The Feeder Effects of Cultured Rice Cells on the Early Development of Rice Zygotes.

Feeder cells and the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in a culture medium promote mitosis and cell division in cultured cells. These are also added to nutrient medium for the cultivation of highly active in mitosis and dividing zygotes, produced in vitro or isolated from pollinated ovaries. In the study, an in vitro fertilization (IVF) system was used to study the precise effects of feeder cells and 2,4-D on the growth and development of rice (Oryza sativa L.) zygote. The elimination of 2,4-D from the culture medium did not affect the early developmental profiles of the zygotes, but decreased the division rates of multicellular embryos. The omission of feeder cells resulted in defective karyogamy, fusion between male and female nuclei, and the subsequent first division of the cultured zygotes. The culture of zygotes in a conditioned medium corrected developmental disorders. Proteome analyses of the conditioned medium revealed the presence of abundant hydrolases possibly released from the feeder cells. Exogenously applied α-amylase ameliorated karyogamy and promoted zygote development. It is suggested that hydrolytic enzymes, including α-amylase, released from feeder cells may be involved in the progression of zygotic development.

A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei.

Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.

An ionic liquid-assisted sample preparation method for sensitive integral-membrane proteome analysis.

Many ion channels and receptor proteins are potential targets for new drugs. However, standard methods for profiling these integral membrane proteins (IMPs) have not been fully established, especially when applied to rare and quantity-limited biological samples. We previously demonstrated that a mixture containing 1-butyl-3-methylimidazolium cyanate, an ionic liquid (IL), and NaOH (termed i-soln) is an excellent solubilizer for insoluble aggregates. In this study, we present a combined i-soln-assisted proteomic sample preparation platform (termed pTRUST), which is compatible with starting materials in the sub-microgram range, using our previously reported i-soln-based sample preparation strategy (iBOPs) and an in-StageTip technique. This novel and straightforward approach allows for the rapid solubilization and processing of a variety of IMPs from human samples to support highly sensitive mass spectrometry analysis. We also demonstrated that the performance of this technology surpasses that of conventional methods such as filter-aided sample preparation methods, FASP and i-FASP. The convenience and availability of pTRUST technology using the IL system have great potential for proteomic identification and characterization of novel drug targets and disease biology in research and clinical settings.

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

Structure of mitoribosome reveals mechanism of mRNA binding, tRNA interactions with L1 stalk, roles of cofactors and rRNA modifications.

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNA Val . The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transition in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide the most complete description so far of the structure and function of the human mitoribosome.

PDGF-B secreted from skeletal muscle enhances myoblast proliferation and myotube maturation via activation of the PDGFR signaling cascade.

Myokines, secreted factors from skeletal muscle, act locally on muscle cells or satellite cells, which is important in regulating muscle mass and function. Here, we found platelet-derived growth factor subunit B (PDGF-B) is constitutively secreted from muscle cells without muscle contraction. Furthermore, PDGF-B secretion increased with myoblast to myotube differentiation. To examine the role of PDGF-B as a paracrine or autocrine myokine, myoblasts or myotubes were treated with PDGF-B. As a result, myoblast proliferation was significantly enhanced via several signaling pathways. Intriguingly, myotubes treated with PDGF-B showed enhanced maturation as indicated by their increased myotube diameter, myosin heavy chain expression, and strengthened contractile force. These findings suggest that PDGF-B is constitutively secreted by myokines to enhance myoblast proliferation and myotube maturation, which may contribute to skeletal muscle regeneration.

Liquid Chromatography-Mass Spectrometry-Based Qualitative Profiling of mRNA Therapeutic Reagents Using Stable Isotope-Labeled Standards Followed by the Automatic Quantitation Software Ariadne.

mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.

Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies.

Many members of the tripartite motif (TRIM) family of ubiquitin ligases localize in spherical, membrane-free structures collectively referred to as cytoplasmic bodies (CBs) in a concentration-dependent manner. These CBs may function as aggresome precursors or storage compartments that segregate potentially harmful excess TRIM molecules from the cytosolic milieu. However, the manner in which TRIM proteins accumulate into CBs is unclear. In the present study, using TRIM32, TRIM5α and TRIM63 as examples, we demonstrated that CBs are in a liquid droplet state, resulting from liquid-liquid phase separation (LLPS). This finding is based on criteria that defines phase-separated structures, such as recovery after photobleaching, sensitivity to hexanediol, and the ability to undergo fusion. CB droplets, which contain cyan fluorescent protein (CFP)-fused TRIM32, were purified from HEK293 cells using a fluorescence-activated cell sorter and analyzed by LC-MS/MS. We found that in addition to TRIM32, these droplets contain a variety of endogenous proteins and enzymes including ubiquitin. Localization of ubiquitin within CBs was further verified by fluorescence microscopy. We also found that the activation of the intracellular ubiquitination cascade promotes the assembly of TRIM32 molecules into CBs, whereas inhibition causes suppression. Regulation is dependent on the intrinsic E3 ligase activity of TRIM32. Similar regulation by ubiquitination on the TRIM assembly was also observed with TRIM5α and TRIM63. Our findings provide a novel mechanical basis for the organization of CBs that couples compartmentalization through LLPS with ubiquitination.

Cryo-EM structure and rRNA modification sites of a plant ribosome.

Protein synthesis in crop plants contributes to the balance of food and fuel on our planet, which influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via the deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and a quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins and 71 plant-specific rRNA modifications, and it re-annotates 30 rRNA residues in the available sequence. At the protein level, isoAsp is found in position 137 of uS11, and a zinc finger previously believed to be universal is missing from eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. At the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation, highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.

The ER cholesterol sensor SCAP promotes CARTS biogenesis at ER-Golgi membrane contact sites.

In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here, we show that in cholesterol-fed cells, ER-localized SCAP interacts through Sac1 phosphatidylinositol 4-phosphate (PI4P) phosphatase with a VAP-OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER-Golgi membrane contact sites (MCSs). SCAP knockdown inhibited the turnover of PI4P, perhaps due to a cholesterol transport defect, and altered the subcellular distribution of the VAP-OSBP complex. As in the case of perturbation of lipid transfer complexes at ER-Golgi MCSs, SCAP knockdown inhibited the biogenesis of the trans-Golgi network-derived transport carriers CARTS, which was reversed by expression of wild-type SCAP or a Golgi transport-defective mutant, but not of cholesterol sensing-defective mutants. Altogether, our findings reveal a new role for SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis via ER-Golgi MCSs, depending on the ER cholesterol.

DKC1 Overexpression Induces a More Aggressive Cellular Behavior and Increases Intrinsic Ribosomal Activity in Immortalized Mammary Gland Cells.

Dyskerin is a nucleolar protein involved in the small nucleolar RNA (snoRNA)-guided pseudouridylation of specific uridines on ribosomal RNA (rRNA), and in the stabilization of the telomerase RNA component (hTR). Loss of function mutations in DKC1 causes X-linked dyskeratosis congenita, which is characterized by a failure of proliferating tissues and increased susceptibility to cancer. However, several tumors show dyskerin overexpression. We observed that patients with primary breast cancers with high dyskerin levels are more frequently characterized by shorter survival rates and positive lymph node status than those with tumors with a lower dyskerin expression. To functionally characterize the effects of high dyskerin expression, we generated stably overexpressing DKC1 models finding that increased dyskerin levels conferred a more aggressive cellular phenotype in untransformed immortalized MCF10A cells. Contextually, DKC1 overexpression led to an upregulation of some snoRNAs, including SNORA67 and a significantly increased U1445 modification on 18S rRNA, the known target of SNORA67. Lastly, we found that dyskerin overexpression strongly enhanced the synthetic activity of ribosomes increasing translational efficiency in MCF10A. Altogether, our results indicate that dyskerin may sustain the neoplastic phenotype from an early stage in breast cancer endowing ribosomes with an augmented translation efficiency.

Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis.

Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.

Direct Determination of Pseudouridine in RNA by Mass Spectrometry Coupled with Stable Isotope Labeling.

Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

Method for Direct Mass-Spectrometry-Based Identification of Monomethylated RNA Nucleoside Positional Isomers and Its Application to the Analysis of Leishmania rRNA.

RNA post-transcriptional modifications are common in all kingdoms of life and are predominantly affiliated with methylations at various nucleobase positions. Methylations occur frequently at specific sites on the RNA nucleobases and appear to regulate site-specific intermolecular/intramolecular interactions. Herein, we present a method that utilizes liquid chromatography-mass spectrometry (LC-MS) to identify positional monomethylated RNA nucleoside isomers. The method produces profiles of in-source fragmentation and subsequent tandem mass spectrometry (MS2) (pseudo-MS3) of RNase-digested fragments of an RNA and distinguishes between positional methylated nucleobase isomers by comparing their intranucleobase fragment ion profiles with signature profiles derived from authentic isomers. For method validation, we independently determined the positions of all known monomethylated nucleoside isomers in the Escherichia coli 16S/23S rRNAs. As proof of concept, we further applied this technology to fully characterize the base-modified nucleoside positional isomers, in rRNAs derived from Leishmania donovani, a human blood parasite afflicting millions around the globe. The method described herein will be highly beneficial for the delineation of RNA modification profiles in various cellular RNAs, and as it only requires a subpicomole amount of RNA, it could also be used for the structure-function studies of RNA populations represented in minute amounts in the cell.

An Ionic Liquid-Based Sample Preparation Method for Next-Stage Aggregate Proteomic Analysis.

A wide variety of proteomic methods have been applied for protein profiling of insoluble aggregates or inclusion bodies deposited in various cells or tissues. However, these are essentially optimized or modified classical protein chemistry techniques using conventional denaturing agents such as formic acid, urea, and sodium dodecyl sulfate (SDS). The use of these denaturants has several shortcomings, including limited solubilization, contamination, and restrictions on absolute sample quantity and throughput. Here, we describe an alternative proteomic sample preparation platform for widespread aggregation analysis. This approach combines two techniques, (1) the use of ionic liquid for protein solubilization and (2) the recently published microbead-based and organic-media-assisted proteolysis strategy (BOPs), into a single-tube workflow. We demonstrate that the combined approach (iBOPs) enabled the successful solubilization of heat-aggregated hen egg whites within 10 min and supported sensitive mass spectrometry (MS) analysis. The performance of the iBOPs system surpassed those of conventional detergents and chaotropes. Moreover, this technology enabled ultrasensitive proteomic characterization of protein aggregates deposited in individual Caenorhabditis elegans nematodes. We identified ubiquitin and other molecules as candidate stochastic factors whose accumulation levels varied among aging nematode individuals. The sensitivity and applicability of the present iBOPs make it especially attractive for next-stage aggregate proteomic analysis of various biological processes.

Sequential two-step chromatographic purification of infectious poliovirus using ceramic fluoroapatite and ceramic hydroxyapatite columns.

Infectious virus purification techniques are important for vaccine development and gene therapy applications. However, the standardized one-step purification technique using ceramic hydroxyapatite (CHAp) has proven unsuitable for poliovirus. Therefore, we designed a sequential two-step chromatographic technique for purification of the infectious Sabin type 2 vaccine strain of poliovirus from the cell culture supernatant. In the first step, we removed protein contaminants from the Sabin type 2 virus fraction by pH gradient elution on a ceramic fluoroapatite column. In the second step, we removed double-stranded DNA derived from host cells by diluting the virus fraction, directly loading it on a CHAp column, and purifying it using a phosphate gradient with 1 M sodium chloride. This process achieved removal rates of more than 99.95% and 99.99% for proteins and double-stranded DNA, respectively, and was highly reproducible and scalable. Furthermore, it is likely that it will be applicable to other virus species.

Low-fluence blue light-induced phosphorylation of Zmphot1 mediates the first positive phototropism.

Phototropin1 (phot1) perceives low- to high-fluence blue light stimuli and mediates both the first and second positive phototropisms. High-fluence blue light is known to induce autophosphorylation of phot1, leading to the second positive phototropism. However, the phosphorylation status of phot1 by low-fluence blue light that induces the first positive phototropism had not been observed. Here, we conducted a phosphoproteomic analysis of maize coleoptiles to investigate the fluence-dependent phosphorylation status of Zmphot1. High-fluence blue light induced phosphorylation of Zmphot1 at several sites. Notably, low-fluence blue light significantly increased the phosphorylation level of Ser291 in Zmphot1. Furthermore, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be more abundant in the low-fluence blue light-irradiated sides than in the shaded sides of coleoptiles. The roles of these phosphorylation events in phototropism were explored by heterologous expression of ZmPHOT1 in the Arabidopsis thaliana phot1phot2 mutant. The first positive phototropism was restored in wild-type ZmPHOT1-expressing plants; however, plants expressing S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 showed significantly reduced complementation rates. All transgenic plants tested in this study exhibited a normal second positive phototropism. These findings provide the first indication that low-fluence blue light induces phosphorylation of Zmphot1 and that this induced phosphorylation is crucial for the first positive phototropism.

TDP-43 regulates site-specific 2'-O-methylation of U1 and U2 snRNAs via controlling the Cajal body localization of a subset of C/D scaRNAs.

TDP-43 regulates cellular levels of Cajal bodies (CBs) that provide platforms for the assembly and RNA modifications of small nuclear ribonucleoproteins (snRNPs) involved in pre-mRNA splicing. Alterations in these snRNPs may be linked to pathogenesis of amyotrophic lateral sclerosis. However, specific roles for TDP-43 in CBs remain unknown. Here, we demonstrate that TDP-43 regulates the CB localization of four UG-rich motif-bearing C/D-box-containing small Cajal body-specific RNAs (C/D scaRNAs; i.e. scaRNA2, 7, 9 and 28) through the direct binding to these scaRNAs. TDP-43 enhances binding of a CB-localizing protein, WD40-repeat protein 79 (WDR79), to a subpopulation of scaRNA2 and scaRNA28; the remaining population of the four C/D scaRNAs was localized to CB-like structures even with WDR79 depletion. Depletion of TDP-43, in contrast, shifted the localization of these C/D scaRNAs, mainly into the nucleolus, as well as destabilizing scaRNA2, and reduced the site-specific 2'-O-methylation of U1 and U2 snRNAs, including at 70A in U1 snRNA and, 19G, 25G, 47U and 61C in U2 snRNA. Collectively, we suggest that TDP-43 and WDR79 have separate roles in determining CB localization of subsets of C/D and H/ACA scaRNAs.

FANCD2 protects genome stability by recruiting RNA processing enzymes to resolve R-loops during mild replication stress.

R-loops, which consist of DNA : RNA hybrids and displaced single-strand DNA, are a major threat to genome stability. We have previously reported that a key Fanconi anemia protein, FANCD2, accumulates on large fragile genes during mild replication stress in a manner depending on R-loops. In this study, we found that FANCD2 suppresses R-loop levels. Furthermore, we identified FANCD2 interactions with RNA processing factors, including hnRNP U and DDX47. Our data suggest that FANCD2, which accumulates with R-loops in chromatin, recruits these factors and thereby promotes efficient processing of long RNA transcripts. This may lead to a reduction in transcription-replication collisions, as detected by PLA between PCNA and RNA Polymerase II, and hence, lowered R-loop levels. We propose that this mechanism might contribute to maintenance of genome stability during mild replication stress.