Quantitative Proteomics Identifies Profilin-1 as a Pseudouridine-Binding Protein.

Pseudouridine (Ψ) is the most abundant RNA modification in nature; however, not much is known about the biological functions of this modified nucleoside. Employing an unbiased quantitative proteomics method, we identified multiple candidate reader proteins of Ψ in RNA, including a cytoskeletal protein profilin-1 (PFN1). We demonstrated that PFN1 binds directly and selectively to Ψ-containing RNA.

DDX50 cooperates with STAU1 to effect stabilization of pro-differentiation RNAs.

Glucose binding can alter protein oligomerization to enable differentiation. Here, we demonstrate that glucose binding is a general capacity of DExD/H-box RNA helicases, including DDX50, which was found to be essential for the differentiation of diverse cell types. Glucose binding to conserved DDX50 ATP binding sequences altered protein conformation and dissociated DDX50 dimers.

Cancer mutations rewire the RNA methylation specificity of METTL3-METTL14.

Chemical modification of RNAs is important for posttranscriptional gene regulation. The METTL3-METTL14 complex generates most -methyladenosine (mA) modifications in messenger RNAs (mRNAs), and dysregulated methyltransferase expression has been linked to cancers. Here we show that a changed sequence context for mA can promote oncogenesis.

Glucose binds and activates NSUN2 to promote translation and epidermal differentiation.

Elevations in intracellular glucose concentrations are essential for epithelial cell differentiation by mechanisms that are not fully understood. Glucose has recently been found to directly bind several proteins to alter their functions to enhance differentiation. Among the newly identified glucose-binding proteins is NSUN2, an RNA-binding protein that we identified as indispensable for epidermal differentiation.

Mass Spectrometry-Based Proteomics for Assessing Epitranscriptomic Regulations.

Epitranscriptomics is a rapidly evolving field that explores chemical modifications in RNA and how they contribute to dynamic and reversible regulations of gene expression. These modifications, for example, N-methyladenosine (mA), are crucial in various RNA metabolic processes, including splicing, stability, subcellular localization, and translation efficiency of mRNAs. Mass spectrometry-based proteomics has become an indispensable tool in unraveling the complexities of epitranscriptomics, offering high-throughput, precise protein identification, and accurate quantification of differential protein expression.

Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells.

Human genomes are susceptible to damage by a variety of endogenous and exogenous agents. If not repaired, the resulting DNA lesions can potentially lead to mutations, genome instability, and cell death. While existing in vitro experiments allow for characterizing replication outcomes from the use of purified translesion synthesis (TLS) DNA polymerases, such studies often lack the sophistication and dynamic nature of cellular contexts.

N 2-Alkyl-dG lesions elicit R-loop accumulation in the genome.

Humans are exposed to DNA alkylating agents through endogenous metabolism, environmental exposure and cancer chemotherapy. The resulting alkylated DNA adducts may elicit genome instability by perturbing DNA replication and transcription. R-loops regulate various cellular processes, including transcription, DNA repair, and telomere maintenance.

Characterizations of Protein Arginine Deiminase 1 as a Substrate of NTMT1: Implications of Nα-Methylation in Protein Stability and Interaction.

α-N-Methylation (Nα-methylation), catalyzed by protein N-terminal methyltransferases (NTMTs), constitutes a crucial post-translational modification involving the transfer of a methyl group from -adenosyl-l-methionine (SAM) to the Nα-terminal amino group of substrate proteins. NTMT1/2 are known to methylate canonical Nα sequences, such as X-P-K/R. With over 300 potential human protein substrates, only a small fraction has been validated, and even less is known about the functions of Nα-methylation.

HMGB3 and SUB1 Bind to and Facilitate the Repair of -Alkylguanine Lesions in DNA.

-Alkyl-2'-deoxyguanosine (-alkyl-dG) is a major type of minor-groove DNA lesions arising from endogenous metabolic processes and exogenous exposure to environmental contaminants. The -alkyl-dG lesions, if left unrepaired, can block DNA replication and transcription and induce mutations in these processes. Nevertheless, the repair pathways for -alkyl-dG lesions remain incompletely elucidated.

Structural basis for the H2AK119ub1-specific DNMT3A-nucleosome interaction.

Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1-nucleosome interaction remains elusive.

DNA polymerase κ participates in early S-phase DNA replication in human cells.

Cycling cells replicate their DNA during the S phase through a defined temporal program known as replication timing. Mutation frequencies, epigenetic chromatin states, and transcriptional activities are different for genomic regions that are replicated early and late in the S phase. Here, we found from ChIP-Seq analysis that DNA polymerase (Pol) κ is enriched in early-replicating genomic regions in HEK293T cells.

Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Perturb mRNA Splicing through Aberrant Interactions with SRSF2.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that results in the degeneration of neurons in the brain and spinal cord. Although a substantial number of studies have been conducted, much remains to be learned about the cellular mechanisms underlying ALS. In this study, we employed an engineered ascorbate peroxidase (APEX)-based proximity biotinylation, together with affinity pull-down of the ensuing biotinylated peptides, to investigate the proximity proteomes of human SOD1 and its two ALS-linked mutants, G85R and G93A.

Structure-guided functional suppression of AML-associated DNMT3A hotspot mutations.

DNA methyltransferases DNMT3A- and DNMT3B-mediated DNA methylation critically regulate epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote polymerization, leading to aberrant DNA methylation that may contribute to the pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the mutation-induced functional misregulation of DNMT3A remains unclear.

Optimising HIV drug resistance testing laboratory networks in Kenya: insights from systems engineering modelling.

Background: HIV drug resistance (DR) is a growing threat to the durability of current and future HIV treatment success. DR testing (DRT) technologies are very expensive and specialised, relying on centralised laboratories in most low and middle-income countries. Modelling for laboratory network with point-of-care (POC) DRT assays to minimise turnaround time (TAT), is urgently needed to meet the growing demand.

Replication Studies of Alkyl Phosphotriester Lesions in Human Cells.

Alkyl phosphotriester (alkyl-PTE) lesions in DNA are shown to be poorly repaired; however, little is known about how these lesions impact DNA replication in human cells. Here, we investigated how the and diastereomers of four alkyl-PTE lesions (alkyl = Me, Et, Pr, or Bu) at the TT site perturb DNA replication in HEK293T cells. We found that these lesions moderately impede DNA replication and that their replicative bypass is accurate.

Meeting Report on the 3rd Chinese American Society for Mass Spectrometry Conference-Advancing Biological and Pharmaceutical Mass Spectrometry.

Following the highly successful Chinese American Society for Mass Spectrometry (CASMS) conferences in the previous 2 years, the 3rd CASMS Conference was held virtually on August 28-31, 2023, using the Gather.Town platform to bring together scientists in the MS field. The conference offered a 4-day agenda with a scientific program consisting of two plenary lectures, and 14 parallel symposia in which a total of 70 speakers presented technological innovations and their applications in proteomics and biological MS and metabo-lipidomics and pharmaceutical MS.

Using queueing models as a decision support tool in allocating point-of-care HIV viral load testing machines in Kisumu County, Kenya.

Point-of-care (POC) technologies-including HIV viral load (VL) monitoring-are expanding globally, including in resource-limited settings. Modelling could allow decision-makers to consider the optimal strategy(ies) to maximize coverage and access, minimize turnaround time (TAT) and minimize cost with limited machines. Informed by formative qualitative focus group discussions with stakeholders focused on model inputs, outputs and format, we created an optimization model incorporating queueing theory and solved it using integer programming methods to reflect HIV VL monitoring in Kisumu County, Kenya.

mA in CAG repeat RNA binds to TDP-43 and induces neurodegeneration.

Microsatellite repeat expansions within genes contribute to a number of neurological diseases. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology. Here we reveal that the adenosine in CAG repeat RNA can be methylated to N-methyladenosine (mA) by TRMT61A, and that mA can be demethylated by ALKBH3.

Exploring the brain epitranscriptome: perspectives from the NSAS summit.

Increasing evidence reinforces the essential function of RNA modifications in development and diseases, especially in the nervous system. RNA modifications impact various processes in the brain, including neurodevelopment, neurogenesis, neuroplasticity, learning and memory, neural regeneration, neurodegeneration, and brain tumorigenesis, leading to the emergence of a new field termed neuroepitranscriptomics. Deficiency in machineries modulating RNA modifications has been implicated in a range of brain disorders from microcephaly, intellectual disability, seizures, and psychiatric disorders to brain cancers such as glioblastoma.

Proximity Proteomics Revealed Aberrant mRNA Splicing Elicited by ALS-Linked Profilin-1 Mutants.

Profilin 1 (PFN1) is a cytoskeleton protein that modulates actin dynamics through binding to monomeric actin and polyproline-containing proteins. Mutations in PFN1 have been linked to the pathogenesis of familial amyotrophic lateral sclerosis (ALS). Here, we employed an unbiased proximity labeling strategy in combination with proteomic analysis for proteome-wide profiling of proteins that differentially interact with mutant and wild-type (WT) PFN1 proteins in human cells.

Single-Nucleotide Resolution Mapping of -Methyladenine in Genomic DNA.

-Methyladenine (6mA) is a naturally occurring DNA modification in both prokaryotes and eukaryotes. Herein, we developed a deaminase-mediated sequencing (DM-seq) method for genome-wide mapping of 6mA at single-nucleotide resolution. The method capitalizes on the selective deamination of adenine, but not 6mA, in DNA mediated by an evolved adenine deaminase, ABE8e.

Promoter R-Loops Recruit U2AF1 to Modulate Its Phase Separation and RNA Splicing.

R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells and are enriched in promoter regions of genes. By employing a bioinformatic approach based on overlap analysis of transcription factor chromatin immunoprecipitation sequencing (ChIP-seq) data sets, we found that many splicing factors, including U2AF1 whose recognition of the 3' splicing site is crucial for pre-mRNA splicing, exhibit pronounced enrichment at endogenous R-loop- and DNA G4-structure loci in promoter regions of human genes. We also revealed that U2AF1 binds directly to R-loops and DNA G4 structures at a low-nM binding affinity.