RNA clamping by Vasa assembles a piRNA amplifier complex on transposon transcripts.

Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5' sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons.

Characterization of the REC114-MEI4-IHO1 complex regulating meiotic DNA double-strand break formation.

Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs), essential for fertility and genetic diversity. In the mouse, DSBs are formed by the catalytic TOPOVIL complex consisting of SPO11 and TOPOVIBL. To preserve genome integrity, the activity of the TOPOVIL complex is finely controlled by several meiotic factors including REC114, MEI4, and IHO1, but the underlying mechanism is poorly understood. Here, we report that mouse REC114 forms homodimers, that it associates with MEI4 as a 2:1 heterotrimer that further dimerizes, and that IHO1 forms coiled-coil-based tetramers. Using AlphaFold2 modeling combined with biochemical characterization, we uncovered the molecular details of these assemblies. Finally, we show that IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and another meiotic factor ANKRD31. These results provide strong evidence for the existence of a ternary IHO1-REC114-MEI4 complex and suggest that REC114 could act as a potential regulatory platform mediating mutually exclusive interactions with several partners.

PRDM9 Methyltransferase Activity Is Essential for Meiotic DNA Double-Strand Break Formation at Its Binding Sites.

The programmed formation of hundreds of DNA double-strand breaks (DSBs) is essential for proper meiosis and fertility. In mice and humans, the location of these breaks is determined by the meiosis-specific protein PRDM9, through the DNA-binding specificity of its zinc-finger domain. PRDM9 also has methyltransferase activity. Here, we show that this activity is required for H3K4me3 and H3K36me3 deposition and for DSB formation at PRDM9-binding sites. By analyzing mice that express two PRDM9 variants with distinct DNA-binding specificities, we show that each variant generates its own set of H3K4me3 marks independently from the other variant. Altogether, we reveal several basic principles of PRDM9-dependent DSB site determination, in which an excess of sites are designated through PRDM9 binding and subsequent histone methylation, from which a subset is selected for DSB formation.

The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization.

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

Mutations in PRDM15 Are a Novel Cause of Galloway-Mowat Syndrome.

BACKGROUND: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease. METHODS: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified. RESULTS: Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes. CONCLUSIONS: Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.

Structural analysis of the KANSL1/WDR5/KANSL2 complex reveals that WDR5 is required for efficient assembly and chromatin targeting of the NSL complex.

The subunits of the nonspecific lethal (NSL) complex, which include the histone acetyltransferase MOF (males absent on the first), play important roles in various cellular functions, including transcription regulation and stem cell identity maintenance and reprogramming, and are frequently misregulated in disease. Here, we provide the first biochemical and structural insights into the molecular architecture of this large multiprotein assembly. We identified several direct interactions within the complex and show that KANSL1 acts as a scaffold protein interacting with four other subunits, including WDR5, which in turn binds KANSL2. Structural analysis of the KANSL1/WDR5/KANSL2 subcomplex reveals how WDR5 is recruited into the NSL complex via conserved linear motifs of KANSL1 and KANSL2. Using structure-based KANSL1 mutants in transgenic flies, we show that the KANSL1-WDR5 interaction is required for proper assembly, efficient recruitment of the NSL complex to target promoters, and fly viability. Our data clearly show that the interactions of WDR5 with the MOF-containing NSL complex and MLL/COMPASS histone methyltransferase complexes are mutually exclusive. We propose that rather than being a shared subunit, WDR5 plays an important role in assembling distinct histone-modifying complexes with different epigenetic regulatory roles.

Selective termination of lncRNA transcription promotes heterochromatin silencing and cell differentiation.

Long non-coding RNAs (lncRNAs) regulating gene expression at the chromatin level are widespread among eukaryotes. However, their functions and the mechanisms by which they act are not fully understood. Here, we identify new fission yeast regulatory lncRNAs that are targeted, at their site of transcription, by the YTH domain of the RNA-binding protein Mmi1 and degraded by the nuclear exosome. We uncover that one of them, nam1, regulates entry into sexual differentiation. Importantly, we demonstrate that Mmi1 binding to this lncRNA not only triggers its degradation but also mediates its transcription termination, thus preventing lncRNA transcription from invading and repressing the downstream gene encoding a mitogen-activated protein kinase kinase kinase (MAPKKK) essential to sexual differentiation. In addition, we show that Mmi1-mediated termination of lncRNA transcription also takes place at pericentromeric regions where it contributes to heterochromatin gene silencing together with RNA interference (RNAi). These findings reveal an important role for selective termination of lncRNA transcription in both euchromatic and heterochromatic lncRNA-based gene silencing processes.

Msl1-mediated dimerization of the dosage compensation complex is essential for male X-chromosome regulation in Drosophila.

The Male-Specific Lethal (MSL) complex regulates dosage compensation of the male X chromosome in Drosophila. Here, we report the crystal structure of its MSL1/MSL2 core, where two MSL2 subunits bind to a dimer formed by two molecules of MSL1. Analysis of structure-based mutants revealed that MSL2 can only interact with the MSL1 dimer, but MSL1 dimerization is MSL2 independent. We show that Msl1 is a substrate for Msl2 E3 ubiquitin ligase activity. ChIP experiments revealed that Msl1 dimerization is essential for targeting and spreading of the MSL complex on X-linked genes; however, Msl1 binding to promoters of male and female cells is independent of the dimer status and other MSL proteins. Finally, we show that loss of Msl1 dimerization leads to male-specific lethality. We propose that Msl1-mediated dimerization of the entire MSL complex is required for Msl2 binding, X chromosome recognition, and spreading along the X chromosome.

The nonspecific lethal complex is a transcriptional regulator in Drosophila.

Here, we report the biochemical characterization of the nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) that associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, we conclude that the NSL complex acts as a major transcriptional regulator in Drosophila.

Pushing the limits of sulfur SAD phasing: de novo structure solution of the N-terminal domain of the ectodomain of HCV E1.

Single-wavelength anomalous dispersion of S atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy-atom incorporation or selenomethionine derivatization. Nevertheless, this technique has been limited by the paucity of the signal at the usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here, the data collection and structure solution of the N-terminal domain of the ectodomain of HCV E1 from crystals that diffracted very weakly is reported. By combining the data from 32 crystals, it was possible to solve the sulfur substructure and calculate initial maps at 7 Šresolution, and after density modication and phase extension using a higher resolution native data set to 3.5 Šresolution model building was achievable.

The multiple Tudor domain-containing protein TDRD1 is a molecular scaffold for mouse Piwi proteins and piRNA biogenesis factors.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs expressed in the germline of animals. They associate with Argonaute proteins of the Piwi subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some Piwi proteins contains symmetrically dimethylated arginines. This modification is thought to enable recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methylarginine-containing peptides from murine Piwi protein MILI. The results show a preference of TD2 and TD3 for consecutive MILI peptides, whereas TD4 and TD1 have, respectively, lower and very weak affinity for any peptide. The affinity of TD1 for methylarginine peptides can be restored by a single-point mutation back to the consensus aromatic cage sequence. These observations were confirmed by pull-down experiments with endogenous Piwi and Piwi-associated proteins. The crystal structure of TD3 bound to a methylated MILI peptide shows an unexpected orientation of the bound peptide, with additional contacts of nonmethylated residues being made outside of the aromatic cage, consistent with solution NMR titration experiments. Finally, the molecular envelope of the four tandem Tudor domains of TDRD1, derived from small angle scattering data, reveals a flexible, elongated shape for the protein. Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex.

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that in vivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.

Unusual bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2.

Nonsense-mediated decay (NMD) is a eukaryotic quality control mechanism that degrades mRNAs carrying premature stop codons. In mammalian cells, NMD is triggered when UPF2 bound to UPF3 on a downstream exon junction complex interacts with UPF1 bound to a stalled ribosome. We report structural studies on the interaction between the C-terminal region of UPF2 and intact UPF1. Crystal structures, confirmed by EM and SAXS, show that the UPF1 CH-domain is docked onto its helicase domain in a fixed configuration. The C-terminal region of UPF2 is natively unfolded but binds through separated alpha-helical and beta-hairpin elements to the UPF1 CH-domain. The alpha-helical region binds sixfold more weakly than the beta-hairpin, whereas the combined elements bind 80-fold more tightly. Cellular assays show that NMD is severely affected by mutations disrupting the beta-hairpin binding, but not by those only affecting alpha-helix binding. We propose that the bipartite mode of UPF2 binding to UPF1 brings the ribosome and the EJC in close proximity by forming a tight complex after an initial weak encounter with either element.

Characterization of N-demethyllincosamide methyltransferases LmbJ and CcbJ.

Chemical diversity: Two SAM-dependent N-methyltransferases-LmbJ from the biosynthesis of the antibiotic lincomycin and CcbJ from celesticetin biosynthesis-have been characterized and compared. Both tested enzymes form multimers and are able to utilize N-demethyllincomycin, the natural substrate of LmbJ, with comparable efficiency.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks.

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identify their conserved interacting domains by structural analysis. We then analyse the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity is strongly reduced genome-wide in oocytes, and only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity is delayed in autosomes. These results suggest that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity.

Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex.

To eliminate specific or aberrant transcripts, eukaryotes use nuclear RNA-targeting complexes that deliver them to the exosome for degradation. S. pombe MTREC, and its human counterpart PAXT, are key players in this mechanism but inner workings of these complexes are not understood in sufficient detail. Here, we present an NMR structure of an MTREC scaffold protein Red1 helix-turn-helix domain bound to the Iss10 N-terminus and show this interaction is required for proper cellular growth and meiotic mRNA degradation. We also report a crystal structure of a Red1-Ars2 complex explaining mutually exclusive interactions of hARS2 with various ED/EGEI/L motif-possessing RNA regulators, including hZFC3H1 of PAXT, hFLASH or hNCBP3. Finally, we show that both Red1 and hZFC3H1 homo-dimerize via their coiled-coil regions indicating that MTREC and PAXT likely function as dimers. Our results, combining structures of three Red1 interfaces with in vivo studies, provide mechanistic insights into conserved features of MTREC/PAXT architecture.

Pharmacokinetics of Intramuscularly Administered Thermoresponsive Polymers.

Aqueous solutions of some polymers exhibit a lower critical solution temperature (LCST); that is, they form phase-separated aggregates when heated above a threshold temperature. Such polymers found many promising (bio)medical applications, including in situ thermogelling with controlled drug release, polymer-supported radiotherapy (brachytherapy), immunotherapy, and wound dressing, among others. Yet, despite the extensive research on medicinal applications of thermoresponsive polymers, their biodistribution and fate after administration remained unknown. Thus, herein, they studied the pharmacokinetics of four different thermoresponsive polyacrylamides after intramuscular administration in mice. In vivo, these thermoresponsive polymers formed depots that subsequently dissolved with a two-phase kinetics (depot maturation, slow redissolution) with half-lives 2 weeks to 5 months, as depot vitrification prolonged their half-lives. Additionally, the decrease of TCP of a polymer solution increased the density of the intramuscular depot. Moreover, they detected secondary polymer depots in the kidneys and liver; these secondary depots also followed two-phase kinetics (depot maturation and slow dissolution), with half-lives 8 to 38 days (kidneys) and 15 to 22 days (liver). Overall, these findings may be used to tailor the properties of thermoresponsive polymers to meet the demands of their medicinal applications. Their methods may become a benchmark for future studies of polymer biodistribution.

Improving diffraction by humidity control: a novel device compatible with X-ray beamlines.

Dehydration of protein crystals is rarely used, despite being a post-crystallization method that is useful for the improvement of crystal diffraction properties, as it is difficult to reproduce and monitor. A novel device for hydration control of macromolecular crystals in a standard data-collection environment has been developed. The device delivers an air stream of precise relative humidity that can be used to alter the amount of water in macromolecular crystals. The device can be rapidly installed and is fully compatible with most standard synchrotron X-ray beamlines. Samples are mounted in cryoloops and the progress of dehydration can be monitored both optically and by the acquisition of diffraction images. Once the optimal hydration level has been obtained, cryocooling is easy to achieve by hand or by using a sample changer. The device has been thoroughly tested on several ESRF beamlines and is available to users.

The structural basis for the interaction between nonsense-mediated mRNA decay factors UPF2 and UPF3.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism by which eukaryotic cells detect and degrade transcripts containing premature termination codons. Three 'up-frameshift' proteins, UPF1, UPF2 and UPF3, are essential for this process in organisms ranging from yeast to human. We present a crystal structure at a resolution of 1.95 A of the complex between the interacting domains of human UPF2 and UPF3b, which are, respectively, a MIF4G (middle portion of eIF4G) domain and an RNP domain (ribonucleoprotein-type RNA-binding domain). The protein-protein interface is mediated by highly conserved charged residues in UPF2 and UPF3b and involves the beta-sheet surface of the UPF3b RNP domain, which is generally used by these domains to bind nucleic acids. We show that the UPF3b RNP does not bind RNA, whereas the UPF2 construct and the complex do. Our results advance understanding of the molecular mechanisms underlying the NMD quality control process.