RNA degradation in human mitochondria: the journey is not finished.

Mitochondria are vital organelles present in almost all eukaryotic cells. Although most of the mitochondrial proteins are nuclear-encoded, mitochondria contain their own genome, whose proper expression is necessary for mitochondrial function. Transcription of the human mitochondrial genome results in the synthesis of long polycistronic transcripts that are subsequently processed by endonucleases to release individual RNA molecules, including precursors of sense protein-encoding mRNA (mt-mRNA) and a vast amount of antisense noncoding RNAs. Because of mitochondrial DNA (mtDNA) organization, the regulation of individual gene expression at the transcriptional level is limited. Although transcription of most protein-coding mitochondrial genes occurs with the same frequency, steady-state levels of mature transcripts are different. Therefore, post-transcriptional processes are important for regulating mt-mRNA levels. The mitochondrial degradosome is a complex composed of the RNA helicase SUV3 (also known as SUPV3L1) and polynucleotide phosphorylase (PNPase, PNPT1). It is the best-characterized RNA-degrading machinery in human mitochondria, which is primarily responsible for the decay of mitochondrial antisense RNA. The mechanism of mitochondrial sense RNA decay is less understood. This review aims to provide a general picture of mitochondrial genome expression, with a particular focus on mitochondrial RNA (mtRNA) degradation.

PKLR mutations in pyruvate kinase deficient Polish patients: Functional characteristics of c.101-1G > A and c.1058delAAG variants.

Pyruvate kinase (PK) deficiency is a rare autosomal recessive disorder characterized by chronic hemolytic anemia of variable severity. Nine Polish patients with severe hemolytic anemia but normal PK activity were found to carry mutations in the PKLR gene encoding PK, five already known ones and one novel (c.178C > T). We characterized two of the known variants by molecular modeling (c.1058delAAG) and minigene splicing analysis (c.101-1G > A). The former gives a partially destabilized PK tetramer, likely of suboptimal activity, and the c.101-1G > A variant gives alternatively spliced mRNA carrying a premature stop codon, encoding a severely truncated PK and likely undergoing nonsense-mediated decay.

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. RESULTS: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. CONCLUSION: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability.

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. Consequently, TNFα may increase the proliferation and metastatic potential of cancer cells. Furthermore, the TNFα-driven increase in metastasis is a result of the ability of this cytokine to induce the epithelial-to-mesenchymal transition (EMT). Overcoming the resistance of cancer cells to TNFα may have a potential therapeutic benefit. NF-κB is a crucial transcription factor mediating inflammatory signals and has a wide-ranging role in tumour progression. NF-κB is strongly activated in response to TNFα and contributes to cell survival and proliferation. The pro-inflammatory and pro-survival function of NF-κB can be disrupted by blocking macromolecule synthesis (transcription, translation). Consistently, inhibition of transcription or translation strongly sensitises cells to TNFα-induced cell death. RNA polymerase III (Pol III) synthesises several essential components of the protein biosynthetic machinery, such as tRNA, 5S rRNA, and 7SL RNA. No studies, however, directly explored the possibility that specific inhibition of Pol III activity sensitises cancer cells to TNFα. Here we show that in colorectal cancer cells, Pol III inhibition augments the cytotoxic and cytostatic effects of TNFα. Pol III inhibition enhances TNFα-induced apoptosis and also blocks TNFα-induced EMT. Concomitantly, we observe alterations in the levels of proteins related to proliferation, migration, and EMT. Finally, our data show that Pol III inhibition is associated with lower NF-κB activation upon TNFα treatment, thus potentially suggesting the mechanism of Pol III inhibition-driven sensitisation of cancer cells 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. However, the mechanisms that govern Pol III activation are not fully elucidated. LPS engage the Toll-like receptor (TLR) 4 and induce various signalling pathways, including mitogen-activated protein kinases (MAPK). MAPKs are serine/threonine kinases that catalyse the phosphorylation of transcription factors, protein kinases, and many other substrates including functional proteins, play a central role in mediating cellular responses to extracellular signals, including inflammatory cues. Here we show that ERK and p38 MAP kinases contribute to the activation of Pol III in macrophages stimulated with LPS. We also demonstrate that MAP kinases effector MSK1/2 kinases are involved in tRNA upregulation. Our data show that ERK, p38, and MSK kinases are required for upregulation of Pol III activity in macrophages stimulated by LPS. The possible modes of their action are discussed.

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 de novo 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. Here, we investigated the mechanism whereby MPA inhibits RNA polymerase III (Pol III) activity, in both yeast and mammalian cells. We show that MPA rapidly inhibits Pol III by depleting GTP. Although MPA treatment can activate p53, this is not required for Pol III transcriptional inhibition. The Pol III repressor MAF1 is also not responsible for inhibiting Pol III in response to MPA treatment. We show that upon MPA treatment, the levels of selected Pol III subunits decrease, but this is secondary to transcriptional inhibition. Chromatin immunoprecipitation (ChIP) experiments show that Pol III does not fully dissociate from tRNA genes in yeast treated with MPA, even though there is a sharp decrease in the levels of newly transcribed tRNAs. We propose that in yeast, GTP depletion may lead to Pol III stalling.