rRNA Binding Research Articles

Two dynamic, N-terminal regions are required for function in Ribosomal RNA Adenine Dimethylase family members

Prominent members of the Ribosomal RNA Adenine Dimethylase (RRAD) family of enzymes facilitate ribosome maturation by dimethylating two nucleotides of small subunit rRNA including the human DIMT1 and bacterial KsgA enzymes. A sub-group of RRAD enzymes, named erythromycin resistance methyltransferases (Erm) dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays we found that the N-terminal region is critical for activity in RRAD enzymes: the N-terminal basic region promotes rRNA binding and the conserved motif likely assists in juxtaposing the adenosine substrate and the SAM cofactor. Our results and emerging structural data suggest this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding forming a cap on the active site required for methylation.

Proteomics revealed an association between ribosome-associated proteins and amyloid beta deposition in Alzheimer's disease.

Most scholars believe that amyloid-beta (Aβ) has the potential to induce apoptosis, stimulate an inflammatory cascade, promote oxidative stress and exacerbate the pathological progression of Alzheimer's disease (AD). Therefore, it is crucial to investigate the deposition of Aβ in AD. At approximately 6months of age, APP/PS1 double transgenic mice gradually exhibit the development of plaques, as well as spatial and learning impairment. Notably, the hippocampus is specifically affected in the course of AD. Herein, 6-month-old APP/PS1 double transgenic mice were utilized, and the differentially expressed (DE) proteins in the hippocampus were identified and analyzed using 4D label-free quantitative proteomics technology and parallel reaction monitoring (PRM). Compared to wild-type mice, 29 proteins were upregulated and 25 proteins were downregulated in the AD group. Gene Ontology (GO) enrichment analysis of biological processes (BP) indicated that the DE proteins were mainly involved in 'ribosomal large subunit biogenesis'. Molecular function (MF) analysis results were primarily associated with '5.8S rRNA binding' and 'structural constituent of ribosome'. In terms of cellular components (CC), the DE proteins were mainly found in 'polysomal ribosome', 'cytosolic large ribosomal subunit', 'cytosolic ribosome', and 'large ribosomal subunit', among others. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the results were mainly enriched in the 'Ribosome signaling pathway'. The key target proteins identified were ribosomal protein (Rp)l18, Rpl17, Rpl19, Rpl24, Rpl35, and Rpl6. The PRM verification results were consistent with the findings of the 4D label-free quantitative proteomics analysis. Overall, these findings suggest that Rpl18, Rpl17, Rpl19, Rpl24, Rpl35, and Rpl6 may have potential therapeutic value for the treatment of AD by targeting Aβ.

CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology.

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.

Noncatalytic regulation of 18S rRNA methyltransferase DIMT1 in acute myeloid leukemia.

Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region.

Morphological characterization and transcriptome analysis of leaf angle mutant bhlh112 in maize [Zea mays L.

Leaf angle is an important agronomic trait in maize [Zea mays L.]. The compact plant phenotype, with a smaller leaf angle, is suited for high-density planting and thus for increasing crop yields. Here, we studied the ethyl methane sulfonate (EMS)-induced mutant bhlh112. Leaf angle and plant height were significantly decreased in bhlh112 compared to the wild-type plants. After treatment of seedlings with exogenous IAA and ABA respectively, under the optimal concentration of exogenous hormones, the variation of leaf angle of the mutant was more obvious than that of the wild-type, which indicated that the mutant was more sensitive to exogenous hormones. Transcriptome analysis showed that the ZmbHLH112 gene was related to the biosynthesis of auxin and brassinosteroids, and involved in the activation of genes related to the auxin and brassinosteroid signal pathways as well as cell elongation. Among the GO enrichment terms, we found many differentially expressed genes (DEGs) enriched in the cell membrane and ribosomal biosynthesis, hormone biosynthesis and signaling pathways, and flavonoid biosynthesis, which could influence cell growth and the level of endogenous hormones affecting leaf angle. Therefore, ZmbHLH112 might regulate leaf angle development through the auxin signaling and the brassinosteroid biosynthesis pathways. 12 genes related to the development of leaf were screened by WGCNA; In GO enrichment and KEGG pathways, the genes were mainly enriched in rRNA binding, ribosome biogenesis, Structural constituent of ribosome; Arabidopsis ribosome RNA methyltransferase CMAL is involved in plant development, likely by modulating auxin derived signaling pathways; The free 60s ribosomes and polysomes in the functional defective mutant rice minute-like1 (rml1) were significantly reduced, resulting in plant phenotypic diminution, narrow leaves, and growth retardation; Hence, ribosomal subunits may play an important role in leaf development. These results provide a foundation for further elucidation of the molecular mechanism of the regulation of leaf angle in maize.

Postnatal microcephaly and retinal involvement expand the phenotype of RPL10-related disorder.

Hemizygous missense variants in the RPL10 gene encoding a ribosomal unit are responsible for an X‐linked syndrome presenting with intellectual disability (ID), autism spectrum disorder, epilepsy, dysmorphic features, and multiple congenital anomalies. Among 15 individuals with RPL10‐related disorder reported so far, only one patient had retinitis pigmentosa and microcephaly was observed in approximately half of the cases. By exome sequencing, three Italian and one Spanish male children, from three independent families, were found to carry the same hemizygous novel missense variant p.(Arg32Leu) in RPL10, inherited by their unaffected mother in all cases. The variant, not reported in gnomAD, is located in the 28S rRNA binding region, affecting an evolutionary conserved residue and predicted to disrupt the salt‐bridge between Arg32 and Asp28. In addition to features consistent with RPL10‐related disorder, all four boys had retinal degeneration and postnatal microcephaly. Pathogenic variants in genes responsible for inherited retinal degenerations were ruled out in all the probands. A novel missense RPL10 variant was detected in four probands with a recurrent phenotype including ID, dysmorphic features, progressive postnatal microcephaly, and retinal anomalies. The presented individuals suggest that retinopathy and postnatal microcephaly are clinical clues of RPL10‐related disorder, and at least the retinal defect might be more specific for the p.(Arg32Leu) RPL10 variant, suggesting a specific genotype/phenotype correlation.

Synergistic interaction network between the snR30 RNP, Utp23, and ribosomal RNA during ribosome synthesis

ABSTRACT snR30/U17 is a highly conserved H/ACA RNA that is required for maturation of the small ribosomal subunit in eukaryotes. By base-pairing to the expansion segment 6 (ES6) of 18S ribosomal RNA (rRNA), the snR30 H/ACA Ribonucleoprotein (RNP) indirectly facilitates processing of the precursor rRNA (pre-rRNA) together with other proteins such as Utp23 and other RNAs acting as ribosome assembly factors. However, the details of the molecular interaction network of snR30 and its binding partners and how these interactions contribute to pre-rRNA processing remains unknown. Here, we report the in vitro reconstitution of a Saccharomyces cerevisiae snR30 RNP and quantitative characterization of the interactions of snR30, H/ACA proteins, the Utp23 protein and ES6 of the 18S rRNA. The snR30 RNA is bound tightly by both H/ACA proteins and Utp23. We dissected the importance of different 18S rRNA regions for snR30 RNP binding and demonstrated that the snR30 complex is tightly anchored on the pre-rRNA through base-pairing to ES6 whereas other reported rRNA binding sites do not contribute to the affinity of the snR30 RNP. On its own, the ribosome assembly factor Utp23 binds in a tight, but unspecific manner to RNA. However, in complex with the snR30 RNP, Utp23 increases the affinity of the RNP for rRNA revealing synergies between snR30 RNP and Utp23 which are enhancing specificity and affinity for rRNA, respectively. Together, these findings provide mechanistic insights how the snR30 RNP and Utp23 cooperate to interact tightly and specifically with rRNA during the early stages of ribosome biogenesis.

Ppan is essential for preimplantation development in mice†.

PETER PAN (PPAN), located to nucleoli and mitochondria, is a member of the Brix domain protein family, involved in rRNA processing through its rRNA binding motif and mitochondrial apoptosis by protecting mitochondria structure and suppressing basal autophagic flux. Ppan is important for cell proliferation and viability, and mutation of Ppan in Drosophila caused larval lethality and oogenesis failure. Yet, its role in mammalian reproduction remains unclear. In this study, we explored the function of Ppan in oocyte maturation and early embryogenesis using conditional knockout mouse model. Deficiency of maternal Ppan significantly downregulated the expression level of 5.8S rRNA, 18S rRNA, and 28S rRNA, though it had no effect on oocyte maturation or preimplantation embryo development. However, depletion of both maternal and zygotic Ppan blocked embryonic development at morula stage. Similar phenotype was obtained when only zygotic Ppan was depleted. We further identified no DNA binding activity of PPAN in mouse embryonic stem cells, and depletion of Ppan had minimum impact on transcriptome but decreased expression of 5.8S rRNA, 18S rRNA, and 28S rRNA nevertheless. Our findings demonstrate that Ppan is indispensable for early embryogenesis in mice.

Differential network analysis of bovine muscle reveals changes in gene coexpression patterns in response to changes in maternal nutrition

BackgroundCoexpression network analysis is a powerful tool to reveal transcriptional regulatory mechanisms, identify transcription factors, and discover gene functions. It can also be used to investigate changes in coexpression patterns in response to environmental insults or changes in experimental conditions. Maternal nutrition is considered a major intrauterine regulator of fetal developmental programming. The objective of this study was to investigate structural changes in gene coexpression networks in the muscle of bull beef calves gestated under diets with or without methionine supplementation. Both muscle transcriptome and methylome were evaluated using next generation sequencing.ResultsMaternal methionine supplementation significantly perturbed coexpression patterns in the offspring’s muscle. Indeed, we found that neither the connection strength nor the connectivity pattern of six modules (subnetworks) detected in the control diet were preserved in the methionine-rich diet. Functional characterization revealed that some of the unpreserved modules are implicated in myogenesis, adipogenesis, fibrogenesis, canonical Wnt/β-catenin pathway, ribosome structure, rRNA binding and processing, mitochondrial activities, ATP synthesis and NAD(P) H oxidoreductases, among other functions. The bisulfite sequencing analysis showed that nearly 2% of all evaluated cytosines were differentially methylated between maternal diets. Interestingly, there were significant differences in the levels of gene body DNA methylation between preserved and unpreserved modules.ConclusionsOverall, our findings provide evidence that maternal nutrition can significantly alter gene coexpression patterns in the offspring, and some of these perturbations are mediated by changes in DNA methylation.

The anti-MRSA compound 3-O-alpha-L-(2″,3″-di-p-coumaroyl)rhamnoside (KCR) inhibits protein synthesis in Staphylococcus aureus

Methicillin-resistant S aureus (MRSA) contributes to patient mortality and extended hospital stays. 3-O-alpha-L-(2″,3″-di-p-coumaroyl)rhamnoside (KCR) is a natural product antibiotic that is effective against MRSA but has no known mechanism of action (MOA). We used proteomics to identify the MOA for KCR.Methicillin sensitive S aureus and a mixture of four KCR stereoisomers were tested. A time-kill assay was used to choose a 4 h treatment using KCR at 5× its MIC for proteomic analysis. S aureus was treated in triplicate with KCR, oxacillin or vehicle and quantitative proteomic analysis was carried out using isobaric tags and mass spectrometry. 1190 proteins were identified and 552 were affected by KCR (q < 0.01). Ontology analysis identified 6 distinct translation-related categories that were affected by KCR (PIANO, 10% false-discovery rate) including structural constituent of ribosome, translation, rRNA binding, tRNA binding, tRNA processing and aminoacyl-tRNA ligase activity. Median fold changes (KCR vs Control) for small and large ribosomal components were 1.46 and 1.43 respectively. KCR inhibited the production of luciferase protein in an in vitro assay (IC50 39.6 μg/ml).Upregulation of translation-related proteins in response to KCR indicates that KCR acts to disrupt S aureus protein synthesis. This was confirmed with an in vitro transcription/translation assay. SignificanceMethicillin-resistant S aureus (MRSA) contributes to patient mortality and extended hospital stays. 3-O-alpha-L-(2″,3″-di-p-coumaroyl)rhamnoside (KCR) is a natural product antibiotic that is effective against MRSA but has no known mechanism of action (MOA). Using proteomic analysis we determined that KCR acts by inhibiting protein synthesis. KCR is an exciting novel antibiotic and this work represents an important step in its development towards clinical use.

Ribosomal RNA-Selective Light-Up Fluorescent Probe for Rapidly Imaging the Nucleolus in Live Cells.

RNA-based fluorescent probes are currently limited by their low selectivity toward RNA versus DNA, and low specificity to different RNA structures. Poor membrane permeability is another defect of existing fluorogenic RNA probes for intracellular imaging. In this work, a naphthalimide derivative, probe 1, was developed for the rapid and selective detection of intracellular rRNA (rRNA). Probe 1 exhibited a 32-fold fluorescent enhancement in response to rRNA binding and showed desirable selectivity for rRNA versus DNA and other nucleic acids in phosphate buffer at pH 7.2. Importantly, probe 1 displayed excellent permeability of the nucleolus, could be taken up in 1 min by four different cell lines, and may be the fastest nucleolus dye. The excellent selectivity of probe 1 toward rRNA is attributed to the specific interaction between the complicated 3D structures of rRNA, which was confirmed by quantum calculations using molecular docking simulations. An appropriate lipophilic balance in 1 with the hydrophilic amine group and hydrophobic naphthalimide, as well as its high water solubility, guarantees the high permeability of 1 in cell membranes and nucleolus pores, compared to other analogues (e.g., probes 2-8 in this work). Furthermore, enlarged confocal laser micro images of nucleoli and RNase digestion tests revealed that 1 remained highly selective toward rRNA, even for intracellular imaging. As a live cell probe, 1 also exhibited better photostability than the commercial RNA dye, SYTO RNA select.

Transcriptomic and proteomic host response to Aspergillus fumigatus conidia in an air-liquid interface model of human bronchial epithelium.

Aspergillus fumigatus (A. fumigatus) is a wide-spread fungus that is a potent allergen in hypersensitive individuals but also an opportunistic pathogen in immunocompromised patients. It reproduces asexually by releasing airborne conidiospores (conidia). Upon inhalation, fungal conidia are capable of reaching the airway epithelial cells (AECs) in bronchial and alveolar tissues. Previous studies have predominantly used submerged monolayer cultures for studying this host-pathogen interaction; however, these cultures do not recapitulate the mucocililary differentiation phenotype of the in vivo epithelium in the respiratory tract. Thus, the aim of this study was to use well-differentiated primary human bronchial epithelial cells (HBECs) grown at the air-liquid interface (ALI) to determine their transcriptomic and proteomic responses following interaction with A. fumigatus conidia. We visualized conidial interaction with HBECs using confocal laser scanning microscopy (CLSM), and applied NanoString nCounter and shotgun proteomics to assess gene expression changes in the human cells upon interaction with A. fumigatus conidia. Western blot analysis was used to assess the expression of top three differentially expressed proteins, CALR, SET and NUCB2. CLSM showed that, unlike submerged monolayer cultures, well-differentiated ALI cultures of primary HBECs were estimated to internalize less than 1% of bound conidia. Nevertheless, transcriptomic and proteomic analyses revealed numerous differentially expressed host genes; these were enriched for pathways including apoptosis/autophagy, translation, unfolded protein response and cell cycle (up-regulated); complement and coagulation pathways, iron homeostasis, nonsense mediated decay and rRNA binding (down-regulated). CALR and SET were confirmed to be up-regulated in ALI cultures of primary HBECs upon exposure to A. fumigatus via western blot analysis. Therefore, using transcriptomics and proteomics approaches, ALI models recapitulating the bronchial epithelial barrier in the conductive zone of the respiratory tract can provide novel insights to the molecular response of bronchial epithelial cells upon exposure to A. fumigatus conidia.

Tetracyclines Modify Translation by Targeting Key Human rRNA Substructures

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

Interaction of CacyBP/SIP with NPM1 and its influence on NPM1 localization and function in oxidative stress.

Calcyclin (S100A6) binding protein/Siah-1 interacting protein (CacyBP/SIP) is mainly a cytoplasmic protein; however, some literature data suggested its presence in the nucleus. In this work we examined more precisely the nuclear localization and function of CacyBP/SIP. By applying mass spectrometry, we have identified several nuclear proteins, among them is nucleophosmin (NPM1), that may interact with CacyBP/SIP. Subsequent assays revealed that CacyBP/SIP forms complexes with NPM1 in the cell and that the interaction between these two proteins is direct. Interestingly, although CacyBP/SIP exhibits phosphatase activity, we have found that its overexpression favors phosphorylation of NPM1 on S125. In turn, the RNA immunoprecipitation assay indicated that the altered CacyBP/SIP level has an impact on the amount of 28S and 18S rRNA bound to NPM1. The overexpression of CacyBP/SIP resulted in a significant increase in the binding of 28S and 18S rRNA to NPM1, whereas silencing of CacyBP/SIP expression decreased 28S rRNA binding and had no effect on the binding of 18S rRNA. Further studies have shown that under oxidative stress, CacyBP/SIP overexpression alters NPM1 distribution in cell nuclei. In addition, staining for a nucleolar marker, fibrillarin, revealed that CacyBP/SIP is indispensable for maintaining the nucleolar structure. These results are in agreement with data obtained by western blot analysis, which show that upon oxidative stress the NPM1 level decreases but that CacyBP/SIP overexpression counteracts the effect of stress. Altogether, our results show for the first time that CacyBP/SIP binds to and affects the properties of a nuclear protein, NPM1, and that it is indispensable for preserving the structure of nucleoli under oxidative stress.

Mitochondrial Dysfunctions Contribute to Hypertrophic Cardiomyopathy in Patient iPSC-Derived Cardiomyocytes with MT-RNR2 Mutation.

SummaryHypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young individuals. A potential role of mtDNA mutations in HCM is known. However, the underlying molecular mechanisms linking mtDNA mutations to HCM remain poorly understood due to lack of cell and animal models. Here, we generated induced pluripotent stem cell-derived cardiomyocytes (HCM-iPSC-CMs) from human patients in a maternally inherited HCM family who carry the m.2336T>C mutation in the mitochondrial 16S rRNA gene (MT-RNR2). The results showed that the m.2336T>C mutation resulted in mitochondrial dysfunctions and ultrastructure defects by decreasing the stability of 16S rRNA, which led to reduced levels of mitochondrial proteins. The ATP/ADP ratio and mitochondrial membrane potential were also reduced, thereby elevating the intracellular Ca2+ concentration, which was associated with numerous HCM-specific electrophysiological abnormalities. Our findings therefore provide an innovative insight into the pathogenesis of maternally inherited HCM.

Tetracyclines Modify Translation By Targeting Key Human rRNA Substructures

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in cell click selective crosslinking with RNA sequence profiling (icCL-Seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

ITRAQ-based proteomic analysis of the viable but nonculturable state of Vibrio parahaemolyticus ATCC 17802 induced by food preservative and low temperature

Vibrio parahaemolyticus is a significant food-borne pathogen which can cause serious acute gastroenteritis. The viable but nonculturable (VBNC) state is a survival strategy of some bacteria when they are exposed to environmental stresses. In this study, V. parahaemolyticus ATCC 17802 was induced into VBNC state by food preservative at low temperature and oligotrophic condition, and the aim of the study is to compare differentially expressed proteins (DEPs) between the VBNC state and the exponential phase of V. parahaemolyticus ATCC 17802 using isobaric tags for relative and absolute quantitation (iTRAQ) technique. Functional annotations were carried out using protein sequence similarity, GO and KEGG pathway analysis. The results showed that V. parahaemolyticus ATCC 17802 was induced into the VBNC state at 4°C in seawater containing 10 mmol/L potassium sorbate within 40 days. A total of 1139 proteins were identified,1088 proteins were quantified. Of the DEPs under the VBNC state, 36 were significantly down-regulated and 15 up-regulated. The remarkable down-regulated proteins were type III secretion host injection and negative regulator protein, polar flagellin protein, ribosomal proteins, multidrug efflux pump component MtrF, and other key components involved in metabolism. The notable up-regulated proteins were mainly focused on transporter proteins, outer membrane protein, ferritin and amino acid synthase. The majorities of the significant DEPs were associated with translation, structural constituent of ribosome, rRNA binding, siderophore transmembrane transporter activity, receptor activity, and bacterial-type flagellum organization. This study contributes to a better understanding of the adaptation mechanism of the VBNC state of V. parahaemolyticus under food processing and environmental stresses.

Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP).

RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains’ interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

When a ribosomal protein grows up - the ribosome assembly path of Rps3.

The biogenesis of ribosomes is a central process in all dividing cells. Eukaryotic ribosomes are composed of a large 60S and a small 40S subunit, each comprising a complex assembly of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). The synthesis of these constituents is spatially separated, with r-proteins being produced by translation in the cytoplasm, while rRNA is generated by transcription in the nucleus. Hence, the arrangement of r-proteins and rRNA into large ribonucleoprotein complexes requires dedicated mechanisms ensuring their encounter in the same compartment. To this end, r-proteins need to be safely delivered to the nucleus where they assemble with the rRNA. Beyond these initial challenges, the synthesis of ribosomes does not merely comprise the joining of r-proteins with rRNA, but occurs in a complex assembly line involving multiple maturation steps, including the processing and folding of rRNA. R-proteins usually have composite rRNA binding sites, with several different rRNA helices contributing to the full interaction. Not all of these interaction sites may already be accessible at the point when an r-protein is incorporated, necessitating that some of the r-protein-rRNA contacts are formed at later maturation stages. In our two recent studies, we investigated the ribosome assembly path of r-proteins in the yeast Saccharomyces cerevisiae using the small subunit r-protein S3 (Rps3) as a model. Our studies revealed intricate mechanisms to protect the protein, transport it into the nucleus, integrate it into pre-ribosomal precursor particles and promote its final stable association with 40S subunits.

Transcriptome changes of blue-green algae, Arthrospira sp. in response to sulfate stress

Arthrospira platensis, a protein-rich blue-green alga is utilized as feed supplements for human and animals, which primarily depend upon nutrients such as carbon, nitrogen, phosphorus and sulfur in the presence of sunlight for their growth and survival. Cyanobacteria depend on sulfur for the synthesis and modification of a variety of biomolecules but, the exact mechanism of sulfur metabolism and impact of sulfate deprivation on A. platensis remain unclear. In this study, we analysed the change in growth and difference in the expression of key molecules of A. platensis during sulfate deprivation by comparing the transcriptome profiles of A. platensis grown at normal and sulfur deprived culture conditions. Observations showed that sulfate stress affected the levels of pigments in spirulina with slightly reduced growth. Transcriptome profile showed that expression profiles of different genes involved in various sulfur-dependent pathways were altered due to sulfur depletion. Major genes that were down-regulated include iron-sulfur clusters biosynthesis due to the absence of sulfur. Expression of genes involved in pathways such as translation, amino acid biosynthesis, protein folding, rRNA binding, were down-regulated, whereas genes involved in carbohydrate metabolism, phosphor relay sensor kinase activity, integral components of membrane, plasma membrane, thylakoid membrane, metal ion binding and DNA repair, were up-regulated during sulfur stress. Genes involved in transcription were up-regulated and hence there is a higher number of transcripts in A. platensis grown in sulfur depleted state. However, genes involved in translation were down-regulated; thus, there was a reduction in total protein content. Based on this analysis, it could be concluded that A. platensis was able to sustain sulfate stress, by its ability to alter the expression of genes that are specifically involved in sulfur metabolism and various other sulfur-dependent pathways.