Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number

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Genetic Analysis of the Ribosome Biogenesis Factor Ltv1 of Saccharomyces cerevisiae

Ribosome biogenesis has been studied extensively in the yeast Saccharomyces cerevisiae. Yeast Ltv1 is a conserved 40S-associated biogenesis factor that has been proposed to function in small subunit nuclear export. Here we show that Ltv1 has a canonical leucine-rich nuclear export signal (NES) at its extreme C terminus that is both necessary for Crm1 interaction and Ltv1 export. The C terminus of Ltv1 can substitute for the NES in the 60S-export adapter Nmd3, demonstrating that it is a functional NES. Overexpression of an Ltv1 lacking its NES (Ltv1∆C13) was strongly dominant negative and resulted in the nuclear accumulation of RpS3-GFP; however, export of the pre-40S was not affected. In addition, expression of endogenous levels of Ltv1∆C protein complemented both the slow-growth phenotype and the 40S biogenesis defect of an ltv1 deletion mutant. Thus, if Ltv1 is a nuclear export adapter for the pre-40S subunit, its function must be fully redundant with additional export factors. The dominant negative phenotype of Ltv1∆NES overexpression was suppressed by co-overexpressing RpS3 and its chaperone, Yar1, or by deletion of the RpS3-binding site in Ltv1∆NES, suggesting that titration of RpS3 by Ltv1∆NES is deleterious in yeast. The dominant-negative phenotype did not correlate with a decrease in 40S levels but rather with a reduction in the polysome-to-monosome ratio, indicating reduced rates of translation. We suggest that titration of RpS3 by excess nuclear Ltv1 interferes with 40S function or with a nonribosomal function of RpS3.     

Looking through the Lens of the Ribosome Biogenesis Evolutionary History: Possible Implications for Archaeal Phylogeny and Eukaryogenesis

Our understanding of microbial diversity and its evolutionary relationships has increased substantially over the last decade. Such an understanding has been greatly fueled by culture-independent metagenomics analyses. However, the outcome of some of these studies and their biological and evolutionary implications, such as the origin of the eukaryotic lineage from the recently discovered archaeal Asgard superphylum, is debated. The sequences of the ribosomal constituents are amongst the most used phylogenetic markers. However, the functional consequences underlying the analysed sequence diversity and their putative evolutionary implications are essentially not taken into consideration. Here, we propose to exploit additional functional hallmarks of ribosome biogenesis to help disentangle competing evolutionary hypotheses. Using selected examples, such as the multiple origins of halophily in archaea or the evolutionary relationship between the Asgard archaea and Eukaryotes, we illustrate and discuss how function-aware phylogenetic framework can contribute to refining our understanding of archaeal phylogeny and the origin of eukaryotic cells.     

Formation and nuclear export of preribosomes are functionally linked to the small-ubiquitin-related modifier pathway

Ribosomal precursor particles are initially assembled in the nucleolus prior to their transfer to the nucleoplasm and export to the cytoplasm. In a screen to identify thermosensitive (ts) mutants defective in the export of pre-60S ribosomal subunit, we isolated the rix16-1 mutant. In this strain, nucleolar accumulation of the Rpl25-eGFP reporter was complemented by UBA2 (a subunit of the E1 sumoylation enzyme). Mutations in UBC9 (E2 enzyme), ULP1 [small-ubiquitin-related modifier (SUMO) isopeptidase] and SMT3 (SUMO-1) caused 60S export defects. A directed analysis of the SUMO proteome revealed that many ribosome biogenesis factors are sumoylated. Importantly, preribosomal particles along both the 60S and the 40S synthesis pathways were decorated with SUMO, showing its direct involvement. Consistent with this, early 60S assembly factors were genetically linked to SUMO conjugation. Notably, the SUMO deconjugating enzyme Ulp1, which localizes to the nuclear pore complex (NPC), was functionally linked to the 60S export factor Mtr2. Together our data suggest that sumoylation of preribosomal particles in the nucleus and subsequent desumoylation at the NPC is necessary for efficient ribosome biogenesis and export in eukaryotes.     

NONU-1 Encodes a Conserved Endonuclease Required for mRNA Translation Surveillance

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A Computationally Guided Protein-Interaction Screen Uncovers Coiled-Coil Interactions Involved in Vesicular Trafficking

Mapping protein-protein interactions at a domain or motif level can provide structural annotation of the interactome. The α-helical coiled coil is among the most common protein-interaction motifs, and proteins predicted to contain coiled coils participate in diverse biological processes. Here, we introduce a combined computational/experimental screening strategy that we used to uncover coiled-coil interactions among proteins involved in vesicular trafficking in Saccharomyces cerevisiae. A number of coiled-coil complexes have already been identified and reported to play important roles in this important biological process. We identify additional examples of coiled coils that can form physical associations. The computational strategy used to prioritize coiled-coil candidates for testing dramatically improved the efficiency of discovery in a large experimental screen. As assessed by comprehensive yeast two-hybrid assays, computational prefiltering retained 90% of positive interacting pairs and eliminated > 60% of negatives from a set of interaction candidates. The coiled-coil-mediated interaction network elucidated using the combined computational/experimental approach comprises 80 coiled-coil associations between 58 protein pairs, among which 21 protein interactions have not been previously reported in interaction databases and 26 interactions were previously known at the protein level but have now been localized to the coiled-coil motif. The coiled-coil-mediated interactions were specific rather than promiscuous, and many interactions could be recapitulated in a green fluorescent protein complementation assay. Our method provides an efficient route to discovering new coiled-coil interactions and uncovers a number of associations that may have functional significance for vesicular trafficking.     

植物核糖体应激响应机制研究进展

核仁是真核细胞中重要的核结构,核糖体发生最初在核仁中进行,该过程涉及一系列复杂的反应,需要许多核仁相关因子参与。核糖体生物发生出现异常通常引起核仁结构紊乱,并导致细胞周期阻滞、细胞衰老甚至凋亡。核糖体应激响应机制在哺乳动物细胞中研究得较为深入,但在植物细胞中尚不明晰。尽管如此,人们逐渐发现某些植物特有的NAC转录因子家族成员在植物细胞中可能参与包括核糖体应激在内的多种胞内应激响应过程。此外,前期研究发现生长素系统与核糖体生物合成之间存在一种相互协调机制来调控植物发育。该文结合哺乳动物细胞中已知的核糖体应激响应通路,探讨植物细胞潜在的核糖体应激机制。     

生物传感器法测定麦芽汁中赖氨酸含量的研究

本研究利用固定化赖氨酸氧化酶和生物传感器,建立了麦芽汁中赖氨酸含量的测定方法。研究了该方法的线性范围、检出限、最适pH值范围、稳定性及专一性。以大生产麦芽汁为样品,与其它检测方法进行了比较。结果表明,生物传感器法测定赖氨酸含量操作简单而准确,加标回收率为97．41％～103．23％,RSD为2．23％;线性范围为2～100 mg/100 mL,检出限为2 mg/100 mL;稳定性及专一性强,不受其他氨基酸的干扰,能够用于氨基酸种类复杂的麦汁赖氨酸的检测。本方法与氨基酸分析仪、DBL、HPLC等方法相比较,测定结果无显著差异（P＞0．05）。     

Nmd3 is a structural mimic of eIF5A,and activates the cpGTPase Lsg1 during 60S ribosome biogenesis

During ribosome biogenesis in eukaryotes, nascent subunits are exported to the cytoplasm in a functionally inactive state. 60S subunits are activated through a series of cytoplasmic maturation events. The last known events in the cytoplasm are the release of Tif6 by Efl1 and Sdo1 and the release of the export adapter, Nmd3, by the GTPase Lsg1. Here, we have used cryo-electron microscopy to determine the structure of the 60S subunit bound by Nmd3, Lsg1, and Tif6. We find that a central domain of Nmd3 mimics the translation elongation factor eIF5A, inserting into the E site of the ribosome and pulling the L1 stalk into a closed position. Additional domains occupy the P site and extend toward the sarcin–ricin loop to interact with Tif6. Nmd3 and Lsg1 together embrace helix 69 of the B2a intersubunit bridge, inducing base flipping that we suggest may activate the GTPase activity of Lsg1.     

Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii

Upon infection, apicomplexan parasites quickly invade host cells and begin a replicative cycle rapidly increasing in number over a short period of time, leading to tissue lysis and disease. The secretory pathway of these highly polarized protozoan parasites tightly controls, in time and space, the biogenesis of specialized structures and organelles required for invasion and intracellular survival. In other systems, regulation of protein trafficking can occur by phosphorylation of vesicle fusion machinery. Previously, we have shown that Toxoplasma gondii αSNAP – a protein that controls the disassembly of cis‐SNARE complexes – is phosphorylated. Here, we show that this post‐translational modification is required for the correct function of αSNAP in controlling secretory traffic. We demonstrate that during intracellular development conditional expression of a non‐phosphorylatable form of αSNAP results in Golgi fragmentation and vesiculation of all downstream secretory organelles. In addition, we show that the vestigial plastid (termed apicoplast), although reported not to be reliant on Golgi trafficking for biogenesis, is also affected upon overexpression of αSNAP and is much more sensitive to the levels of this protein than targeting to other organelles. This work highlights the importance of αSNAP and its phosphorylation in Toxoplasma organelle biogenesis and exposes a hereto fore‐unexplored mechanism of regulation of vesicle fusion during secretory pathway trafficking in apicomplexan parasites.     

SARS-CoV-2 Nucleocapsid Protein Targets a Conserved Surface Groove of the NTF2-like Domain of G3BP1

Stress granule (SG) formation mediated by Ras GTPase-activating protein-binding protein 1 (G3BP1) constitutes a key obstacle for viral replication, which makes G3BP1 a frequent target for viruses. For instance, the SARS-CoV-2 nucleocapsid (N) protein interacts with G3BP1 directly to suppress SG assembly and promote viral production. However, the molecular basis for the SARS-CoV-2 N ? G3BP1 interaction remains elusive. Here we report biochemical and structural analyses of the SARS-CoV-2 N ? G3BP1 interaction, revealing differential contributions of various regions of SARS-CoV-2 N to G3BP1 binding. The crystal structure of the NTF2-like domain of G3BP1 (G3BP1_NTF2) in complex with a peptide derived from SARS-CoV-2 N (residues 1–25, N_1–25) reveals that SARS-CoV-2 N_1–25 occupies a conserved surface groove of G3BP1_NTF2 via surface complementarity. We show that a φ-x-F (φ, hydrophobic residue) motif constitutes the primary determinant for G3BP1_NTF2-targeting proteins, while the flanking sequence underpins diverse secondary interactions. We demonstrate that mutation of key interaction residues of the SARS-CoV-2 N_1–25 ? G3BP1_NTF2 complex leads to disruption of the SARS-CoV-2 N ? G3BP1 interaction in vitro. Together, these results provide a molecular basis of the strain-specific interaction between SARS-CoV-2 N and G3BP1, which has important implications for the development of novel therapeutic strategies against SARS-CoV-2 infection.     

Dual Strategies for Argonaute2-Mediated Biogenesis of Erythroid miRNAs Underlie Conserved Requirements for Slicing in Mammals

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A Similarity-Based Method for Predicting Enzymatic Functions in Yeast Uncovers a New AMP Hydrolase

Despite decades of research and the availability of the full genomic sequence of the baker’s yeast Saccharomyces cerevisiae, still a large fraction of its genome is not functionally annotated. This hinders our ability to fully understand cellular activity and suggests that many additional processes await discovery. The recent years have shown an explosion of high-quality genomic and structural data from multiple organisms, ranging from bacteria to mammals. New computational methods now allow us to integrate these data and extract meaningful insights into the functional identity of uncharacterized proteins in yeast. Here, we created a database of sensitive sequence similarity predictions for all yeast proteins. We use this information to identify candidate enzymes for known biochemical reactions whose enzymes are unidentified, and show how this provides a powerful basis for experimental validation. Using one pathway as a test case we pair a new function for the previously uncharacterized enzyme Yhr202w, as an extra-cellular AMP hydrolase in the NAD degradation pathway. Yhr202w, which we now term Smn1 for Scavenger MonoNucleotidase 1, is a highly conserved protein that is similar to the human protein E5NT/CD73, which is associated with multiple cancers. Hence, our new methodology provides a paradigm, that can be adopted to other organisms, for uncovering new enzymatic functions of uncharacterized proteins.     

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

In all three domains of life ribosomal RNAs are extensively modified at functionally important sites of the ribosome. These modifications are believed to fine-tune the ribosome structure for optimal translation. However, the precise mechanistic effect of modifications on ribosome function remains largely unknown. Here we show that a cluster of methylated nucleotides in domain IV of 25S rRNA is critical for integrity of the large ribosomal subunit. We identified the elusive cytosine-5 methyltransferase for C2278 in yeast as Rcm1 and found that a combined loss of cytosine-5 methylation at C2278 and ribose methylation at G2288 caused dramatic ribosome instability, resulting in loss of 60S ribosomal subunits. Structural and biochemical analyses revealed that this instability was caused by changes in the structure of 25S rRNA and a consequent loss of multiple ribosomal proteins from the large ribosomal subunit. Our data demonstrate that individual RNA modifications can strongly affect structure of large ribonucleoprotein complexes.     

A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development

Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development.     

A novel synthesis and antimicrobial activity of 1-[(Substituted-phenyl) sulfonyl]pyrrolidin-2-ones

Novel cyclization of 4-(substituted-phenylsulfonamido)butanoic acids to their corresponding 1-[(substituted-phenyl)sulfonyl]pyrrolidin-2-ones was successfully achieved by using polyphosphate ester (PPE). The reaction time was considerably reduced with corresponding increase in the yields, when polyphosphate ester (PPE) was used in combination with 4-(N,N-dimethylamino)pyridine (DMAP). All the synthesized compounds were screened for their antimicrobial activity. Minimum Inhibitory Concentration (MIC) values of synthesized compounds were also determined, and were found to be in the range of 0.09–1.0 mg.