Kinetics of Translating Ribosomes Determine the Efficiency of Programmed Stop Codon Readthrough.

During translation, a stop codon on the mRNA signals the ribosomes to terminate the process. In certain mRNAs, the termination fails due to the recoding of the canonical stop codon, and ribosomes continue translation to generate C-terminally extended protein. This process, termed stop codon readthrough (SCR), regulates several cellular functions. SCR is driven by elements/factors that act immediately downstream of the stop codon. Here, we have analysed the process of SCR using a simple mathematical model to investigate how the kinetics of translating ribosomes influences the efficiency of SCR. Surprisingly, the analysis revealed that the rate of translation inversely regulates the efficiency of SCR. We tested this prediction experimentally in mammalian AGO1 and MTCH2 mRNAs. Reduction in translation either globally by harringtonine or locally by rare codons caused an increase in the efficiency of SCR. Thus, our study has revealed a hitherto unknown mode of regulation of SCR.

Involvement of epigenetics in affecting host immunity during SARS-CoV-2 infection.

Coronavirus disease 19 (COVID-19) is caused by a highly contagious RNA virus Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2), originated in December 2019 in Wuhan, China. Since then, it has become a global public health concern and leads the disease table with the highest mortality rate, highlighting the necessity for a thorough understanding of its biological properties. The intricate interaction between the virus and the host immune system gives rise to diverse implications of COVID-19. RNA viruses are known to hijack the host epigenetic mechanisms of immune cells to regulate antiviral defence. Epigenetics involves processes that alter gene expression without changing the DNA sequence, leading to heritable phenotypic changes. The epigenetic landscape consists of reversible modifications like chromatin remodelling, DNA/RNA methylation, and histone methylation/acetylation that regulates gene expression. The epigenetic machinery contributes to many aspects of SARS-CoV-2 pathogenesis, like global DNA methylation and receptor angiotensin-converting enzyme 2 (ACE2) methylation determines the viral entry inside the host, viral replication, and infection efficiency. Further, it is also reported to epigenetically regulate the expression of different host cytokines affecting antiviral response. The viral proteins of SARS-CoV-2 interact with various host epigenetic enzymes like histone deacetylases (HDACs) and bromodomain-containing proteins to antagonize cellular signalling. The central role of epigenetic factors in SARS-CoV-2 pathogenesis is now exploited as promising biomarkers and therapeutic targets against COVID-19. This review article highlights the ability of SARS-CoV-2 in regulating the host epigenetic landscape during infection leading to immune evasion. It also discusses the ongoing therapeutic approaches to curtail and control the viral outbreak.

ESAT-6 impedes IL-18 mediated phagosome lysosome fusion via microRNA-30a upon Calcimycin treatment in mycobacteria infected macrophages.

The weaponry possessed by Mycobacterium tuberculosis (M. tb) in the form of immunodominant antigens hijack the host immune system to give a survival advantage to this intracellular fiend, but the mechanism of this control is not entirely known. Since we have previously reported the mechanism of autophagy inhibition by early secreted antigenic target 6 kDa (ESAT-6) through microRNA (miR)-30a-3p in Calcimycin treated differentiated THP-1 (dTHP-1) cells, the present study was undertaken to deduce the effect of miR-30a on the immunomodulatory profile of ESAT-6 treated cells and the mechanism involved thereof, if any. Initially, the effect of recombinant ESAT-6 (rESAT-6) on the immunomodulatory profile in Calcimycin-treated phorbol 12-myristate 13-acetate (PMA) dTHP-1 cells was checked. Later, transfection studies using miR-30a-3p inhibitor or -5p mimic highlighted the contrary roles of different arms of the same miRNA in regulating IL-18 response by ESAT-6 in dTHP-1 cells after Calcimycin treatment. By using either IL-18 neutralizing antibody or inhibitors of phosphoinositide 3-kinase (PI3K)/NF-κB/phagosome-lysosome fusion in the miRNA-30a transfected background, IL-18 mediated signaling and intracellular killing of mycobacteria was reversed in the presence of ESAT-6. Overall, the results of this study conclusively prove the contrary roles of miR-30a-3p and miR-30a-5p in regulating IL-18 signaling by ESAT-6 in dTHP-1 cells upon Calcimycin treatment that affected phagosome-lysosome fusion and intracellular survival of mycobacteria.

ESAT-6 modulates Calcimycin-induced autophagy through microRNA-30a in mycobacteria infected macrophages.

OBJECTIVE: Mycobacterium tuberculosis (M. tb) has a sumptuous repertoire of effector molecules to counter host defenses. Some of these antigens inhibit autophagy but the exact mechanism of this inhibition is poorly understood. METHODS: Purified protein derivative (PPD) was fractionated using 10 (PPD 10, antigenic molecular weight > 10 kDa) and 3 (PPD 3, mol. weight > 3 kDa) kDa cutters. Effect of these fractions on Calcimycin-induced autophagy and intracellular mycobacterial viability was then studied using different experimental approaches. RESULT: We found significant downregulation of autophagy by PPD 3 pre-treatment in Calcimycin-treated dTHP-1 cells compared to PPD 10. This reduction in autophagy also corroborated with the enhanced survival of mycobacteria in macrophages. We demonstrate that recombinant early secreted antigenic target 6 (rESAT-6) is responsible to inhibit Calcimycin-induced autophagy and enhance intracellular survival of mycobacteria. We also show that pre-treatment with rESAT-6 upregulates microRNA (miR)-30a-3p expression and vis-a-vis downregulates miR-30a-5p expression in Calcimycin-treated dTHP-1 cells. Transfection studies with either miR-30a-3p inhibitor or miR-30a-5p mimic clearly elucidated the opposing roles of miR-30a-3p and miR-30a-5p in rESAT-6 mediated mycobacterial survival through autophagy inhibition. CONCLUSION: Taken together, our result evidently highlights that rESAT-6 enhances intracellular survival of mycobacteria by modulating miR-30a-3p and miR-30a-5p expression.

Structure-function and application of plant lectins in disease biology and immunity.

Lectins are proteins with a high degree of stereospecificity to recognize various sugar structures and form reversible linkages upon interaction with glyco-conjugate complexes. These are abundantly found in plants, animals and many other species and are known to agglutinate various blood groups of erythrocytes. Further, due to the unique carbohydrate recognition property, lectins have been extensively used in many biological functions that make use of protein-carbohydrate recognition like detection, isolation and characterization of glycoconjugates, histochemistry of cells and tissues, tumor cell recognition and many more. In this review, we have summarized the immunomodulatory effects of plant lectins and their effects against diseases, including antimicrobial action. We found that many plant lectins mediate its microbicidal activity by triggering host immune responses that result in the release of several cytokines followed by activation of effector mechanism. Moreover, certain lectins also enhance the phagocytic activity of macrophages during microbial infections. Lectins along with heat killed microbes can act as vaccine to provide long term protection from deadly microbes. Hence, lectin based therapy can be used as a better substitute to fight microbial diseases efficiently in future.