A Broad Response to Intracellular Long-Chain Polyphosphate in Human Cells.

Polyphosphates (polyPs) are long chains of inorganic phosphates linked by phosphoanhydride bonds. They are found in all kingdoms of life, playing roles in cell growth, infection, and blood coagulation. Unlike in bacteria and lower eukaryotes, the mammalian enzymes responsible for polyP metabolism are largely unexplored. We use RNA sequencing (RNA-seq) and mass spectrometry to define a broad impact of polyP produced inside of mammalian cells via ectopic expression of the E. coli polyP synthetase PPK. We find that multiple cellular compartments can support accumulation of polyP to high levels. Overproduction of polyP is associated with reprogramming of both the transcriptome and proteome, including activation of the ERK1/2-EGR1 signaling axis. Finally, fractionation analysis shows that polyP accumulation results in relocalization of nuclear/cytoskeleton proteins, including targets of non-enzymatic lysine polyphosphorylation. Our work demonstrates that internally produced polyP can activate diverse signaling pathways in human cells.

SIRT3 controls brown fat thermogenesis by deacetylation regulation of pathways upstream of UCP1.

OBJECTIVE: Brown adipose tissue (BAT) is important for thermoregulation in many mammals. Uncoupling protein 1 (UCP1) is the critical regulator of thermogenesis in BAT. Here we aimed to investigate the deacetylation control of BAT and to investigate a possible functional connection between UCP1 and sirtuin 3 (SIRT3), the master mitochondrial lysine deacetylase. METHODS: We carried out physiological, molecular, and proteomic analyses of BAT from wild-type and Sirt3KO mice when BAT is activated. Mice were either cold exposed for 2 days or were injected with the ß3-adrenergic agonist, CL316,243 (1 mg/kg; i.p.). Mutagenesis studies were conducted in a cellular model to assess the impact of acetylation lysine sites on UCP1 function. Cardiac punctures were collected for proteomic analysis of blood acylcarnitines. Isolated mitochondria were used for functional analysis of OXPHOS proteins. RESULTS: Our findings showed that SIRT3 absence in mice resulted in impaired BAT lipid use, whole body thermoregulation, and respiration in BAT mitochondria, without affecting UCP1 expression. Acetylome profiling of BAT mitochondria revealed that SIRT3 regulates acetylation status of many BAT mitochondrial proteins including UCP1 and crucial upstream proteins. Mutagenesis work in cells suggested that UCP1 activity was independent of direct SIRT3-regulated lysine acetylation. However, SIRT3 impacted BAT mitochondrial proteins activities of acylcarnitine metabolism and specific electron transport chain complexes, CI and CII. CONCLUSIONS: Our data highlight that SIRT3 likely controls BAT thermogenesis indirectly by targeting pathways upstream of UCP1.

Nonhistone targets of KAT2A and KAT2B implicated in cancer biology 1.

Lysine acetylation is a critical post-translation modification that can impact a protein's localization, stability, and function. Originally thought to only occur on histones, we now know thousands of nonhistone proteins are also acetylated. In conjunction with many other proteins, lysine acetyltransferases (KATs) are incorporated into large protein complexes that carry out these modifications. In this review we focus on the contribution of two KATs, KAT2A and KAT2B, and their potential roles in the development and progression of cancer. Systems biology demands that we take a broad look at protein function rather than focusing on individual pathways or targets. As such, in this review we examine KAT2A/2B-directed nonhistone protein acetylations in cancer in the context of the 10 "Hallmarks of Cancer", as defined by Hanahan and Weinberg. By focusing on specific examples of KAT2A/2B-directed acetylations with well-defined mechanisms or strong links to a cancer phenotype, we aim to reinforce the complex role that these enzymes play in cancer biology.

A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy.

Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.

A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis.

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.

RNA binding protein RALY promotes Protein Arginine Methyltransferase 1 alternatively spliced isoform v2 relative expression and metastatic potential in breast cancer cells.

Aberrant expression of Protein Arginine Methyltransferases (PRMTs) has been observed in several cancer types, including breast cancer. We previously reported that the PRMT1v2 isoform, which is generated through inclusion of alternative exon 2, is overexpressed in breast cancer cells and promotes their invasiveness. However, the precise mechanism by which expression of this isoform is controlled and how it is dysregulated in breast cancer remains unknown. Using a custom RNA interference-based screen, we identified several RNA binding proteins (RBP) which, when knocked down, altered the relative abundance of the alternatively spliced PRMT1v2 isoform. Amongst the top hits were SNW Domain containing 1 (SNW1) and RBP-associated with lethal yellow mutation (RALY), which both associated with the PRMT1 pre-mRNA and upon depletion caused an increase or decrease in the relative abundance of PRMT1v2 isoform mRNA and protein. Most importantly, a significant decrease in invasion was observed upon RALY knockdown in aggressive breast cancer cells, consistent with targeting PRMT1v2 directly, and this effect was rescued by the exogenous re-expression of PRMT1v2. We show that SNW1 expression is decreased, while RALY expression is increased in breast cancer cells and tumours, which correlates with decreased patient survival. This work revealed crucial insight into the mechanisms regulating the expression of the PRMT1 alternatively spliced isoform v2 and its dysregulation in breast cancer. It also provides proof-of-concept support for the development of therapeutic strategies where regulators of PRMT1 exon 2 alternative splicing are targeted as an approach to selectively reduce PRMT1v2 levels and metastasis in breast cancer.

Muscle-specific expression of the RNA-binding protein Staufen1 induces progressive skeletal muscle atrophy via regulation of phosphatase tensin homolog.

Converging lines of evidence have now highlighted the key role for post-transcriptional regulation in the neuromuscular system. In particular, several RNA-binding proteins are known to be misregulated in neuromuscular disorders including myotonic dystrophy type 1, spinal muscular atrophy and amyotrophic lateral sclerosis. In this study, we focused on the RNA-binding protein Staufen1, which assumes multiple functions in both skeletal muscle and neurons. Given our previous work that showed a marked increase in Staufen1 expression in various physiological and pathological conditions including denervated muscle, in embryonic and undifferentiated skeletal muscle, in rhabdomyosarcomas as well as in myotonic dystrophy type 1 muscle samples from both mouse models and humans, we investigated the impact of sustained Staufen1 expression in postnatal skeletal muscle. To this end, we generated a skeletal muscle-specific transgenic mouse model using the muscle creatine kinase promoter to drive tissue-specific expression of Staufen1. We report that sustained Staufen1 expression in postnatal skeletal muscle causes a myopathy characterized by significant morphological and functional deficits. These deficits are accompanied by a marked increase in the expression of several atrophy-associated genes and by the negative regulation of PI3K/AKT signaling. We also uncovered that Staufen1 mediates PTEN expression through indirect transcriptional and direct post-transcriptional events thereby providing the first evidence for Staufen1-regulated PTEN expression. Collectively, our data demonstrate that Staufen1 is a novel atrophy-associated gene, and highlight its potential as a biomarker and therapeutic target for neuromuscular disorders and conditions.

Staufen1s role as a splicing factor and a disease modifier in Myotonic Dystrophy Type I.

In a recent issue of PLOS Genetics, we reported that the double-stranded RNA-binding protein, Staufen1, functions as a disease modifier in the neuromuscular disorder Myotonic Dystrophy Type I (DM1). In this work, we demonstrated that Staufen1 regulates the alternative splicing of exon 11 of the human Insulin Receptor, a highly studied missplicing event in DM1, through Alu elements located in an intronic region. Furthermore, we found that Staufen1 overexpression regulates numerous alternative splicing events, potentially resulting in both positive and negative effects in DM1. Here, we discuss our major findings and speculate on the details of the mechanisms by which Staufen1 could regulate alternative splicing, in both normal and DM1 conditions. Finally, we highlight the importance of disease modifiers, such as Staufen1, in the DM1 pathology in order to understand the complex disease phenotype and for future development of new therapeutic strategies.

Staufen1 Regulates Multiple Alternative Splicing Events either Positively or Negatively in DM1 Indicating Its Role as a Disease Modifier.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an expansion of CUG repeats in the 3' UTR of the DMPK gene. The CUG repeats form aggregates of mutant mRNA, which cause misregulation and/or sequestration of RNA-binding proteins, causing aberrant alternative splicing in cells. Previously, we showed that the multi-functional RNA-binding protein Staufen1 (Stau1) was increased in skeletal muscle of DM1 mouse models and patients. We also showed that Stau1 rescues the alternative splicing profile of pre-mRNAs, e.g. the INSR and CLC1, known to be aberrantly spliced in DM1. In order to explore further the potential of Stau1 as a therapeutic target for DM1, we first investigated the mechanism by which Stau1 regulates pre-mRNA alternative splicing. We report here that Stau1 regulates the alternative splicing of exon 11 of the human INSR via binding to Alu elements located in intron 10. Additionally, using a high-throughput RT-PCR screen, we have identified numerous Stau1-regulated alternative splicing events in both WT and DM1 myoblasts. A number of these aberrant ASEs in DM1, including INSR exon 11, are rescued by overexpression of Stau1. However, we find other ASEs in DM1 cells, where overexpression of Stau1 shifts the splicing patterns away from WT conditions. Moreover, we uncovered that Stau1-regulated ASEs harbour Alu elements in intronic regions flanking the alternative exon more than non-Stau1 targets. Taken together, these data highlight the broad impact of Stau1 as a splicing regulator and suggest that Stau1 may act as a disease modifier in DM1.

A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy.

Loss of 'Survival of Motor Neurons' (SMN) leads to spinal muscular atrophy (SMA), a disease characterized by degeneration of spinal cord alpha motor neurons, resulting in muscle weakness, paralysis and death during early childhood. SMN is required for assembly of the core splicing machinery, and splicing defects were documented in SMA. We previously uncovered that Coactivator-Associated Methyltransferase-1 (CARM1) is abnormally up-regulated in SMA, leading to mis-regulation of a number of transcriptional and alternative splicing events. We report here that CARM1 can promote decay of a premature terminating codon (PTC)-containing mRNA reporter, suggesting it can act as a mediator of nonsense-mediated mRNA decay (NMD). Interestingly, this pathway, while originally perceived as solely a surveillance mechanism preventing expression of potentially detrimental proteins, is now emerging as a highly regulated RNA decay pathway also acting on a subset of normal mRNAs. We further show that CARM1 associates with major NMD factor UPF1 and promotes its occupancy on PTC-containing transcripts. Finally, we identify a specific subset of NMD targets that are dependent on CARM1 for degradation and that are also misregulated in SMA, potentially adding exacerbated targeting of PTC-containing mRNAs to the already complex array of molecular defects associated with this disease.