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A systematic, label-free method for identifying RNA-associated proteins in vivo provides insights into vertebrate ciliary beating machinery.

ABSTRACT: Cell-type specific RNA-associated proteins are essential for development and homeostasis in animals. Despite a massive recent effort to systematically identify RNA-associated proteins, we currently have few comprehensive rosters of cell-type specific RNA-associated proteins in vertebrate tissues. Here, we demonstrate the feasibility of determining the RNA-associated proteome of a defined vertebrate embryonic tissue using DIF-FRAC, a systematic and universal (i.e., label-free) method. Application of DIF-FRAC to cultured tissue explants of Xenopus mucociliary epithelium identified dozens of known RNA-associated proteins as expected, but also several novel RNA-associated proteins, including proteins related to assembly of the mitotic spindle and regulation of ciliary beating. In particular, we show that the inner dynein arm tether Cfap44 is an RNA-associated protein that localizes not only to axonemes, but also to liquid-like organelles in the cytoplasm called DynAPs. This result led us to discover that DynAPs are generally enriched for RNA. Together, these data provide a useful resource for a deeper understanding of mucociliary epithelia and demonstrate that DIF-FRAC will be broadly applicable for systematic identification of RNA-associated proteins from embryonic tissues.

SUBMITTER: Drew K

PROVIDER: S-EPMC7668317 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPrimary ciliary dyskinesia (PCD) is a rare congenital respiratory disorder characterized by abnormal ciliary motility leading to chronic airway infections. Qualitative evaluation of ciliary beat pattern based on digital high-speed videomicroscopy analysis has been proposed in the diagnosis process of PCD. Although this evaluation is easy in typical cases, it becomes difficult when ciliary beating is partially maintained. We postulated that a quantitative analysis of beat pattern would improve PCD diagnosis. We compared quantitative parameters with the qualitative evaluation of ciliary beat pattern in patients in whom the diagnosis of PCD was confirmed or excluded.MethodsNasal nitric oxide measurement, nasal brushings and biopsies were performed prospectively in 34 patients with suspected PCD. In combination with qualitative analysis, 12 quantitative parameters of ciliary beat pattern were determined on high-speed videomicroscopy recordings of beating ciliated edges. The combination of ciliary ultrastructural abnormalities on transmission electron microscopy analysis with low nasal nitric oxide levels was the "gold standard" used to establish the diagnosis of PCD.ResultsThis "gold standard" excluded PCD in 15 patients (non-PCD patients), confirmed PCD in 10 patients (PCD patients) and was inconclusive in 9 patients. Among the 12 parameters, the distance traveled by the cilium tip weighted by the percentage of beating ciliated edges presented 96% sensitivity and 95% specificity. Qualitative evaluation and quantitative analysis were concordant in non-PCD patients. In 9/10 PCD patients, quantitative analysis was concordant with the "gold standard", while the qualitative evaluation was discordant with the "gold standard" in 3/10 cases. Among the patients with an inconclusive "gold standard", the use of quantitative parameters supported PCD diagnosis in 4/9 patients (confirmed by the identification of disease-causing mutations in one patient) and PCD exclusion in 2/9 patients.ConclusionsWhen the beat pattern is normal or virtually immotile, the qualitative evaluation is adequate to study ciliary beating in patients suspected for PCD. However, when cilia are still beating but with moderate alterations (more than 40% of patients suspected for PCD), quantitative analysis is required to precise the diagnosis and can be proposed to select patients eligible for TEM.

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A systematic, label-free method for identifying RNA-associated proteins in vivo provides insights into vertebrate ciliary beating machinery.

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