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In recent years, there has been a high interest in researching RNA modifications, as they are involved in many cellular processes and in human diseases. A substantial set of enzymes within the cell, called RNA writers, place RNA modifications selectively and site-specifically. Another set of enzymes, called readers, recognize these modifications which guide the fate of the modified RNA. Although RNA is a transient molecule and RNA modification could be removed by RNA degradation, a subclass of enzymes, called RNA erasers, remove RNA modifications selectively and site-specifically to alter the characteristics of the RNA. The detection of RNA modifications can be done by various methods including second and next generation sequencing but also mass spectrometry. An approach capable of both qualitative and quantitative RNA modification analysis is liquid chromatography coupled to mass spectrometry of enzymatic hydrolysates of RNA into nucleosides. However, for successful detection and quantification, various factors must be considered to avoid biased identification and inaccurate quantification. In this Account, we identify three classes of errors that may distort the analysis. These classes comprise (I) errors related to chemical instabilities, (II) errors revolving around enzymatic hydrolysis to nucleosides, and (III) errors arising from issues with chromatographic separation and/or subsequent mass spectrometric analysis.A prominent example for class 1 is Dimroth rearrangement of m 1 A to m 6 A, but class 1 also comprises hydrolytic reactions and reactions with buffer components. Here, we also present the conversion of m 3 C to m 3 U under mild alkaline conditions and propose a practical solution to overcome these instabilities. Class 2 errors-such as contaminations in hydrolysis reagents or nuclease specificities-have led to erroneous discoveries of nucleosides in the past and possess the potential for misquantification of nucleosides. Impurities in the samples may also lead to class 3 errors: For instance, issues with chromatographic separation may arise from residual organic solvents, and salt adducts may hamper mass spectrometric quantification. This Account aims to highlight various errors connected to mass spectrometry analysis of nucleosides and presents solutions for how to overcome or circumnavigate those issues. Therefore, the authors anticipate that many scientists, but especially those who plan on doing nucleoside mass spectrometry, will benefit from the collection of data presented in this Account as a raised awareness, toward the variety of potential pitfalls, may further enhance the quality of data.

The sessile lifestyle of plants requires an immediate response to environmental stressors that affect photosynthesis, growth, and crop yield. Here, we showed that three abiotic perturbations-heat, cold, and high light-triggered considerable changes in the expression signatures of 42 epitranscriptomic factors (writers, erasers, and readers) with putative chloroplast-associated functions that formed clusters of commonly expressed genes in Arabidopsis. The expression changes under all conditions were reversible upon deacclimation, identifying epitranscriptomic players as modulators in acclimation processes. Chloroplast dysfunctions, particularly those induced by the oxidative stress-inducing norflurazon in a largely GENOME UNCOUPLED-independent manner, triggered retrograde signals to remodel chloroplast-associated epitranscriptomic expression patterns. N 6 -methyladenosine (m 6 A) is known as the most prevalent RNA modification and impacts numerous developmental and physiological functions in living organisms. During cold treatment, expression of components of the primary nuclear m 6 A methyltransferase complex was upregulated, accompanied by a significant increase in cellular m 6 A mRNA marks. In the cold, the presence of FIP37, a core component of the writer complex, played an important role in positive regulation of thylakoid structure, photosynthetic functions, and accumulation of photosystem I, the Cytb 6 f complex, cyclic electron transport proteins, and Curvature Thylakoid1 but not that of photosystem II components and the chloroplast ATP synthase. Downregulation of FIP37 affected abundance, polysomal loading, and translation of cytosolic transcripts related to photosynthesis in the cold, suggesting m 6 A-dependent translational regulation of chloroplast functions. In summary, we identified multifaceted roles of the cellular m 6 A RNA methylome in coping with cold; these were predominantly associated with chloroplasts and served to stabilize photosynthesis., (Copyright © 2023 LMU Munich. Published by Elsevier Inc. All rights reserved.)

Background: We assessed whether participation in Healthy Start Brooklyn's By My Side Birth Support Program-a maternal-health program providing community-based doula support during pregnancy, labor and delivery, and the early postpartum period-was associated with improved birth outcomes. By My Side takes a strength-based approach that aligns with the doula principles of respecting the client's autonomy, providing culturally appropriate care without judgment or conditions, and promoting informed decision making., Methods: Using a matched cohort design, birth certificate records for By My Side participants from 2010 through 2017 (n = 603) were each matched to three controls who also lived in the program area (n = 1809). Controls were matched on maternal age, race/ethnicity, education level, and trimester of prenatal-care initiation, using the simple random sampling method. The sample was restricted to singleton births. The odds of preterm birth, low birthweight, and cesarean birth were estimated, using conditional logistic regression., Results: By My Side participants had lower odds of having a preterm birth (5.6% vs 11.9%, P < .0001) or a low-birthweight baby (5.8% vs 9.7%, P = .0031) than controls. There was no statistically significant difference in the odds of cesarean delivery., Conclusion: Participation in the By My Side Birth Support Program was associated with lower odds of preterm birth and low birthweight for participants, who were predominantly Black and Hispanic. Investing in doula services is an important way to address birth inequities among higher risk populations such as birthing people of color and those living in poverty. It could also help shape a new vision of the maternal-health system, placing the needs and well-being of birthing people at the center., (© 2023 The Authors. Birth published by Wiley Periodicals LLC.)

RNA is dynamically modified and has the potential to respond to environmental changes and tune translation. The objective of this work is to uncover the temporal limitation of our recently developed cell culture NAIL-MS (nucleic acid isotope labelling coupled mass spectrometry) technology and overcome it. Actinomycin D (AcmD), an inhibitor of transcription, was used in the NAIL-MS context to reveal the origin of hybrid nucleoside signals composed of unlabelled nucleosides and labelled methylation marks. We find that the formation of these hybrid species depends exclusively on transcription for Poly-A RNA and rRNA but is partly transcription-independent for tRNA. This finding suggests that tRNA modifications adapt and are dynamically regulated by cells to overcome e.g. stress. Future studies on the tRNA modification mediated stress response are now accessible and the temporal resolution of NAIL-MS is improved by the use of AcmD., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3'end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m 6 A, the most abundant RNA modification. RBM15 positively regulates m 6 A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes., (© 2023. The Author(s).)

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N 6 -methyladenosine (m 6 A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m 6 A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m 6 A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt + regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m 6 A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m 6 A modification impacts on TCR signal transduction and determines activation and survival of T cells., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2'-O-methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Organisms from all domains of life invest a substantial amount of energy for the introduction of RNA modifications into nearly all transcripts studied to date. Instrumental analysis of RNA can focus on the modified residues and reveal the function of these epitranscriptomic marks. Here, we will review recent advances and breakthroughs achieved by NMR spectroscopy, sequencing, and mass spectrometry of the epitranscriptome.

Backgrounded membrane imaging (BMI) is a novel automated, 96-well plate-based microscopic approach for subvisible particle analysis. We scientifically evaluated BMI with respect to sizing and counting accuracy, working range, impact of refractive index, and interferences by silicone oil droplets, and compared BMI to state-of-the-art dynamic image analysis (DIA). Image quality was found to be comparable to current DIA methodologies. However, with the first versions of BMI image analysis software, an undersizing of polystyrene beads was observed. BMI linear concentration range was found to reach an upper limit (7.1 × 10 5 particles/mL) similar to DIA. In the absence of silicone oil droplets, BMI and DIA showed good agreement in total particle concentrations (particle diameter ≥2 μm) but differences in size distributions for particle sizes ≥4 μm. Analyses of prefilled syringe products and silicone oil emulsions demonstrated the removal of silicone oil in BMI sample processing. In contrast to DIA, particle counting by BMI remained unaffected by changes in refractive index. Overall, we demonstrated BMI to be a promising orthogonal method for subvisible particle characterization. Aspects like low required sample volume, high throughput, and ease of handling can make BMI a valuable alternative or complement to DIA in particular for formulation screening., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Purpose: Women of color in the United States, particularly in high-poverty neighborhoods, experience high rates of poor birth outcomes, including cesarean section, preterm birth, low birthweight, and infant mortality. Doula care has been linked to improvements in many perinatal outcomes, but women of color and low-income women often face barriers in accessing doula support., Description: To address this issue, the New York City Department of Health and Mental Hygiene's Healthy Start Brooklyn introduced the By My Side Birth Support Program in 2010. The goal was to complement other maternal home-visiting programs by providing doula support during labor and birth, along with prenatal and postpartum visits. Between 2010 and 2015, 489 infants were born to women enrolled in the program., Assessment: Data indicate that By My Side is a promising model of support for Healthy Start projects nationwide. Compared to the project area, program participants had lower rates of preterm birth (6.3 vs. 12.4%, p < 0.001) and low birthweight (6.5 vs. 11.1%, p = 0.001); however, rates of cesarean birth did not differ significantly (33.5 vs. 36.9%, p = 0.122). Further research is needed to explore possible reasons for this finding, and to examine the influence of doula support on birth outcomes among populations with high rates of chronic disease and stressors such as poverty, racism, and exposure to violence. However, feedback from participants indicates that doula support is highly valued and helps give women a voice in consequential childbirth decisions., Conclusion: Available evidence suggests that doula services may be an important component of an effort to address birth inequities.