Production of Fully Capped mRNA for Transfection into Mammalian Cells: A Protocol for Enzymatic Degradation of Uncapped Transcripts After In Vitro mRNA Synthesis.

The functionality of messenger RNA, such as stability and translation, is determined by several elements. In Eukaryotes, the 5' end of the mRNA is modified to contain a 5' cap structure, the presence of which protects the mRNA from degradation by 5' to 3' exoribonucleases and promotes mRNA translation. The in vitro synthesis of RNA has recently attracted ample attention for its application as a source of therapeutic agents or research tools.

The strain-dependent cytostatic activity of Lactococcus lactis on CRC cell lines is mediated through the release of arginine deiminase.

Background: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics.

Inhibition of RNA Polymerase III Augments the Anti-Cancer Properties of TNFα.

Tumour necrosis factor alpha (TNFα) is a multifunctional cytokine that plays a pivotal role in apoptosis, cell survival, as well as in inflammation and immunity. Although named for its antitumor properties, TNFα also has tumour-promoting properties. TNFα is often present in large quantities in tumours, and cancer cells frequently acquire resistance to this cytokine.

MAP kinases are involved in RNA polymerase III regulation upon LPS treatment in macrophages.

Macrophages are transcriptionally highly dynamic cell type, rapidly adapting to a changing environment to execute innate immune functions. Activation of macrophages with lipopolysaccharides (LPS), a major component of the outer membrane of most Gram-negative bacteria, induces rapid transcriptional changes and within a few hours transcription of several hundred genes is altered. Within these genes are tRNAs, which are synthesised by RNA Polymerase (Pol) III, and whose expression is rapidly upregulated in response to LPS.

Molecular and Cellular Mechanisms Influenced by Postbiotics.

In recent years, commensal bacteria colonizing the human body have been recognized as important determinants of health and multiple pathologic conditions. Among the most extensively studied commensal bacteria are the gut microbiota, which perform a plethora of functions, including the synthesis of bioactive products, metabolism of dietary compounds, and immunomodulation, both through attenuation and immunostimulation. An imbalance in the microbiota population, i.

Inhibition of tRNA Gene Transcription by the Immunosuppressant Mycophenolic Acid.

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, a drug that is widely used for immunosuppression in organ transplantation and autoimmune diseases, as well as anticancer chemotherapy. It inhibits IMP dehydrogenase, a rate-limiting enzyme in synthesis of guanidine nucleotides. MPA treatment interferes with transcription elongation, resulting in a drastic reduction of pre-rRNA and pre-tRNA synthesis, the disruption of the nucleolus, and consequently cell cycle arrest.

Regulation of tRNA synthesis by the general transcription factors of RNA polymerase III - TFIIIB and TFIIIC, and by the MAF1 protein.

The synthesis of transfer RNA (tRNA) is directed by RNA polymerase III (Pol III) specialized in high-level transcription of short DNA templates. Pol III recruitment to tRNA genes is controlled by two general initiation factors, TFIIIB and TFIIIC. They are multi-protein complexes regulated at the level of expression of individual subunits, as well as through phosphorylation and interaction with partner proteins.

Involvement of RNA Polymerase III in Immune Responses.

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis.

Maf1, repressor of tRNA transcription, is involved in the control of gluconeogenetic genes in Saccharomyces cerevisiae.

Maf1 is a negative regulator of RNA polymerase III (Pol III) in yeast. Maf1-depleted cells manifest elevated tRNA transcription and inability to grow on non-fermentable carbon source, such as glycerol. Using genomic microarray approach, we examined the effect of Maf1 deletion on expression of Pol II-transcribed genes in yeast grown in medium containing glycerol.

RNA polymerase III under control: repression and de-repression.

The synthesis of tRNA by yeast RNA polymerase III (Pol III) is regulated in response to changing environmental conditions. This control is mediated by Maf1, the global negative regulator of Pol III transcription conserved from yeast to humans. Details regarding the molecular basis of Pol III repression by Maf1 are now emerging from recently reported structural and biochemical data on Pol III and Maf1.

Casein kinase II-mediated phosphorylation of general repressor Maf1 triggers RNA polymerase III activation.

Maf1 protein is a global negative regulator of RNA polymerase (Pol) III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2.

Yeast prion [PSI+] lowers the levels of mitochondrial prohibitins.

We report proteomic analyses that establish the effect of cytoplasmic prion [PSI(+)] on the protein complement of yeast mitochondria. A set of 44 yeast mitochondrial proteins whose levels were affected by [PSI(+)] was identified by two methods of gel-free and label-free differential proteomics. From this set we focused on prohibitins, Phb1 and Phb2, and the mitochondrially synthesized Cox2 subunit of cytochrome oxidase.

Derepression of RNA polymerase III transcription by phosphorylation and nuclear export of its negative regulator, Maf1.

Maf1 is the global repressor of RNA polymerase III (Pol III) in yeast Saccharomyces cerevisiae. Transcription regulation by Maf1 is important under stress conditions and during the switch between fermentation and respiration. Under repressive conditions on nonfermentable carbon sources, Maf1 is dephosphorylated and located predominantly in the nucleus.

Maf1 is involved in coupling carbon metabolism to RNA polymerase III transcription.

RNA polymerase III (Pol III) produces essential components of the biosynthetic machinery, and therefore its activity is tightly coupled with cell growth and metabolism. In the yeast Saccharomyces cerevisiae, Maf1 is the only known global and direct Pol III transcription repressor which mediates numerous stress signals. Here we demonstrate that transcription regulation by Maf1 is not limited to stress but is important for the switch between fermentation and respiration.