Genetic interaction between RLM1 and F-box motif encoding gene SAF1 contributes to stress response in Saccharomyces cerevisiae

Background Stress response is mediated by the transcription of stress-responsive genes. The F-box motif protein Saf1p is involved in SCF-E3 ligase mediated degradation of the adenine deaminase, Aah1p upon nutrient stress. The four transcription regulators, BUR6, MED6, SPT10, SUA7, are listed for SAF1 in the genome database of Saccharomyces cerevisiae. Here in this study, we carried out an in-silico analysis of gene expression and transcription factor databases to understand the regulation of SAF1 expression during stress for hypothesis and experimental analysis. Result An analysis of the GEO profile database indicated an increase in SAF1 expression when cells were treated with stress agents such as Clioquinol, Pterostilbene, Gentamicin, Hypoxia, Genotoxic, desiccation, and heat. The increase in expression of SAF1 during stress conditions correlated positively with the expression of RLM1, encoding the Rlm1p transcription factor. The expression of AAH1 encoding Aah1p, a Saf1p substrate for ubiquitination, appeared to be negatively correlated with the expression of RLM1 as revealed by an analysis of the Yeastract expression database. Based on analysis of expression profile and regulatory association of SAF1 and RLM1, we hypothesized that inactivation of both the genes together may contribute to stress tolerance. The experimental analysis of cellular growth response of cells lacking both SAF1 and RLM1 to selected stress agents such as cell wall and osmo-stressors, by spot assay indicated stress tolerance phenotype similar to parental strain however sensitivity to genotoxic and microtubule depolymerizing stress agents. Conclusions Based on in-silico and experimental data we suggest that SAF1 and RLM1 both interact genetically in differential response to genotoxic and general stressors.

Developing PspCas13b-based enhanced RESCUE system, eRESCUE, with efficient RNA base editing

RNA base editing is potential for cellular function research and genetic diseases treating. There are two main RNA base editors, REPAIR and RESCUE, for in vitro use. REPAIR was developed by fusing inactivated Cas13 (dCas13) with the adenine deaminase domain of ADAR2, which efficiently performs adenosine-to-inosine (A-to-I) RNA editing. RESCUE, which performs both cytidine-to-uridine (C-to-U) and A-to-I RNA editing, was developed by fusing inactivated Cas13 (dCas13) with the evolved ADAR2. However, the relatively low editing efficiency of the RESCUE system limits its broad application. Here, we constructed an enhanced RESCUE (eRESCUE) system; this dPspCas13b-RESCUE-NES system was generated by fusing inactivated PspCas13b with the evolved ADAR2. We determined the endogenous mRNA A-to-I and C-to-U editing efficiency mediated by the dPspCas13b-RESCUE-NES system in HEK-293T cells. This new RNA base editor was then used to induce 177Ser/Gly conversion of inhibitor kappa B kinase β (IKKβ) by changing the genetic code from AGU to GGU. The results showed that the eRESCUE editor mediates more efficient A-to-I and C-to-U RNA editing than the RESCUE RNA editor, as was previously reported. The 177Ser/Gly conversion of IKKβ, accomplished by converting the genetic code from AGU to GGU, resulted in a decrease in the phosphorylation of IKKβ and downregulation of downstream IKKβ-related genes. In summary, we developed a more efficient RNA base editor, eRESCUE, which may provide a useful tool for biomedical research and genetic disease treatment.

Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing

Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.

Co-administration of HAART and antikoch triggers cardiometabolic dysfunction through an oxidative stress-mediated pathway

Background Antikoch and highly active anti-retroviral therapy are effective drugs in the management of tuberculosis and Human Immunodeficiency Virus, respectively. However, these cocktails have been independently associated with the aetiopathogenesis of metabolic syndrome. This study investigated whether or not the co-administration of antikoch and anti-retroviral, as seen in tuberculosis/Human Immunodeficiency Virus co-infection, will produce a similar effect. Also, it evaluated the role of glutathione and adenine deaminase/xanthine oxidase/uric acid signaling in antikoch/anti-retroviral-induced cardiometabolic dysfunction. Methods Male rats of Wistar strain were randomized into four groups: the control, which had 0.5 mL of distilled water as a vehicle, anti-Koch-treated rats that were administered a cocktail of anti-Koch, HAART-treated rats that had a combination of anti-retroviral drugs, and anti-Koch + HAART-treated rats that had treatments as anti-Koch-treated and HAART-treated rats. The treatment was once daily and lasted for eight weeks. One way-analysis of variance followed by Tukey’s posthoc test was used to test for significance and pairwise comparisons respectively. Results Although no changes in body weight gain and cardiac weight were noted, it was found that antikoch and/or HAART caused insulin resistance and elevated blood glucose level. In addition, antikoch and/or HAART led to dyslipidaemia, increased atherogenic indices, and elevated cardiac injury markers. These were accompanied by increased plasma and cardiac concentrations of malondialdehyde and nitric oxide, C-reactive protein, and myeloperoxidase activity, as well as suppressed activities of glutathione peroxidase and glutathione-S-transferase, and a fall in reduced glutathione level. The observed alterations were more pronounced in animals that received a combination of antikoch and HAART. Conclusions This study provides the first evidence that antikoch and/or HAART induce cardiometabolic dysfunction via glutathione suppression and up-regulation of adenine deaminase/xanthine oxidase/uric acid-dependent oxidative stress and inflammatory response. These events were associated with dyslipidaemia and increased atherogenic indices. This infers that regular monitoring of glucose level, insulin sensitivity, lipid profile, and oxido-inflammatory markers is important in patients on antikoch and/or HAART for prompt diagnosis and management of cardiometabolic disorder if it ensues.

Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti

Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia.

Programmable adenine deamination in bacteria using a Cas9–adenine-deaminase fusion

We report a pABE system which enables highly efficient adenine to guanine conversion in bacteria. Key residues of a staphylopine/metal complex transporter cntBC were systematically screened via the pABE system.

Genetic Interaction between RLM1 and F-box Motif Encoding gene SAF1 Contributes to Stress Response in Saccharomyces cerevisiae

Stress response is mediated by transcription of stress responsive genes. F-box motif protein Saf1 involves in SCF-E3 ligase mediated degradation of the adenine deaminase, Aah1 upon nutrient stress. Four transcription regulators, BUR6, MED6, SPT10, SUA7, have been reported for SAF1 gene in genome database of Saccharomyces cerevisiae. Here in this study an in-silco analysis of gene expression and transcription factor databases was carried out to understand the regulation of SAF1 gene expression during stress for hypothesis generation and experimental analysis. The GEO profile database analysis showed increased expression of SAF1 gene when treated with clioquinol, pterostilbene, gentamicin, hypoxia, genotoxic, desiccation, and heat stress, in WT cells. SAF1 gene expression in stress conditions correlated positively whereas AAH1 expression negatively with RLM1 transcription factor, which was not reported earlier. Based on analysis of expression profile and regulatory association of SAF1 and RLM1, we hypothesized that inactivation of both the genes may contribute to stress tolerance. The experimental analysis with the double mutant, saf1Δrlm1Δ for cellular growth response to stress causing agents, showed tolerance to calcofluor white, SDS, and hydrogen peroxide. On the contrary, saf1Δrlm1Δ showed sensitivity to MMS, HU, DMSO, Nocodazole, Benomyl stress. Based on in-silico and experimental data we suggest that SAF1 and RLM1 both interact genetically in differential response to genotoxic and general stressors.

A cytosine deaminase for programmable single-base RNA editing

Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive β-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.

Absence of Replication fork associated factor CTF4 and F-box motif Encoding Gene SAF1 leads to reduction in Cell Size and Stress Tolerance Phenotype in S. cerevisiae

Chromosome transmission fidelity factor, Ctf4 in S. cerevisiae associates with replication fork and helps in the sister chromatid cohesion. At the replication fork, Ctf4 links DNA helicase with the DNA polymerase. The absence of Ctf4 invokes replication checkpoint in the cells. The Saf1 of S.cerevisiae interacts with Skp1 of SCF-E3 ligase though F box-motif and ubiquitinates the adenine deaminase Aah1 during phase transition due to nutrient stress. The genetic interaction between the CTF4 and SAF1 has not been studied. Here we report genetic interaction between CTF4 and SAF1 which impacts the growth fitness and response to stress. The single and double gene deletions of SAF1 and CTF4 were constructed in the BY4741 genetic background. The strains were tested for growth on rich media and media containing stress causing agents. The saf1Δctf4Δ cells with reduced cell size showed the fastest growth phenotype on YPD medium when compared with the saf1Δ, ctf4Δ, and WT. The saf1Δctf4Δ cells also showed the tolerance to MMS, NaCl, Glycerol, SDS, Calcofluor white, H2O2, DMSO, Benomyl, and Nocodazole when compared with the saf1Δ, ctf4Δ, and WT cells. However, saf1Δctf4Δ cells showed the sensitivity to HU when compared with WT and saf1Δ. Based on these observations we suggest that SAF1 and CTF4 interact genetically to regulate the cell size, growth and stress response.