Uncovering the Localization and Function of a Novel Read-Through Transcript 'TOMM40-APOE'.

Recent advancements in genome analysis technology have revealed the presence of read-through transcripts in which transcription continues by skipping the polyA signal. We here identified and characterized a new read-through transcript, TOMM40-APOE. With cDNA amplification from THP-1 cells, the TOMM40-APOE3 product was successfully generated. We also generated TOMM40-APOE4, another isoform, by introducing point mutations. Notably, while APOE3 and APOE4 exhibited extracellular secretion, both TOMM40-APOE3 and TOMM40-APOE4 were localized exclusively to the mitochondria. But functionally, they did not affect mitochondrial membrane potential. Cell death induction studies illustrated increased cell death with TOMM40-APOE3 and TOMM40-APOE4, and we did not find any difference in cellular function between the two isoforms. These findings indicated that the new mitochondrial protein TOMM40-APOE has cell toxic ability.

The hidden diversity of ancient bornaviral sequences from X and P genes in vertebrate genomes.

Endogenous bornavirus-like elements (EBLs) are heritable sequences derived from bornaviruses in vertebrate genomes that originate from transcripts of ancient bornaviruses. EBLs have been detected using sequence similarity searches such as tBLASTn, whose technical limitations may hinder the detection of EBLs derived from small and/or rapidly evolving viral X and P genes. Indeed, no EBLs derived from the X and P genes of orthobornaviruses have been detected to date in vertebrate genomes. Here, we aimed to develop a novel strategy to detect such 'hidden' EBLs. To this aim, we focused on the 1.9-kb read-through transcript of orthobornaviruses, which encodes a well-conserved N gene and small and rapidly evolving X and P genes. We show a series of evidence supporting the existence of EBLs derived from orthobornaviral X and P genes (EBLX/Ps) in mammalian genomes. Furthermore, we found that an EBLX/P is expressed as a fusion transcript with the cellular gene, ZNF451, which potentially encodes the ZNF451/EBLP fusion protein in miniopterid bat cells. This study contributes to a deeper understanding of ancient bornaviruses and co-evolution between bornaviruses and their hosts. Furthermore, our data suggest that endogenous viral elements are more abundant than those previously appreciated using BLAST searches alone, and further studies are required to understand ancient viruses more accurately.

Identification of aberrant transcription termination at specific gene loci with DNA hypomethylated transcription termination sites caused by DNA methyltransferase deficiency.

CpG methylation of genomic DNA is a well-known repressive epigenetic marker in eukaryotic transcription, and DNA methylation of promoter regions is correlated with gene silencing. In contrast to the promoter regions, the function of DNA methylation during transcription termination remains to be elucidated. A recent study revealed that mouse DNA methyltransferase 3a (Dnmt3a) mainly functions in de novo methylation in the promoter and gene body regions, including transcription termination sites (TTSs), during development. To investigate the relationship between DNA methylation overlapping the TTSs and transcription termination, we performed bioinformatics analysis using six pre-existing Dnmt-/- mouse cell datasets: four types of neurons (three Dnmt3a-/- and one Dnmt1-/- mutants) and two types of embryonic fibroblasts (MEFs) (Dnmt3a-/- and Dnmt3b-/- mutants). Combined analyses using methylome and transcriptome data revealed that read counts downstream of hypomethylated TTSs were increased in three types of neurons (two Dnmt3a-/- and one Dnmt1-/- mutants). Among these, an increase in chimeric transcripts downstream of the TTSs was observed in Dnmt3a-/- mature olfactory sensory neurons and Dnmt3a-/- agouti-related peptide (protein)-producing neurons, thereby indicating that read-through occurs in hypomethylated TTSs at specific gene loci in these two mutants. Conversely, in Dnmt3a-/- MEFs, we detected reductions in read counts downstream of hypomethylated TTSs. These results indicate that the hypomethylation of TTSs can both positively and negatively regulate transcription termination, dependent on Dnmt and cell types. This study is the first to identify the aberrant termination of transcription at specific gene loci with DNA hypomethylated TTSs attributable to Dnmt deficiency.

Using TIF-Seq2 to investigate association between 5´ and 3´mRNA ends.

The development of high-throughput technologies has revealed pervasive transcription in all genomes that have been investigated so far. This has uncovered a highly interleaved transcriptome organization involving thousands of overlapping coding and non-coding RNA isoforms that challenge our traditional definitions of genes and functional regions of the genome. In this chapter, we discuss the application of an improved Transcript Isoform Sequencing approach (TIF-Seq2) able to concurrently determine the start and end sites of individual RNA molecules. We exemplify its use for the investigation of the human transcriptome and show how it is especially well suited to discriminate between overlapping molecules and accurately define their boundaries.