The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies' association with histone locus bodies.

Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3'-end processing factors for RDH genes are enriched; however, this association's role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs' association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)-Cap binding complex (CBC)-Negative elongation factor (NELF), components of CBs, interferes with CBs' association with HLBs and 3' Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator's involvement in CBs' association with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing of RDH genes by supplying 3'-end processing factors.

A linear five-ring pyrrole-imidazole polyamide-triphenylphosphonium conjugate targeting a mitochondrial DNA mutation efficiently induces apoptosis of HeLa cybrid cells carrying the mutation.

Somatic mutations in mitochondrial DNA may provide a new avenue for cancer therapy due to their associations to a number of cancers and a tendency of homoplasmicity. In consideration of mitochondrial features and its relatively small genome size, a nucleotide-based targeting approach is a considerably more promising option. To explore the efficacy of short linear N-methylpyrrole-N-methylimidazole polyamide (PI polyamide), we synthesized a five-ring short PI polyamide that provided sequence-specific homing for the A3243G mitochondrial mutation upon conjugation with triphenylphosphonium cation (TPP). This PI polyamide-TPP was able to induce cytotoxicity in HeLamtA3243G cybrid cells, while preserving preferential binding for oligonucleotides containing the A3243G motif from melting temperature assays. The PI polyamide-TPP also localized in the mitochondria in HeLamtA3243G cells and induced mitochondrial reactive oxygen species production, mitophagy and apoptosis in a mutation-specific fashion compared to the wild-type HeLamtHeLa cybrids; normal human dermal fibroblasts were also relatively unaffected to suggest discriminating selectivity for the mutant mitochondria, offering a novel outlook for cancer therapy via mitochondrial homing of short linear PIP-TPPs.

A PI polyamide-TPP conjugate targeting a mtDNA mutation induces cell death of cancer cells with the mutation.

Mitochondrial DNA (mtDNA) mutations occur frequently in cancer cells, and some of them are often homoplasmic. Targeting such mtDNA mutations could be a new method for killing cancer cells with minimal impact on normal cells. Pyrrole-imidazole polyamides (PIPs) are cell-permeable minor groove binders that show sequence-specific binding to double-stranded DNA and inhibit the transcription of target genes. PIP conjugated with the lipophilic triphenylphosphonium (TPP) cation can be delivered to mitochondria without uptake into the nucleus. Here, we investigated the feasibility of the use of PIP-TPP to target a mtDNA mutation in order to kill cancer cells that harbor the mutation. We synthesized hairpin-type PIP-TPP targeting the A3243G mutation and examined its effects on the survival of HeLa cybrid cells with or without the mutation (HeLamtA3243G cells or HeLamtHeLa cells, respectively). A surface plasmon resonance assay demonstrated that PIP-TPP showed approximately 60-fold higher binding affinity for the mutant G-containing synthetic double-stranded DNA than for the wild-type A-containing DNA. When added to cells, it localized in mitochondria and induced mitochondrial reactive oxygen species production, extensive mitophagy, and apoptosis in HeLamtA3243G cells, while only slightly exerting these effects in HeLamtHeLa cells. These results suggest that PIP-TPPs targeting mtDNA mutations could be potential chemotherapeutic drugs to treat cancers without severe adverse effects.