Padi6 expression patterns in buffalo oocytes and preimplantation embryos.

The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the activation of the zygote genome. One such gene, known as peptidyl-arginine deiminase VI (Padi6), is involved in the oocyte maturation, fertilization and embryonic development. However, the precise function of Padi6 gene in buffalo is still unclear and requires further investigation. In this study, the sequence, mRNA and protein expression patterns of Padi6 gene were analyzed in oocytes, preimplantation embryos and somatic tissues of buffalo. The coding sequence of gene was successfully cloned and characterized. Real-time quantitative PCR results indicated an absence of Padi6 transcripts in somatic tissues. Notably, the expression levels of Padi6 in oocytes showed an increased from the germinal vesicle stage to metaphase II stage, followed by a rapid decrease during the morula and blastocyst stages. Immunofluorescence analysis confirmed these findings, revealing a noticeable decline in protein expression levels. Our research provides the initial comprehensive expression profile of Padi6 in buffalo oocytes and preimplantation embryos, serving as a solid foundation for further investigations into the functionality of maternal-effect genes in buffalo.

Identification of Hammerhead-variant ribozyme sequences in SARS-CoV-2.

The SARS-CoV-2 RNA virus and variants, responsible for the COVID-19 pandemic has become endemic, raised a need for further understanding of the viral genome and biology. Despite vast research on SARS-CoV-2, no ribozymes have been found in the virus genome. Here we report the identification of 39 Hammerhead-variant ribozyme sequences (CoV-HHRz) in SARS-CoV-2. These sequences are highly conserved within SARS-CoV-2 variants but show large diversity among other coronaviruses. In vitro CoV-HHRz sequences possess the characteristics of typical ribozymes; cleavage is pH and ion dependent, although their activity is relatively low and Mn2+ is required for cleavage. The cleavage sites of four CoV-HHRz coincide with the breakpoint of expressed subgenomic RNA (sgRNAs) in SARS-CoV-2 transcriptome data suggesting in vivo activity. The CoV-HHRz are involved in processing sgRNAs for ORF7b, ORF 10 and ORF1ab nsp13 which are essential for viral packaging and life cycle.

Genome-wide identification of Kanamycin B binding RNA in Escherichia coli.

BACKGROUND: The aminoglycosides are established antibiotics that inhibit bacterial protein synthesis by binding to ribosomal RNA. Additional non-antibiotic aminoglycoside cellular functions have also been identified through aminoglycoside interactions with cellular RNAs. The full extent, however, of genome-wide aminoglycoside RNA interactions in Escherichia coli has not been determined. Here, we report genome-wide identification and verification of the aminoglycoside Kanamycin B binding to Escherichia coli RNAs. Immobilized Kanamycin B beads in pull-down assays were used for transcriptome-profiling analysis (RNA-seq). RESULTS: Over two hundred Kanamycin B binding RNAs were identified. Functional classification analysis of the RNA sequence related genes revealed a wide range of cellular functions. Small RNA fragments (ncRNA, tRNA and rRNA) or small mRNA was used to verify the binding with Kanamycin B in vitro. Kanamycin B and ibsC mRNA was analysed by chemical probing. CONCLUSIONS: The results will provide biochemical evidence and understanding of potential extra-antibiotic cellular functions of aminoglycosides in Escherichia coli.

Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 µM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5' UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.

Nlrp5 and Tle6 expression patterns in buffalo oocytes and pre-implantation embryos.

Maternal-effect genes (MEGs) accumulate in oocytes during oogenesis and mediate the pre-implantation embryo developmental programme until activation of the zygote genome. Nlrp5 and Tle6 are required for normal pre-implantation and embryonic development. However, the precise function of these MEGs in buffalo (Bubalus bubalis) remains to be elucidated. The aim of this study was to characterize Nlrp5 and Tle6 sequences and analyse their mRNA and protein expression patterns in somatic tissues, oocytes and pre-implantation embryos of buffalo. The coding sequences of each gene were successfully cloned and characterized. Real-time quantitative reverse transcription PCR results revealed an absence of Nlrp5 or Tle6 transcripts in somatic tissues, with the exception of ovary. Expression levels of Nlrp5 and Tle6 in oocytes increased from the germinal vesicle stage to metaphase II stage and then gradually decreased during morula and blastocyst stages. Protein expression patterns were confirmed by immunofluorescence analysis. This study lays a foundation for further validation of the function of MEGs in buffalo.

The function of twister ribozyme variants in non-LTR retrotransposition in Schistosoma mansoni.

The twister ribozyme is widely distributed over numerous organisms and is especially abundant in Schistosoma mansoni, but has no confirmed biological function. Of the 17 non-LTR retrotransposons known in S. mansoni, none have thus far been associated with ribozymes. Here we report the identification of novel twister variant (T-variant) ribozymes and their function in S. mansoni non-LTR retrotransposition. We show that T-variant ribozymes are located at the 5' end of Perere-3 non-LTR retrotransposons in the S. mansoni genome. T-variant ribozymes were demonstrated to be catalytically active in vitro. In reporter constructs, T-variants were shown to cleave in vivo, and cleavage of T-variants was sufficient for the translation of downstream reporter genes. Our analysis shows that the T-variants and Perere-3 are transcribed together. Target site duplications (TSDs); markers of target-primed reverse transcription (TPRT) and footmarks of retrotransposition, are located adjacent to the T-variant cleavage site and suggest that T-variant cleavage has taken place inS. mansoni. Sequence heterogeneity in the TSDs indicates that Perere-3 retrotransposition is not site-specific. The TSD sequences contribute to the 5' end of the terminal ribozyme helix (P1 stem). Based on these results we conclude that T-variants have a functional role in Perere-3 retrotransposition.

Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage.

Interactions between aminoglycoside antibiotics and the twister ribozyme were investigated in this study. An initial screen of 17 RNA-binding antibiotics showed that a number of aminoglycosides inhibit the ribozyme, while a subset of aminoglycosides enhances twister cleavage. Initial kinetic analysis of the twister ribozyme showed a sevenfold inhibition of ribozyme cleavage by paromomycin and a fivefold enhancement of cleavage by sisomicin. Direct binding between the twister ribozyme RNA and paromomycin or sisomicin was measured by microscale thermophoresis. Selective 2'-hydroxyl acylation analysed by primer extension shows that both paromomycin and sisomicin induce distinctive tertiary structure changes to the twister ribozyme. Published crystal structures and mechanistic analysis of the twister ribozyme have deduced a nucleobase-mediated general acid-base catalytic mechanism, in which a conserved guanine plays a key role. Here, we show that paromomycin binding induces a structural transition to the twister ribozyme such that a highly conserved guanine in the active site becomes displaced, leading to inhibition of cleavage. In contrast, sisomicin binding appears to change interactions between P3 and L2, inducing allosteric changes to the active site that enhance twister RNA cleavage. Therefore, we show that small-molecule binding can modulate twister ribozyme activity. These results suggest that aminoglycosides may be used as molecular tools to study this widely distributed ribozyme.

Proteomic analysis demonstrates that parthenogenetically activated swamp buffalo embryos have dysregulated energy metabolism.

The comprehensive understanding of early embryo development is essential to optimize in vitro culture conditions. Protein expression landscape of parthenogenetically produced embryo remains unexplored. This study aimed to investigate the protein expression dynamics with a particular focus on energy metabolism throughout the early developmental stages of parthenogenetic buffalo embryos. For this purpose, we performed iTRAQ-based quantitative mass spectrometry and identified 280 proteins common in all stages. A total of 933 proteins were identified during the proteomics analysis. The data depicted that morula and blastocyst had distinct protein expression dynamics as compared to 2- to 16-cell-stage embryo. KEGG pathway analysis showed 23 proteins belonging to energy metabolism appeared in the data. Study of energy metabolism-related protein's expression pattern demonstrated that there was asynchrony in proteins related to glycolysis throughout the examined developmental stages. The expression pattern of pyruvate kinase mutase (PKM), an essential protein of glycolysis, indicated a slightly decreasing trend from 2-cell-stage embryo to blastocyst, and it was supported by expression of proteins involved in lactate production (LDHA and LDHB) suggesting the decreasing rate of aerobic glycolysis (Warburg Effect) at morula and blastocyst stage. The increased Warburg Effect is considered as the hallmark of proliferating cells or embryo at the blastocyst stage. Furthermore, the proteins involved in the citric acid cycle also showed down-regulation at the blastocyst stage, indicating a lesser role of oxidative phosphorylation at this stage. Therefore, it could be divulged from the study that there may be an irregular pattern of energy metabolism in early parthenogenetic embryos. Further studies are recommended to understand this phenomenon.

Aminoglycoside riboswitch control of the expression of integron associated aminoglycoside resistance adenyltransferases.

The proliferation of antibiotic resistance has its origins in horizontal gene transfer. The class 1 integrons mediate gene transfer by assimilating antibiotic-resistance genes through site-specific recombination. For the class 1 integrons the first assimilated gene normally encodes an aminoglycoside antibiotic resistance protein which is either an aminoglycoside acetyltransferase (AAC), nucleotidyltransferase - (ANT), or adenyl transferase (AAD). An aminoglycoside-sensing riboswitch RNA in the leader RNA of AAC/AAD that controls the expression of aminoglycoside resistance genes has been previously described. Here we explore the relationship between the recombinant products of integron recombination and a series of candidate riboswitch RNAs in the 5' UTR of aad (aminoglycoside adenyltransferases) genes. The RNA sequences from the 5' UTR of the aad genes from pathogenic strains that are the products of site-specific DNA recombination by class 1 integrons were investigated. Reporter assays, MicroScale Thermophoresis (MST) and covariance analysis revealed that a functional aminoglycoside-sensing riboswitch was selected at the DNA level through integron-mediated site-specific recombination. This study explains the close association between integron recombination and the aminoglycoside-sensing riboswitch RNA.

Interactions between the 5' UTR mRNA of the spe2 gene and spermidine regulate translation in S. pombe.

The 5' untranslated regions (5' UTR) of mRNAs play an important role in the eukaryotic translation initiation process. Additional levels of translational regulation may be mediated through interactions between structured mRNAs that can adopt interchangeable secondary or tertiary structures and the regulatory protein/RNA factors or components of the translational apparatus. Here we report a regulatory function of the 5' UTR mRNA of the spe2 gene (SAM decarboxylase) in polyamine metabolism of the fission yeast Schizosaccharomyces pombe Reporter assays, biochemical experiments, and mutational analysis demonstrate that this 5' UTR mRNA of spe2 can bind to spermidine to regulate translation. A tertiary structure transition in the 5' UTR RNA upon spermidine binding is essential for translation regulation. This study provides biochemical evidence for spermidine binding to regulate translation of the spe2 gene through interactions with the 5' UTR mRNA. The identification of such a regulatory RNA that is directly associated with an essential eukaryotic metabolic process suggests that other ligand-binding RNAs may also contribute to eukaryotic gene regulation.

Interactions between SAM and the 5' UTR mRNA of the sam1 gene regulate translation in S. pombe.

The 5' untranslated region (5' UTR) of eukaryotic mRNA plays an important role in translation. Here we report the function of the 5' UTR mRNA of S-adenosylmethionine synthetase (sam1) in translational modulation in the presence of SAM in fission yeast Schizosaccharomyces pombe Reporter assays, binding and chemical probing experiments, and mutational analysis show that the 5' UTR mRNA of sam1 binds to SAM to effect translation. Translational modulation is dependent on a tertiary structure transition in the RNA upon SAM binding. The characterization of such an RNA that is directly associated with an essential metabolic process in eukaryotes provides additional evidence that ligand binding by RNAs plays an important role in eukaryotic gene regulation.

Identification of the Sex of Pre-implantation Mouse Embryos Using a Marked Y Chromosome and CRISPR/Cas9.

Although numerous attempts have been made to alter the sex ratio of the progeny of mammals, the limitations of current technologies have prevented their widespread use in farm animals. The presence or absence of a Y chromosome determines whether a mammalian embryo develops as a male or female, and non-invasive genetic reporters such as fluorescence protein markers have been intensively applied in a variety of fields of research. To develop a non-invasive and instantaneous method for advance determination of the sex of embryos, we developed a Y chromosome-linked eGFP mouse line that stably expresses green fluorescent protein under the control of the CAG promoter. The development of the CRISPR/Cas9 system has made it easy to deliver an exogenous gene to a specific locus of a genome, and linking a tracer to the Y chromosome has simplified the process of predicting the sex of embryos collected by mating a Y-Chr-eGFP transgenic male with a wild-type female. XY embryos appeared green, under a fluorescence microscope, and XX embryos did not. Y chromosome-linked genes were amplified by nested PCR to further confirm the accuracy of this method, and the simultaneous transplantation of green and non-green embryos into foster mothers indicated that 100% accuracy was achieved by this method. Thus, the Y-Chr-eGFP mouse line provides an expeditious and accurate approach for sexing pre-implantation embryos and can be efficiently used for the pre-selection of sex.

Integron-Derived Aminoglycoside-Sensing Riboswitches Control Aminoglycoside Acetyltransferase Resistance Gene Expression.

Class 1 integrons accumulate antibiotic resistance genes by site-specific recombination at aatI-1 sites. Captured genes are transcribed from a promoter located within the integron; for class 1 integrons, the first gene to be transcribed and translated normally encodes an aminoglycoside antibiotic resistance protein (either an acetyltransferase [AAC] or adenyltransferase [AAD]). The leader RNA from the Pseudomonas fluorescens class 1 integron contains an aminoglycoside-sensing riboswitch RNA that controls the expression of the downstream aminoglycoside resistance gene. Here, we explore the relationship between integron-dependent DNA recombination and potential aminoglycoside-sensing riboswitch products of recombination derived from a series of aminoglycoside-resistant clinical strains. Sequence analysis of the clinical strains identified a series of sequence variants that were associated with class I integron-derived aminoglycoside-resistant (both aac and aad) recombinants. For the aac recombinants, representative sequences showed up to 6-fold aminoglycoside-dependent regulation of reporter gene expression. Microscale thermophoresis (MST) confirmed RNA binding. Covariance analysis generated a secondary-structure model for the RNA that is an independent verification of previous models that were derived from mutagenesis and chemical probing data and that was similar to that of the P. fluorescens riboswitch RNA. The aminoglycosides were among the first antibiotics to be used clinically, and the data suggest that in an aminoglycoside-rich environment, functional riboswitch recombinants were selected during integron-mediated recombination to regulate aminoglycoside resistance. The incorporation of a functional aminoglycoside-sensing riboswitch by integron recombination confers a selective advantage for the expression of resistance genes of diverse origins.

WITHDRAWN: Comparative proteomic identification of capacitation and noncapacitation swamp buffalo spermatozoa.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Theriogenology, 126C (1 March 2019) 303-309, http://dx.doi.org/10.1016/j.theriogenology.2018.12.025. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Comparative proteomic identification buffalo spermatozoa during in vitro capacitation.

To investigate the proteomic profiling in buffalo spermatozoa before and after capacitation, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with Tandem Mass Tag (TMT) labeling strategy was applied. As a result, 1461 proteins were identified, 93 of them were found to be differentially expressed (>1.5-fold), including 52 up-regulated proteins and 41 down-regulated proteins during sperm capacitation. 88 out of 93 proteins were annotated and classified. Gene ontology (GO) analysis revealed that most of the differently expressed proteins (DEPs) were involved in the Biological Process of transport, cytoskeleton organization, sexual reproduction, and spermatogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that DEPs were mainly involved in the pathways of metabolic pathways, PPAR signaling pathway, and oxidative phosphorylation. Western blot (WB) assay confirmed the expressional variation of VAMP4 and APOC3 proteins. Our date provided a foundation for studying the changes in protein expression during sperm capacitation, which contributing to identifying marker proteins that may be associated with sperm capacitation.

Integrated Analysis of Quantitative Proteome and Transcriptional Profiles Reveals the Dynamic Function of Maternally Expressed Proteins After Parthenogenetic Activation of Buffalo Oocyte.

Maternal-effect genes are especially critical for early embryonic development after fertilization and until massive activation of the embryonic genome occurs. By applying a tandem mass tag (TMT)-labeled quantitative proteomics combined with RNA sequencing approach, the proteome of the buffalo was quantitatively analyzed during parthenogenesis of mature oocytes and the two-cell stage embryo. Of 1908 quantified proteins, 123 differed significantly. The transcriptome was analyzed eight stages (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst) of Buffalo using the RNA sequencing approach, and a total of 3567 unique genes were identified to be differently expressed between all consecutive stages of pre-implantation development. Validation of proteomics results (TUBB3, CTNNA1, CDH3, MAP2K1), which are involved in tight junction and gap junction, revealing that the maternal expression of the proteins possibly plays a role in the formation of cellular junctions firstly after parthenogenetic activation. Correlation and hierarchical analyses of transcriptional profiles and the expression of NPM2 and NLRP5 mRNA of buffalo in vitro developed oocytes and parthenogenetic embryos indicated that the "maternal-to-zygotic transition" (MZT) process might exist in the model of parthenogenesis, which is similar to a normally fertilized embryo, and may occur between the 8-cell to 16-cell stage. These data provide a rich resource for further studies on maternal proteins and genes and are conducive to improving nuclear transfer technology.

Osmium tetroxide as a probe of RNA structure.

Structured RNAs have a central role in cellular function. The capability of structured RNAs to adopt fixed architectural structures or undergo dynamic conformational changes contributes to their diverse role in the regulation of gene expression. Although numerous biophysical and biochemical tools have been developed to study structured RNAs, there is a continuing need for the development of new methods for the investigation of RNA structures, especially methods that allow RNA structure to be studied in solution close to its native cellular conditions. Here we use osmium tetroxide (OsO4) as a chemical probe of RNA structure. In this method, we have used fluorescence-based sequencing technologies to detect OsO4 modified RNA. We characterized the requirements for OsO4 modification of RNA by investigating three known structured RNAs: the M-box, glycine riboswitch RNAs, and tRNAasp Our results show that OsO4 predominantly modifies RNA at uracils that are conformationally exposed on the surface of the RNA. We also show that changes in OsO4 reactivity at flexible positions in the RNA correlate with ligand-driven conformational changes in the RNA structure. Osmium tetroxide modification of RNA will provide insights into the structural features of RNAs that are relevant to their underlying biological functions.

Substrate recognition and modification by the nosiheptide resistance methyltransferase.

BACKGROUND: The proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR) confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2'O-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR) in complex with the cofactor S-Adenosyl-L-methionine (SAM) are available, the principles behind NHR substrate recognition and catalysis remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA) and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066) and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5') to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation. CONCLUSION/SIGNIFICANCE: Taken together these data suggest that local base-base interactions play an important role in aligning the substrate 2' hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an increasingly important role in fundamental biological processes and we anticipate that the approach outlined in this manuscript may be useful for investigating other classes of nucleic acid methyltransferases.

An aminoglycoside sensing riboswitch controls the expression of aminoglycoside resistance acetyltransferase and adenyltransferases.

The emergence of antibiotic resistance in human pathogens is an increasing threat to public health. The fundamental mechanisms that control the high levels of expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are one of the earliest classes of antibiotics that were introduced in the 1940s. In the clinic aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug although resistance through enzymatic modification of the target rRNA through methylation or the overexpression of efflux pumps is also appearing. An aminoglycoside sensing riboswitch has been identified that controls expression of the aminoglycoside resistance genes that encode the aminoglycoside acetyltransferase (AAC) and aminoglycoside nucleotidyltransferase (ANT) (adenyltransferase (AAD)) enzymes. AAC and ANT cause resistance to aminoglycoside antibiotics through modification of the drugs. Expression of the AAC and ANT resistance genes is regulated by aminoglycoside binding to the 5' leader RNA of the aac/aad genes. The aminoglycoside sensing RNA is also associated with the integron cassette system that captures antibiotic resistance genes. Specific aminoglycoside binding to the leader RNA induces a structural transition in the leader RNA, and consequently induction of resistance protein expression. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrated that the leader RNA functioned as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycoside antibiotic resistance. This article is part of a Special Issue entitled: Riboswitches.