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Multiple-respiratory-chain deficiency represents an important cause of mitochondrial disorders. Hitherto, however, mutations in genes involved in mtDNA maintenance and translation machinery only account for a fraction of cases. Exome sequencing in two siblings, born to consanguineous parents, with severe encephalomyopathy, choreoathetotic movements, and combined respiratory-chain defects allowed us to identify a homozygous PNPT1 missense mutation (c.1160A>G) that encodes the mitochondrial polynucleotide phosphorylase (PNPase). Blue-native polyacrylamide gel electrophoresis showed that no PNPase complex could be detected in subject fibroblasts, confirming that the substitution encoded by c.1160A>G disrupts the trimerization of the protein. PNPase is predominantly localized in the mitochondrial intermembrane space and is implicated in RNA targeting to human mitochondria. Mammalian mitochondria import several small noncoding nuclear RNAs (5S rRNA, MRP RNA, some tRNAs, and miRNAs). By RNA hybridization experiments, we observed a significant decrease in 5S rRNA and MRP-related RNA import into mitochondria in fibroblasts of affected subject 1. Moreover, we found a reproducible decrease in the rate of mitochondrial translation in her fibroblasts. Finally, overexpression of the wild-type PNPT1 cDNA in fibroblasts of subject 1 induced an increase in 5S rRNA import in mitochondria and rescued the mitochondrial-translation deficiency. In conclusion, we report here abnormal RNA import into mitochondria as a cause of respiratory-chain deficiency.
The Polyribonucleotide nucleotidyltransferase 1 gene (PNPT1) encodes polynucleotide phosphorylase (PNPase), a 3'-5' exoribonuclease involved in mitochondrial RNA degradation and surveillance and RNA import into the mitochondrion. Here, we have characterized the PNPT1 promoter by in silico analysis, luciferase reporter assays, electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), siRNA-based mRNA silencing and RT-qPCR. We show that the Specificity protein 1 (SP1) transcription factor and Nuclear transcription factor Y (NFY) bind the PNPT1 promoter, and have a relevant role regulating the promoter activity, PNPT1 expression, and mitochondrial activity. We also found in Kaplan-Meier survival curves that a high expression of either PNPase, SP1 or NFY subunit A (NFYA) is associated with a poor prognosis in liver cancer. In summary, our results show the relevance of SP1 and NFY in PNPT1 expression, and point to SP1/NFY and PNPase as possible targets in anti-cancer therapy.
Extracellular microRNAs (miRNAs) play a critical role in horizontal gene regulation. Uptake of extracellular miRNAs by recipient cells and their intracellular transport, however, remains elusive. Here RNA phase separation is shown as a novel pathway of miRNA uptake. In the presence of serum, synthetic miRNAs rapidly self-assembly into ≈110 nm discrete nanoparticles, which enable miRNAs' entry into different cells. Depleting serum cationic proteins prevents the formation of such nanoparticles and thus blocks miRNA uptake. Different from lipofectamine-mediated miRNA transfection in which majority of miRNAs are accumulated in lysosomes of transfected cells, nanoparticles-mediated miRNA uptake predominantly delivers miRNAs into mitochondria in a polyribonucleotide nucleotidyltransferase 1(PNPT1)-dependent manner. Functional assays further show that the internalized miR-21 via miRNA phase separation enhances mitochondrial translation of cytochrome b (CYB), leading to increase in adenosine triphosphate (ATP) and reactive oxygen species (ROS) reduction in HEK293T cells. The findings thus reveal a previously unrecognized mechanism for uptake and delivery functional extracellular miRNAs into mitochondria.
Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.
Methylation of miRNAs at the 2'-hydroxyl group on the ribose at 3'-end (2'-O-methylation, 2'Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC-MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3'-terminal 2'Ome. Predominant 3'-terminal 2'Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/β-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3'-terminal 2'Ome. We further identify HENMT1 as the methyltransferase responsible for 3'-terminal 2'Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3'→5' exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3'-terminal 2'Ome of mammalian miRNAs and highlight its role in enhancing miRNA's stability and function.
Intervertebral disc (IVD) degeneration is a complicated physiological change involving cellular senescence, inflammation and the degradation of the extracellular matrix. Long non‑coding RNAs (lncRNAs) have been identified as new players in IVD degeneration. The present study aimed to identify lncRNAs implicated in IVD degeneration via the regulation of cellular senescence. In the present study, nucleus pulposus (NP) cells isolated from moderately degenerated IVD tissues exhibited a senescent phenotype with increased senescence rates, detected by senescence‑associated β‑galactosidase (SA‑β‑gal) staining, and reduced growth and migratory abilities. Microarray and target prediction analyses identified 353 differentially expressed lncRNAs, and 251 cis‑ and 2,170 trans‑acting targets in degenerated NP cells. Bioinformatic analyses revealed that these predicted targets were enriched in the regulation of response to DNA damage stimulus, positive regulation of cell cycle processes and interferon‑β production. In addition, a network of the top 10 upregulated and top 10 downregulated lncRNA targets was constructed, and two trans‑acting targets, C‑C motif chemokine ligand 5 (CCL5) and polyribonucleotide nucleotidyltransferase 1 (PNPT1) involved in aging or senescence, and their corresponding lncRNAs, lnc‑ST8SIA5‑1:2 and lnc‑HRK‑2:1, were identified. Reverse transcription‑quantitative PCR validation demonstrated that the two targets and two candidate lncRNAs were significantly upregulated in degenerated NP cells. Overexpression of lnc‑HRK‑2:1, with validated higher expression levels, in normal NP cells induced a senescent phenotype, with enhanced rates of senescence detected by SA‑β‑gal staining in cells, decreased growth and migratory abilities and improved expression levels of CCL5 and PNPT1. Collectively, these results suggested that upregulation of lnc‑HRK‑2:1 prompted NP cell senescence in IVD degeneration, which may be associated with increased expression levels of CCL5 and PNPT1.
Daptomycin is an extensively used anti-staphylococcal agent due to the rise in methicillin-resistant Staphylococcus aureus, but the mechanism(s) of resistance is poorly understood. Comparative genome sequencing, transcriptomics, ultrastructure, and cell envelope studies were carried out on two relatively higher level (4 and 8 µg/ml(-1)) laboratory-derived daptomycin-resistant strains (strains CB1541 and CB1540 respectively) compared to their parent strain (CB1118; MW2). Several mutations were found in the strains. Both strains had the same mutations in the two-component system genes walK and agrA. In strain CB1540 mutations were also detected in the ribose phosphate pyrophosphokinase (prs) and polyribonucleotide nucleotidyltransferase genes (pnpA), a hypothetical protein gene, and in an intergenic region. In strain CB1541 there were mutations in clpP, an ATP-dependent protease, and two different hypothetical protein genes. The strain CB1540 transcriptome was characterized by upregulation of cap (capsule) operon genes, genes involved in the accumulation of the compatible solute glycine betaine, ure genes of the urease operon, and mscL encoding a mechanosensitive chanel. Downregulated genes included smpB, femAB and femH involved in the formation of the pentaglycine interpeptide bridge, genes involved in protein synthesis and fermentation, and spa encoding protein A. Genes altered in their expression common to both transcriptomes included some involved in glycine betaine accumulation, mscL, ure genes, femH, spa and smpB. However, the CB1541 transcriptome was further characterized by upregulation of various heat shock chaperone and protease genes, consistent with a mutation in clpP, and lytM and sceD. Both strains showed slow growth, and strongly decreased autolytic activity that appeared to be mainly due to decreased autolysin production. In contrast to previous common findings, we did not find any mutations in phospholipid biosynthesis genes, and it appears there are multiple pathways to and factors in daptomycin resistance.
RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category 'RNA-binding', have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.
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