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Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non-coding regions, which make up 98% of the genome, cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA-seq a powerful companion diagnostic. Here, we illustrate this point by identifying six patients with a recessive Osteogenesis Imperfecta (OI) and neonatal progeria syndrome. By integrating homozygosity mapping and RNA-seq, we delineated a deep intronic TAPT1 mutation (c.1237-52G>A) that segregated with the disease. Using patients’ fibroblasts, we document that TAPT1’s nascent transcription was not affected, indicating instead that this variant leads to an alteration of pre-mRNA processing. Predicted to serve as an alternative splicing branchpoint, this mutation causes TAPT1 exon 12 skipping, creating a protein-null allele. Additionally, our study reveals dysregulation of pathways involved in collagen and extracellular matrix biology in disease-relevant cells. Overall, our work highlights the power of transcriptomic approaches in deciphering the repercussion of non-coding variants as well as in illuminating the molecular mechanisms and underlying pathways of human diseases.

Pausing of transcribing RNA polymerase is regulated and creates opportunities to control gene expression. Research in metazoans has so far mainly focused on RNA polymerase II (Pol II) promoter-proximal pausing leaving the pervasive nature of pausing and its regulatory potential in mammalian cells unclear. Here, we developed a pause detecting algorithm (PDA) for nucleotide-resolution occupancy data and a new native elongating transcript sequencing approach, termed nested NET-seq, that strongly reduces artifactual peaks commonly misinterpreted as pausing sites. Leveraging PDA and nested NET-seq reveal widespread genome-wide Pol II pausing at single-nucleotide resolution in human cells. Notably, the majority of Pol II pauses occur outside of promoter-proximal gene regions primarily along the gene-body of transcribed genes. Sequence analysis combined with machine learning modeling reveals DNA sequence properties underlying widespread transcriptional pausing including a new pause motif. Interestingly, key sequence determinants of RNA polymerase pausing are conserved between human cells and bacteria. These studies indicate pervasive sequence-induced transcriptional pausing in human cells and the knowledge of exact pause locations implies potential functional roles in gene expression.

PURPOSE. The complete type of X-linked congenital stationary night blindness (CSNB1) in human and mouse is caused by mutations in the NYX gene. The human NYX protein has been predicted to contain an N-terminal endoplasmic reticulum (ER) signaling sequence and a C-terminal glycosylphosphatidylinositol (GPI) anchor. In the current study, these computer predictions were verified experimentally by expression of domainspecific cDNA constructs in COS-7 and HeLa cells. Moreover, computer-based analysis of the orthologue mouse amino acid sequence did not reveal a GPI anchor, which may result in a different protein localization compared with human NYX. Therefore, the cellular localization for the mouse Nyx protein was also examined. METHODS. A new method was established that differentially visualizes both the protein at the surface of the living cell and subsequently in intracellular compartments. The localization of the human and mouse V5-tagged wild-type and mutant NYX protein were studied. RESULTS. Human and mouse V5-NYX proteins were dispersed in the form of speckles over the entire cell surface. Subsequent staining of the same cells after detergent extraction revealed that V5-NYX located to the ER and Golgi apparatus. Deletion of the GPI anchor domain of NYX resulted in a time-dependent loss of V5-NYX from the surface of living cells and accumulation of this truncated protein in the ER and Golgi apparatus. Deletion of the ER signal sequence in Nyx delocalized the intracellular V5-Nyx protein and caused its dispersion in the cytosol. Furthermore, mutations introduced in the leucine-rich repeat (LRR>region, which has been described as a pathogenic variant of NYX, had no effect on subcellular localization of the protein. CONCLUSIONS. These data provide evidence that human and mouse nyctalopin are membrane-bound extracellular proteins and are functionally conserved.

Rad51 is the key enzyme for homologous recombination, an evolutionarily conserved mechanism for the repair of DNA damage and the generation of genetic diversity. Given the observation that many tumors become resistant to radiation therapy and DNA-damaging chemotherapeutics and also that tumor cell populations can acquire a high number of genetic alterations and then expand clonally, dysfunction of the mammalian Rad51 recombinase could play a major role in the multistep process of tumorigenesis. The data we present provide further strong support for this hypothesis. Using anti-Rad51 immunofluorescence staining, widely different tumor cell lines displayed increased numbers of nuclei with focally concentrated Rad51 protein compared with nonmalignant control cell lines. These nuclear foci are thought to represent a repairosome-type assembly of Rad51 and other proteins required for recombinational DNA repair. By Western blot analyses, the net amount of Rad51 protein was increased 2-7-fold in all tested tumor cell lines. Inhibition of de novo protein synthesis by cycloheximide treatment showed a similar half-life of Rad51 protein in normal and tumor cells. Fluorescence in situ hybridization experiments did not detect Rad51 gene amplifications in tumors. Because Northern blot analysis demonstrated highly elevated Rad51 mRNA levels, we conclude that the increases in Rad51 protein and nuclear foci formation in tumor cells are the result of transcriptional up-regulation.