplex (G4) DNA structures accumulate in cell nuclei, which leads to the recruitment of ZRF1 to these G4 sites. ZRF1 binding to G4s ensures genome stability. The absence of ZRF1 triggers an accumulation of G4 structures, improper UV lesion repair, and entry into senescence. On the molecular level loss of ZRF1 as well as high G4 levels lead to the upregulation of DDB2, a protein associated with the UV-damage repair pathway, which drives cells into senescence.

Prioritization of non-coding elements involved in non-syndromic cleft lip with/without cleft palate through genome-wide analysis of de novo mutations.

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a highly heritable facial disorder. To date, systematic investigations of the contribution of rare variants in non-coding regions to nsCL/P etiology are sparse. Here, we re-analyzed available whole-genome sequence (WGS) data from 211 European case-parent trios with nsCL/P and identified 13,522 de novo mutations (DNMs) in nsCL/P cases, 13,055 of which mapped to non-coding regions. We integrated these data with DNMs from a reference cohort, with results of previous genome-wide association studies (GWASs), and functional and epigenetic datasets of relevance to embryonic facial development. A significant enrichment of nsCL/P DNMs was observed at two GWAS risk loci (4q28.1 (p = 8 × 10-4) and 2p21 (p = 0.02)), suggesting a convergence of both common and rare variants at these loci. We also mapped the DNMs to 810 position weight matrices indicative of transcription factor (TF) binding, and quantified the effect of the allelic changes in silico. This revealed a nominally significant overrepresentation of DNMs (p = 0.037), and a stronger effect on binding strength, for DNMs located in the sequence of the core binding region of the TF Musculin (MSC). Notably, MSC is involved in facial muscle development, together with a set of nsCL/P genes located at GWAS loci. Supported by additional results from single-cell transcriptomic data and molecular binding assays, this suggests that variation in MSC binding sites contributes to nsCL/P etiology. Our study describes a set of approaches that can be applied to increase the added value of WGS data.

Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

Detecting G4 unwinding.

DNA can, in addition to the B-DNA conformation, fold into a variety of additional conformations. Among them are G-quadruplex structures that have gained a lot of attention in recent years. G-quadruplex structures (G4s) are highly stable nucleic acid structures that can fold within DNA and RNA molecules. They form in guanine-rich regions that harbor a specific G4 motif. The three-dimensional structure forms via Hoogsteen hydrogen bonding, where the guanines form hydrogen bonds to each other in order to generate G quartets, which stack in order to become G4 structures. The existence and relevance of G4s was controversial as discussed in the past. However, accumulating data was published that supported the model that G4s form in living cells and importantly support biological processes. G4 formation and unfolding is tightly regulated in vivo. If G4s persist in the cell, they can lead to cellular defects such as genome instability. To avoid G4 accumulation in cells, and by this prevent cellular defect, cells has evolved a variety of proteins, mostly helicases, that efficiently unfold G4 DNA and RNA structures. Here, we describe a detailed protocol to monitor G4 structure unfolding by helicases.

Impaired neurogenesis alters brain biomechanics in a neuroprogenitor-based genetic subtype of congenital hydrocephalus.

Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH.