Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3′ splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ~15-fold increase in the frequency of three base pair gaps at 3′ splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.Damon Runyon Cancer Research Foundation (DRG 2032-09)National Science Foundation (U.S.). (no. 0821391)United States. National Institutes of Healt

Contrast Enhanced CT Radiogenomics in a Retrospective NSCLC Cohort: Models, Attempted Validation of a Published Model and the Relevance of the Clinical Context

RATIONALE AND OBJECTIVE: To develop a radiogenomic predictive model for non-small cell lung cancer (NSCLC) patients studied through contrast enhanced chest computed tomography (CE-CT) targeting the most frequent gene alterations. M&M: A retrospective study of patients with NSCLC imaged with CE-CT before treatment and had their tumor genomics sequenced at our institution was performed. Data was gathered from their imaging studies, their electronic medical records and a web-based database search (cBioPortal.ca). All of the patient data was tabulated for analysis. Two predictive models (M1 & M2) were created using different approaches and a third model was extracted from the literature to also be tested in our population. RESULTS: Out of 157 patients, eighty were male (51%) and 124 (79%) had a history of smoking. The three most prevalent genes were KRAS, TP53 and EGFR. The M1 radiomics-only model median AUC were 0.61 (TP53), 0.53 (KRAS) and 0.64 (EGFR) and for M1 radiomics + clinical were 0.61 (TP53), 0.61 (KRAS) and 0.80 (EGFR). The M2 radiomics-only model median AUC were 0.63 (TP53), 0.60 (KRAS) and 0.65 (EGFR) and for M2 radiomics + clinical were 0.64 (TP53), 0.62 (KRAS) and 0.81 (EGFR). The external EGFR radiomic model showed an AUC of 0.69 and 0.86 for the radiomics-only and combined radiomics + clinical respectively. CONCLUSION: Our study was able to provide robust predictive radiomics model evaluation for the detection of TP53, KRAS and EGFR. We also compared our performance with an already published model and observed how impactful clinical variables can be on models' performance. KEYPOINTS • Tumor genomic profiling is critical for treatment selection • CE-CT radiomics produce robust predictive models comparable to those already published • Clinical variables should be considered/included in predictive models

Inter-observer Variability of Expert-derived Morphologic Risk Predictors in Aortic Dissection

OBJECTIVES: Establishing the reproducibility of expert-derived measurements on CTA exams of aortic dissection is clinically important and paramount for ground-truth determination for machine learning. METHODS: Four independent observers retrospectively evaluated CTA exams of 72 patients with uncomplicated Stanford type B aortic dissection and assessed the reproducibility of a recently proposed combination of four morphologic risk predictors (maximum aortic diameter, false lumen circumferential angle, false lumen outflow, and intercostal arteries). For the first inter-observer variability assessment, 47 CTA scans from one aortic center were evaluated by expert-observer 1 in an unconstrained clinical assessment without a standardized workflow and compared to a composite of three expert-observers (observers 2-4) using a standardized workflow. A second inter-observer variability assessment on 30 out of the 47 CTA scans compared observers 3 and 4 with a constrained, standardized workflow. A third inter-observer variability assessment was done after specialized training and tested between observers 3 and 4 in an external population of 25 CTA scans. Inter-observer agreement was assessed with intraclass correlation coefficients (ICCs) and Bland-Altman plots. RESULTS: Pre-training ICCs of the four morphologic features ranged from 0.04 (-0.05 to 0.13) to 0.68 (0.49-0.81) between observer 1 and observers 2-4 and from 0.50 (0.32-0.69) to 0.89 (0.78-0.95) between observers 3 and 4. ICCs improved after training ranging from 0.69 (0.52-0.87) to 0.97 (0.94-0.99), and Bland-Altman analysis showed decreased bias and limits of agreement. CONCLUSIONS: Manual morphologic feature measurements on CTA images can be optimized resulting in improved inter-observer reliability. This is essential for robust ground-truth determination for machine learning models. KEY POINTS: • Clinical fashion manual measurements of aortic CTA imaging features showed poor inter-observer reproducibility. • A standardized workflow with standardized training resulted in substantial improvements with excellent inter-observer reproducibility. • Robust ground truth labels obtained manually with excellent inter-observer reproducibility are key to develop reliable machine learning models

Registry of Aortic Diseases to Model Adverse Events and Progression (ROADMAP) in Uncomplicated Type B Aortic Dissection: Study Design and Rationale

PURPOSE To describe the design and methodological approach of a multicenter, retrospective study to externally validate a clinical and imaging-based model for predicting the risk of late adverse events in patients with initially uncomplicated type B aortic dissection (uTBAD). MATERIALS AND METHODS The Registry of Aortic Diseases to Model Adverse Events and Progression (ROADMAP) is a collaboration between 10 academic aortic centers in North America and Europe. Two centers have previously developed and internally validated a recently developed risk prediction model. Clinical and imaging data from eight ROADMAP centers will be used for external validation. Patients with uTBAD who survived the initial hospitalization between January 1, 2001, and December 31, 2013, with follow-up until 2020, will be retrospectively identified. Clinical and imaging data from the index hospitalization and all follow-up encounters will be collected at each center and transferred to the coordinating center for analysis. Baseline and follow-up CT scans will be evaluated by cardiovascular imaging experts using a standardized technique. RESULTS The primary end point is the occurrence of late adverse events, defined as aneurysm formation (≥6 cm), rapid expansion of the aorta (≥1 cm/y), fatal or nonfatal aortic rupture, new refractory pain, uncontrollable hypertension, and organ or limb malperfusion. The previously derived multivariable model will be externally validated by using Cox proportional hazards regression modeling. CONCLUSION This study will show whether a recent clinical and imaging-based risk prediction model for patients with uTBAD can be generalized to a larger population, which is an important step toward individualized risk stratification and therapy.Keywords: CT Angiography, Vascular, Aorta, Dissection, Outcomes Analysis, Aortic Dissection, MRI, TEVAR© RSNA, 2022See also the commentary by Rajiah in this issue

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies

COMMD1-Mediated Ubiquitination Regulates CFTR Trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis

Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains

BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells. CONCLUSIONS: We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes

Radiation balance and albedo in the Tropical Atlantic during ATEX 1969

This paper presents the components of the radiation balance and the balance itself, which are measured from "Meteor" during A TEX from January 1 to March 2, 1969 in the Atlantic Ocean. For "Planet", also engaged in ATEX, measurements of the global radiation are shown for the period of February 4 to February 28, 1969. From the results obtained by "Meteor" the albedo of the sea surface has been calculated and is presented for two exemplary days