Open reading frame dominance indicates protein-coding potential of RNAs.

Recent studies have identified numerous RNAs with both coding and noncoding functions. However, the sequence characteristics that determine this bifunctionality remain largely unknown. In the present study, we develop and test the open reading frame (ORF) dominance score, which we define as the fraction of the longest ORF in the sum of all putative ORF lengths. This score correlates with translation efficiency in coding transcripts and with translation of noncoding RNAs. In bacteria and archaea, coding and noncoding transcripts have narrow distributions of high and low ORF dominance, respectively, whereas those of eukaryotes show relatively broader ORF dominance distributions, with considerable overlap between coding and noncoding transcripts. The extent of overlap positively and negatively correlates with the mutation rate of genomes and the effective population size of species, respectively. Tissue-specific transcripts show higher ORF dominance than ubiquitously expressed transcripts, and the majority of tissue-specific transcripts are expressed in mature testes. These data suggest that the decrease in population size and the emergence of testes in eukaryotic organisms allowed for the evolution of potentially bifunctional RNAs.

Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

Inhibition of OCT4 binding at the MYCN locus induces neuroblastoma cell death accompanied by downregulation of transcripts with high-open reading frame dominance.

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether inhibition of alternative transcription factor induces NB cell death. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN expression, thereby suppressing MYCN-target genes. After inhibition of OCT4 binding, differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with a high-ORF dominance score were significantly associated with genes whose high expression is associated with a poor prognosis in NB. Because the ORF dominance score correlates with the translation efficiency of transcripts, our findings suggest that MYCN maintains the expression of transcripts with high translation efficiency, contributing to a poor prognosis in NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the OCT4 binding at the MYCN locus may serve as an effective therapeutic strategy for MYCN-amplified NB.

Tyrosyl-DNA phosphodiesterases are involved in mutagenic events at a ribonucleotide embedded into DNA in human cells.

Ribonucleoside triphosphates are often incorporated into genomic DNA during DNA replication. The accumulation of unrepaired ribonucleotides is associated with genomic instability, which is mediated by DNA topoisomerase 1 (Top1) processing of embedded ribonucleotides. The cleavage initiated by Top1 at the site of a ribonucleotide leads to the formation of a Top1-DNA cleavage complex (Top1cc), occasionally resulting in a DNA double-strand break (DSB). In humans, tyrosyl-DNA phosphodiesterases (TDPs) are essential repair enzymes that resolve the trapped Top1cc followed by downstream repair factors. However, there is limited cellular evidence of the involvement of TDPs in the processing of incorporated ribonucleotides in mammals. We assessed the role of TDPs in mutagenesis induced by a single ribonucleotide embedded into DNA. A supF shuttle vector site-specifically containing a single riboguanosine (rG) was introduced into the human lymphoblastoid TK6 cell line and its TDP1-, TDP2-, and TDP1/TDP2-deficient derivatives. TDP1 and TDP2 insufficiency remarkably decreased the mutant frequency caused by an embedded rG. The ratio of large deletion mutations induced by rG was also substantially lower in TDP1/TDP2-deficient cells than wild-type cells. Furthermore, the disruption of TDPs reduced the length of rG-mediated large deletion mutations. The recovery ratio of the propagated plasmid was also increased in TDP1/TDP2-deficient cells after the transfection of the shuttle vector containing rG. The results suggest that TDPs-mediated ribonucleotide processing cascade leads to unfavorable consequences, whereas in the absence of these repair factors, a more error-free processing pathway might function to suppress the ribonucleotide-induced mutagenesis. Furthermore, base substitution mutations at sites outside the position of rG were detected in the supF gene via a TDPs-independent mechanism. Overall, we provide new insights into the mechanism of mutagenesis induced by an embedded ribonucleotide in mammalian cells, which may lead to the fatal phenotype in the ribonucleotide excision repair deficiency.