Canady Helios Cold Plasma Induces Breast Cancer Cell Death by Oxidation of Histone mRNA

Breast cancer is the most common cancer among women worldwide. Its molecular receptor marker status and mutational subtypes complicate clinical therapies. Cold atmospheric plasma is a promising adjuvant therapy to selectively combat many cancers, including breast cancer, but not normal tissue; however, the underlying mechanisms remain unexplored. Here, four breast cancer cell lines with different marker status were treated with Canady Helios Cold Plasma™ (CHCP) at various dosages and their differential progress of apoptosis was monitored. Inhibition of cell proliferation, induction of apoptosis, and disruption of the cell cycle were observed. At least 16 histone mRNA types were oxidized and degraded immediately after CHCP treatment by 8-oxoguanine (8-oxoG) modification. The expression of DNA damage response genes was up-regulated 12 h post-treatment, indicating that 8-oxoG modification and degradation of histone mRNA during the early S phase of the cell cycle, rather than DNA damage, is the primary cause of cancer cell death induced by CHCP. Our report demonstrates for the first time that CHCP effectively induces cell death in breast cancer regardless of subtyping, through histone mRNA oxidation and degradation during the early S phase of the cell cycle.

SMN controls neuromuscular junction integrity through U7 snRNP

The neuromuscular junction (NMJ) is an essential synapse for animal survival whose loss is a key hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). While insights into the function of the causative genes implicate RNA dysregulation in NMJ pathogenesis, the RNA-mediated mechanisms controlling the biology of this specialized synapse that go awry in disease remain elusive. Here, we show that activity of the SMA-determining SMN protein in the assembly of U7 small nuclear ribonucleoprotein (snRNP), which functions in the 3'-end processing of replication-dependent histone mRNAs, is required for NMJ integrity. AAV9-mediated gene delivery of U7-specific Lsm10 and Lsm11 proteins selectively enhances U7 snRNP assembly, corrects histone mRNA processing defects, and rescues key structural and functional abnormalities of neuromuscular pathology in SMA mice - including NMJ denervation, reduced synaptic transmission, and skeletal muscle atrophy. Furthermore, U7 snRNP dysfunction induced by SMN deficiency drives selective loss of the synaptic organizing protein Agrin at NMJs innervating vulnerable axial muscles of SMA mice, revealing an unanticipated link between U7-dependent histone mRNA processing and motor neuron-derived expression of an essential factor for NMJ biology. Together, these findings establish a direct contribution of U7 snRNP dysfunction to the neuromuscular phenotype in SMA and the requirement of RNA-mediated histone gene regulation for maintaining functional synaptic connections between motor neurons and muscles.

A region of SLBP outside the mRNA processing domain is essential for deposition of histone mRNA into the Drosophila egg

Replication-dependent histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending in a stemloop instead of a polyA tail, and are normally regulated coordinately with DNA replication. SLBP binds the 3’ end of histone mRNA, and is required for processing and translation. During Drosophila oogenesis, large amounts of histone mRNAs and proteins are deposited in the developing oocyte.The maternally deposited histone mRNA is synthesized in stage 10B oocytes after the nurse cells complete endoreduplication. We report that in WT stage 10B oocytes, the Histone Locus Bodies (HLBs), formed on the histone genes, produce histone mRNAs in the absence of phosphorylation of Mxc, normally required for histone gene expression in S-phase cells. Two mutants of SLBP, one with reduced expression and another with a 10 aa deletion, fail to deposit sufficient histone mRNA in the oocyte, and don't transcribe the histone genes in stage 10B. Mutations in a putative SLBP nuclear localization sequence overlapping the deletion, phenocopy the deletion. We conclude a high concentration of SLBP in the nucleus of stage 10B oocytes is essential for histone gene transcription.

A region of Drosophila SLBP distinct from the histone pre-mRNA binding and processing domains is essential for deposition of histone mRNA in the oocyte

ABSTRACTDuring Drosophila oogenesis, large amounts of histone mRNA and proteins are deposited in the developing oocyte. These are sufficient for the first 14 embryonic cell cycles and provide the developing embryo with sufficient histone proteins until the zygotic histone genes are activated. The maternally deposited histone mRNA is synthesized in stage 10b of oogenesis after completion of endoreduplication of the nurse cells. Histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending instead in a conserved stemloop instead of a polyA tail. The Stem-loop binding protein (SLBP) binds the 3’ end of histone mRNA and is essential for both the biosynthesis and translation of histone mRNA. We report that a 10 aa region in SLBP, which is not required for processing in vitro, is essential for transcription of histone mRNA in the stage 10b oocyte. In stage 10b the Histone Locus Bodies (HLBs) produce histone mRNAs in the absence of phosphorylation of Mxc, normally required for histone gene expression in S-phase cells. Mutants expressing this SLBP develop normally, produce small amounts of polyadenylated histone mRNA throughout development, but little histone mRNA in stage 10b resulting in death of the embryos in the first hr of development.

Drosophila Histone Locus Body assembly and function involves multiple interactions

The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome has a single locus comprised of ∼100 copies of a tandemly arrayed repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, we used transgenic gene arrays containing 12 copies of the histone repeat unit that functionally complement loss of the ∼200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression.