RESUMEN
We performed long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors to discover new transcript and protein isoforms expressed during immune responses to diverse pathogens. Long-read transcriptome profiling reveals novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. Widespread loss of intron retention occurs as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. RNA expression differences did not result in differences in the amounts of secreted proteins. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and poly(I:C)-stimulated PBMCs. Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.
RESUMEN
Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying ADGRV1-associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in adgrv1 exon 9 (adgrv1rmc22). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1rmc22 zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing adgrv1rmc22 zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the adgrv1rmc22 zebrafish as the first ADGRV1 mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for ADGRV1-associated RP.
Asunto(s)
Retinitis Pigmentosa , Pez Cebra , Animales , Humanos , Pez Cebra/genética , Sistemas CRISPR-Cas/genética , Edición Génica , Retina , Retinitis Pigmentosa/genéticaRESUMEN
Our incomplete knowledge of the human transcriptome impairs the detection of disease-causing variants, in particular if they affect transcripts only expressed under certain conditions. These transcripts are often lacking from reference transcript sets, such as Ensembl/GENCODE and RefSeq, and could be relevant for establishing genetic diagnoses. We present SUsPECT (Solving Unsolved Patient Exomes/gEnomes using Custom Transcriptomes), a pipeline based on the Ensembl Variant Effect Predictor (VEP) to predict variant impact on custom transcript sets, such as those generated by long-read RNA-sequencing, for downstream prioritization. Our pipeline predicts the functional consequence and likely deleteriousness scores for missense variants in the context of novel open reading frames predicted from any transcriptome. We demonstrate the utility of SUsPECT by uncovering potential mutational mechanisms of pathogenic variants in ClinVar that are not predicted to be pathogenic using the reference transcript annotation. In further support of SUsPECT's utility, we identified an enrichment of immune-related variants predicted to have a more severe molecular consequence when annotating with a newly generated transcriptome from stimulated immune cells instead of the reference transcriptome. Our pipeline outputs crucial information for further prioritization of potentially disease-causing variants for any disease and will become increasingly useful as more long-read RNA sequencing datasets become available.