Intensity and retention time prediction improves the rescoring of protein-nucleic acid cross-links.

In protein-RNA cross-linking mass spectrometry, UV or chemical cross-linking introduces stable bonds between amino acids and nucleic acids in protein-RNA complexes that are then analyzed and detected in mass spectra. This analytical tool delivers valuable information about RNA-protein interactions and RNA docking sites in proteins, both in vitro and in vivo. The identification of cross-linked peptides with oligonucleotides of different length leads to a combinatorial increase in search space. We demonstrate that the peptide retention time prediction tasks can be transferred to the task of cross-linked peptide retention time prediction using a simple amino acid composition encoding, yielding improved identification rates when the prediction error is included in rescoring. For the more challenging task of including fragment intensity prediction of cross-linked peptides in the rescoring, we obtain, on average, a similar improvement. Further improvement in the encoding and fine-tuning of retention time and intensity prediction models might lead to further gains, and merit further research.

GPATCH4 regulates rRNA and snRNA 2'-O-methylation in both DHX15-dependent and DHX15-independent manners.

Regulation of RNA helicase activity, often accomplished by protein cofactors, is essential to ensure target specificity within the complex cellular environment. The largest family of RNA helicase cofactors are the G-patch proteins, but the cognate RNA helicases and cellular functions of numerous human G-patch proteins remain elusive. Here, we discover that GPATCH4 is a stimulatory cofactor of DHX15 that interacts with the DEAH box helicase in the nucleolus via residues in its G-patch domain. We reveal that GPATCH4 associates with pre-ribosomal particles, and crosslinks to the transcribed ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated RNAs that guide rRNA and snRNA modifications. Loss of GPATCH4 impairs 2'-O-methylation at various rRNA and snRNA sites leading to decreased protein synthesis and cell growth. We demonstrate that the regulation of 2'-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2'-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings extend knowledge on RNA helicase regulation by G-patch proteins and also provide important new insights into the mechanisms regulating installation of rRNA and snRNA modifications, which are essential for ribosome function and pre-mRNA splicing.

Mammalian oocytes store proteins for the early embryo on cytoplasmic lattices.

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.

Control of intracellular pH and bicarbonate by CO2 diffusion into human sperm.

The reaction of CO2 with H2O to form bicarbonate (HCO3-) and H+ controls sperm motility and fertilization via HCO3--stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3- in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pHi is set by amiloride-sensitive Na+/H+ exchange. The sperm-specific putative Na+/H+ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO3- transport are not detected, and may be present at copy numbers < 10 molecules/sperm cell. Instead, HCO3- is produced by diffusion of CO2 into cells and readjustment of the CO2/HCO3-/H+ equilibrium. The proton channel Hv1 may serve as a unidirectional valve that blunts the acidification ensuing from HCO3- synthesis. This work provides a new framework for the study of male infertility.

A viral ADP-ribosyltransferase attaches RNA chains to host proteins.

The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate1,2. NAD has previously been identified as a 5' modification of cellular RNAs3-5. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an 'RNAylation' reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD-RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host's translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections6, so RNAylation may have far-reaching implications.

Human TRMT2A methylates tRNA and contributes to translation fidelity.

5-Methyluridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme for m5U formation at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by hTRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with cross-linking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that hTRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of hTRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.

Structural basis of mRNA binding by the human FERRY Rab5 effector complex.

The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.

Mammalian oocytes store mRNAs in a mitochondria-associated membraneless compartment.

Full-grown oocytes are transcriptionally silent and must stably maintain the messenger RNAs (mRNAs) needed for oocyte meiotic maturation and early embryonic development. However, where and how mammalian oocytes store maternal mRNAs is unclear. Here, we report that mammalian oocytes accumulate mRNAs in a mitochondria-associated ribonucleoprotein domain (MARDO). MARDO assembly around mitochondria was promoted by the RNA-binding protein ZAR1 and directed by an increase in mitochondrial membrane potential during oocyte growth. MARDO foci coalesced into hydrogel-like matrices that clustered mitochondria. Maternal mRNAs stored in the MARDO were translationally repressed. Loss of ZAR1 disrupted the MARDO, dispersed mitochondria, and caused a premature loss of MARDO-localized mRNAs. Thus, a mitochondria-associated membraneless compartment controls mitochondrial distribution and regulates maternal mRNA storage, translation, and decay to ensure fertility in mammals.

Sleep neuron depolarization promotes protective gene expression changes and FOXO activation.

Sleep is an essential state that allows for recuperation and survival processes. Disturbing sleep triggers stress responses that promote protective gene expression. Sleep and its deprivation grossly impact gene expression, but little is known about how normal or disturbed sleep control gene expression. Central to the induction of sleep are sleep-active neurons, which inhibit wakefulness and promote survival. Sleep and sleep-active neurons are highly conserved. In Caenorhabditis elegans, the sleep-active RIS neuron is crucial for sleep and survival. Here, we show that RIS depolarization promotes the protective gene expression response that occurs during developmental arrest. This response includes the activation of FOXO/DAF-16 and expression of DAF-16 target genes such as HSP-12.6, a small heat-shock protein that is required for starvation survival. Disturbing sleep by mechanical stimulation increases RIS depolarization. RIS activation in turn activates DAF-16 and other genes required for survival. Hence, during normal sleep, RIS depolarization promotes protective gene expression. When sleep is disturbed, protective gene expression gets further increased by raised RIS depolarization. We thus link sleep-active neuron depolarization to protective gene expression changes and suggest that the cellular stress response following sleep deprivation could be understood as a safeguarding process that is caused by the overactivation of sleep-active neurons.

Structural and functional insights into human tRNA guanine transgylcosylase.

The eukaryotic tRNA guanine transglycosylase (TGT) is an RNA modifying enzyme incorporating queuine, a hypermodified guanine derivative, into the tRNAsAsp,Asn,His,Tyr. While both subunits of the functional heterodimer have been crystallized individually, much of our understanding of its dimer interface or recognition of a target RNA has been inferred from its more thoroughly studied bacterial homolog. However, since bacterial TGT, by incorporating queuine precursor preQ1, deviates not only in function, but as a homodimer, also in its subunit architecture, any inferences regarding the subunit association of the eukaryotic heterodimer or the significance of its unique catalytically inactive subunit are based on unstable footing. Here, we report the crystal structure of human TGT in its heterodimeric form and in complex with a 25-mer stem loop RNA, enabling detailed analysis of its dimer interface and interaction with a minimal substrate RNA. Based on a model of bound tRNA, we addressed a potential functional role of the catalytically inactive subunit QTRT2 by UV-crosslinking and mutagenesis experiments, identifying the two-stranded ßEßF-sheet of the QTRT2 subunit as an additional RNA-binding motif.

Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA.

TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m7G) in tRNA. In tRNAs m7G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of B. subtilis TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant Kd of TrmB and tRNAPhe was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNAPhe complex structure.

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry.

Protein-DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein-DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein-RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.

Structural insights into the stimulation of S. pombe Dnmt2 catalytic efficiency by the tRNA nucleoside queuosine.

Dnmt2 methylates cytosine at position 38 of tRNAAsp in a variety of eukaryotic organisms. A correlation between the presence of the hypermodified nucleoside queuosine (Q) at position 34 of tRNAAsp and the Dnmt2 dependent C38 methylation was recently found in vivo for S. pombe and D. discoideum. We demonstrate a direct effect of the Q-modification on the methyltransferase catalytic efficiency in vitro, as Vmax/K0.5 of purified S. pombe Dnmt2 shows an increase for in vitro transcribed tRNAAsp containing Q34 to 6.27 ∗ 10-3 s-1 µM-1 compared to 1.51 ∗ 10-3 s-1 µM-1 for the unmodified substrate. Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA. In order to get insight into the structural basis for the Q-dependency, the crystal structure of S. pombe Dnmt2 was determined at 1.7 Å resolution. It closely resembles the known structures of human and E. histolytica Dnmt2, and contains the entire active site loop. The interaction with tRNA was analyzed by means of mass-spectrometry using UV cross-linked Dnmt2-tRNA complex. These cross-link data and computational docking of Dnmt2 and tRNAAsp reveal Q34 positioned adjacent to the S-adenosylmethionine occupying the active site, suggesting that the observed increase of Dnmt2 catalytic efficiency by queuine originates from optimal positioning of the substrate molecules and residues relevant for methyl transfer.