N1-methylpseudouridine found within COVID-19 mRNA vaccines produces faithful protein products.

Synthetic mRNA technology is a promising avenue for treating and preventing disease. Key to the technology is the incorporation of modified nucleotides such as N1-methylpseudouridine (m1Ψ) to decrease immunogenicity of the RNA. However, relatively few studies have addressed the effects of modified nucleotides on the decoding process. Here, we investigate the effect of m1Ψ and the related modification pseudouridine (Ψ) on translation. In a reconstituted system, we find that m1Ψ does not significantly alter decoding accuracy. More importantly, we do not detect an increase in miscoded peptides when mRNA containing m1Ψ is translated in cell culture, compared with unmodified mRNA. We also find that m1Ψ does not stabilize mismatched RNA-duplex formation and only marginally promotes errors during reverse transcription. Overall, our results suggest that m1Ψ does not significantly impact translational fidelity, a welcome sign for future RNA therapeutics.

Discovery and validation of dominantly inherited Alzheimer's disease mutations in populations from Latin America.

BACKGROUND: In fewer than 1% of patients, AD is caused by autosomal dominant mutations in either the presenilin 1 (PSEN1), presenilin 2 (PSEN2), or amyloid precursor protein (APP) genes. The full extent of familial AD and frequency of these variants remains understudied in Latin American (LatAm) countries. Due to the rare nature of these variants, determining the pathogenicity of a novel variant in these genes can be challenging. Here, we use a systematic approach to assign the likelihood of pathogenicity in variants from densely affected families in Latin American populations. METHODS: Clinical data was collected from LatAm families at risk for DIAD. Symptomatic family members were identified and assessed by local clinicians and referred for genetic counseling and testing. To determine the likelihood of pathogenicity among variants of unknown significance from LatAm populations, we report pedigree information, frequency in control populations, in silico predictions, and cell-based models of amyloid-beta ratios. RESULTS: We identified five novel variants in the presenilin1 (PSEN1) gene from Brazilian and Mexican families. The mean age at onset in newly identified families was 43.5 years (range 36-54). PSEN1 p.Val103_Ser104delinsGly, p.Lys395Ile, p.Pro264Se, p.Ala275Thr, and p.Ile414Thr variants have not been reported in PubMed, ClinVar, and have not been reported in dominantly inherited AD (DIAD) families. We found that PSEN1 p.Val103_Ser104delinsGly, p.Lys395Ile, p.Pro264Se, and p.Ala275Thr produce Aß profiles consistent with known AD pathogenic mutations. PSEN1 p.Ile414Thr did not alter Aß in a manner consistent with a known pathogenic mutation. CONCLUSIONS: Our study provides further insights into the genetics of AD in LatAm. Based on our findings, including clinical presentation, imaging, genetic, segregations studies, and cell-based analysis, we propose that PSEN1 p.Val103_Ser104delinsGly, p.Lys395Ile, p.Pro264Se, and p.Ala275Thr are likely pathogenic variants resulting in DIAD, whereas PSEN1 p.Ile414Thr is likely a risk factor. This report is a step forward to improving the inclusion/engagement of LatAm families in research. Family discovery is of great relevance for the region, as new initiatives are underway to extend clinical trials and observational studies to families living with DIAD.

The C-Terminal SynMuv/DdDUF926 Domain Regulates the Function of the N-Terminal Domain of DdNKAP.

NKAP (NF-κB activating protein) is a highly conserved SR (serine/arginine-rich) protein involved in transcriptional control and splicing in mammals. We identified DdNKAP, the Dictyostelium discoideum ortholog of mammalian NKAP, as interacting partner of the nuclear envelope protein SUN-1. DdNKAP harbors a number of basic RDR/RDRS repeats in its N-terminal domain and the SynMuv/DUF926 domain at its C-terminus. We describe a novel and direct interaction between DdNKAP and Prp19 (Pre mRNA processing factor 19) which might be relevant for the observed DdNKAP ubiquitination. Genome wide analysis using cross-linking immunoprecipitation-high-throughput sequencing (CLIP-seq) revealed DdNKAP association with intergenic regions, exons, introns and non-coding RNAs. Ectopic expression of DdNKAP and its domains affects several developmental aspects like stream formation, aggregation, and chemotaxis. We conclude that DdNKAP is a multifunctional protein, which might influence Dictyostelium development through its interaction with RNA and RNA binding proteins. Mutants overexpressing full length DdNKAP and the N-terminal domain alone (DdN-NKAP) showed opposite phenotypes in development and opposite expression profiles of several genes and rRNAs. The observed interaction between DdN-NKAP and the DdDUF926 domain indicates that the DdDUF926 domain acts as negative regulator of the N-terminus.

NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins.

NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS-NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing.

Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy.

Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calcium-regulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2's role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects.