Rationale: NAA15 (N-alpha-acetyltransferase 15) is a component of the NatA (N-terminal acetyltransferase complex). The mechanism by which NAA15 haploinsufficiency causes congenital heart disease remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective: We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity and identifying proteins dependent upon a full amount of NAA15. Methods and Results: We introduced heterozygous loss of function, compound heterozygous, and missense residues (R276W) in iPSCs using CRISPR/Cas9. Haploinsufficient NAA15 iPSCs differentiate into cardiomyocytes, unlike NAA15 -null iPSCs, presumably due to altered composition of NatA. Mass spectrometry analyses reveal ≈80% of identified iPSC NatA targeted proteins displayed partial or complete N-terminal acetylation. Between null and haploinsufficient NAA15 cells, N-terminal acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W induced pluripotent stem cells. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; 18 were ribosomal-associated proteins. At least 4 proteins were encoded by genes known to cause autosomal dominant congenital heart disease. Conclusions: These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and N-terminal acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated congenital heart disease. Additionally, genetically engineered induced pluripotent stem cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.
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Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies
Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3′ UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.
more » « less- Award ID(s):
- 1661730
- NSF-PAR ID:
- 10308320
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 12
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.
Methods We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.
Results Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12Vand KRASQ61Hcells, respectively, 997 of which were shared (
p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.Conclusion Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.
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The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches, we explored how depletion of cMyBPC altered sarcomere function. We demonstrated that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM), normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation, enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with MYBPC3 mutations.more » « less
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Abstract U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for the evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome, predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA binding motif protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5′ end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron-containing genes have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of minor intron-containing genes disrupted in both species. Mutations in the majority of these orthologous minor intron-containing genes have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.
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Abstract Mutations in the
TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specificTP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.
