Rewriting the message: Harnessing RNA pseudouridylation to tackle disease.

Adachi et al.1 and Song et al.2 demonstrate the feasibility of engineering pseudouridylation at specific sites and its utility to correct disease-causing premature termination codons (PTCs) in human cells.

Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect pre-mRNA processing.

Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.

Why U matters: detection and functions of pseudouridine modifications in mRNAs.

The uridine modifications pseudouridine (Ψ), dihydrouridine, and 5-methyluridine are present in eukaryotic mRNAs. Many uridine-modifying enzymes are associated with human disease, underscoring the importance of uncovering the functions of uridine modifications in mRNAs. These modified uridines have chemical properties distinct from those of canonical uridines, which impact RNA structure and RNA-protein interactions. Ψ, the most abundant of these uridine modifications, is present across (pre-)mRNAs. Recent work has shown that many Ψs are present at intermediate to high stoichiometries that are likely conducive to function and at locations that are poised to influence pre-/mRNA processing. Technological innovations and mechanistic investigations are unveiling the functions of uridine modifications in pre-mRNA splicing, translation, and mRNA stability, which are discussed in this review.

Regulation and Function of RNA Pseudouridylation in Human Cells.

Recent advances in pseudouridine detection reveal a complex pseudouridine landscape that includes messenger RNA and diverse classes of noncoding RNA in human cells. The known molecular functions of pseudouridine, which include stabilizing RNA conformations and destabilizing interactions with varied RNA-binding proteins, suggest that RNA pseudouridylation could have widespread effects on RNA metabolism and gene expression. Here, we emphasize how much remains to be learned about the RNA targets of human pseudouridine synthases, their basis for recognizing distinct RNA sequences, and the mechanisms responsible for regulated RNA pseudouridylation. We also examine the roles of noncoding RNA pseudouridylation in splicing and translation and point out the potential effects of mRNA pseudouridylation on protein production, including in the context of therapeutic mRNAs.

Review of ethical values across the ICRP's system of radiological protection.

In 2018, the International Commission on Radiological Protection (ICRP) released Publication 138, which highlights the ethical values foundational to the system of radiological protection. Additional work, both within and beyond the ICRP, has proposed or recommended ethical values associated with applications of the system in different areas, perhaps most notably in medical, veterinary, and environmental radiological protection. There are also existing ethical frameworks not specifically related to radiological protection that are nonetheless relevant to its practice; for example, the Beauchamp and Childress principles of biomedical ethics are of particular significance when it comes to medical uses of radiation and radioactivity. At first glance, it may seem as if there are unique or isolated sets of ethical values that need to be applied depending on the circumstance. Yet while each area of application will indeed have its own unique aspects and associated value judgements, there are consistent and complementary relationships between these ethical values. This paper reviews the work of the ICRP related to ethics, including brief historical context, and highlights the similarities and differences between sets of ethical values with emphasis on medical, veterinary, and environmental applications of radiological protection.

Tissue-Specific Toxicokinetics of Aqueous Radium-226 in an Estuarine Mussel, Geukensia demissa.

Radiological contamination of coastal habitats poses potential risk for native fauna, but the bioavailability of aqueous radium (Ra) and other dissolved metals to marine bivalves remains unclear. This study was the first to examine the tissue-specific disposition of aqueous 226Ra in a coastal mussel, specifically the Atlantic ribbed mussel Geukensia demissa. Most organ groups reached steady-state concentrations within 7 days during experimental exposure, with an average uptake rate constant of 0.0013 mL g-1 d-1. When moved to Ra-free synthetic seawater, mussels rapidly eliminated aqueous 226Ra (average elimination rate constant 1.56 d-1). The biological half-life for aqueous 226Ra ranged from 8.9 h for the gills and labial palps to 15.4 h for the muscle. Although previous field studies have demonstrated notable 226Ra accumulation in the soft tissues of marine mussels and that, for freshwater mussels, tissue-incorporated 226Ra derives primarily from the aqueous phase, our tissue-specific bioconcentration factors (BCFs) were on the order of (8.3 ± 1.5) × 10-4 indicating low accumulation potential of aqueous 226Ra in estuarine mussels. This suggests marine and estuarine mussels obtain 226Ra from an alternate route, such as particulate-sorbed Ra ingested during filter-feeding or from a contaminated food source.

Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation.

Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ∼25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling.

Use of life cycle assessment (LCA) to advance optimisation of radiological protection and safety.

Life cycle assessment (LCA) is a modelling technique used to determine the cradle-to-grave environmental and human health impacts from the production of a good or the provision of a service. Radiological protection may benefit from employing tools like LCA to obtain a broader perspective and enable comparison with analyses of non-radiological systems. Despite structural similarities to other well-established decision-aiding techniques (DATs), the impact assessment within LCA (i.e. LCIA) is not commonly used in the optimisation of radiological protection process. This paper provides a brief review of LCA, including LCIA, along with more traditional DATs (such as multi-attribute utility analysis) used in the optimisation process for comparison. Basic concrete shielding was considered as a simple, illustrative example; concrete attenuates emissions from a radiation source but is also associated with a financial cost as well as costs with respect to energy, material, and water use. LCA offers quantification of these and other key resources (termed 'impact categories'). Ultimately, we offer that, depending on the circumstance, LCA can be a useful tool in radiological protection decision-making, complementing existing techniques.

Flowthrough of239PU and55FE during RNA extraction.

Analysis of gene expression has become an important tool in understanding low-dose effect mechanisms of ionizing radiation at the cellular level. Metal binding to nucleic acids needs to be considered when interpreting these results, as some radioactive metals, particularly actinides, may produce free radicals and cause oxidative stress damage via chemical means at rates much higher than free radical formation related to their radiological properties. Bacteria exposedin situto low dose rates of plutonium-239 (239Pu) and iron-55 (55Fe) were previously analysed for gene expression. The work herein was motivated by an interest in more precisely identifying the distribution of radionuclides in these bacteria as well as the practical need to ensure appropriate transport and handling of the associated ribonucleic acid (RNA) extractions. RNA extractions were performed on bacteria growth media with and without bacteria cells (i.e. with and without RNA) at several different concentrations of239Pu and55Fe to inform the level of specificity of the extraction membrane as well as provide insight into internal (uptake) vs external (sorption) accumulation of these radionuclides in bacteria cells. Results of the study suggest that239Pu and55Fe detected in RNA extraction samples during long term cell studies is the result of binding to RNA prior to the time of extraction, as opposed to flow through or binding after cell lysis, and it highlights the practical importance of nucleic acid sample characterization to radiation protection more generally.

The three R's of reasonable in radiological protection: relationships, rationale, and resources.

Central to applying the principle of optimisation in the system of radiological protection is the evaluation of what level of radiation exposure should be considered 'as low as reasonably achievable' (ALARA), after taking into account the prevailing circumstances. Determining what is 'reasonable' is an abstract, although somewhat intuitive concept with many potential interpretations depending on both the situation and those involved, whether individuals or organisations. There are common themes in the types of considerations that need to be addressed to determine 'reasonableness' regardless of the exposure situation. However, despite the consistent and agreeable nature of these themes, there remains a gap in how to apply them in real situations. For example, without measurable goalposts (or a clear process for setting such goalposts) for determining what constitutes ALARA, we can find ourselves misinterpreting the optimisation process as keeping exposures 'as low as possible'. We propose herein, by consolidating and building on existing ideas, an easily understandable and actionable 'reasonableness' framework. This simple, yet broadly applicable tool is intended to help radiation protection experts in the systematic, deliberative, and collaborative reflection on all of the factors that make up 'reasonable' before making a decision-whether it be a proposed medical treatment or clean-up of a contaminated site. Each process execution and decision itself will necessarily retain the complexity of the prevailing circumstance. The proposed 'Rs' of Reasonable represent Relationships (stakeholders, empathy, and trust), Rationale (contextual, technical, and ethical), and Resources (technological, financial, and time).

Women in radiation (WiR)-a perspective for the strengthening of radiation protection.

Gender balance refers to the equitable treatment and access to opportunities for all genders. In order to achieve true gender balance, a variety of proactive approaches developed collaboratively, with insight from multiple perspectives, need to be implemented. With that purpose, the participation of women in professions related to radiation and radiation protection was prioritised and given high visibility by allocating a 'Women in Radiation' (WiR) Special Session at the 15th International Congress of the International Radiation Protection Association (IRPA), hosted by South Korea on 20 January 2021. In this session, various issues related to gender balance and equity/equality were highlighted by the panellists, and further elaborated in a subsequent discussion with attendees. The main goal of the WiR Special Session was to convene women from different organisations, career and age stages, disciplines and countries, in particular to consider the Asian-Oceanic vision and status of gender equality, along with other topics to support a 'Call for Action', with concrete recommendations subsequently provided to IRPA. The discussion stressed the main needs and challenges faced by women working in various radiation fields, along with raising awareness of possible professional and employment opportunities. This paper identifies some steps necessary to encourage, enhance and support the inclusion of more diversity in nuclear professions with specific emphasis on women. In conclusion, gender balance and equality must be at the heart of any strategic plan for the future of the radiological protection profession; international cooperation between relevant bodies is essential for success and could serve as a catalyst for specific policy statements aimed at achieving a balanced representation of women in radiological protection.

mRNA structure determines modification by pseudouridine synthase 1.

Pseudouridine (Ψ) is a post-transcriptional RNA modification that alters RNA-RNA and RNA-protein interactions that affect gene expression. Messenger RNA pseudouridylation was recently discovered as a widespread and conserved phenomenon, but the mechanisms responsible for selective, regulated pseudouridylation of specific sequences within mRNAs were unknown. Here, we have revealed mRNA targets for five pseudouridine synthases and probed the determinants of mRNA target recognition by the predominant mRNA pseudouridylating enzyme, Pus1, by developing high-throughput kinetic analysis of pseudouridylation in vitro. Combining computational prediction and rational mutational analysis revealed an RNA structural motif that is both necessary and sufficient for mRNA pseudouridylation. Applying this structural context information predicted hundreds of additional mRNA targets that were pseudouridylated in vivo. These results demonstrate a structure-dependent mode of mRNA target recognition by a conserved pseudouridine synthase and implicate modulation of RNA structure as the probable mechanism to regulate mRNA pseudouridylation.

Feasibility and Acceptability of an Adolescent-Friendly Rap Video to Improve Health Literacy Among HIV-Positive Youth in Urban Peru.

Clinical outcomes among adolescents living with HIV (ALHIV) might be improved by interventions aimed at addressing limited health literacy. We developed a Spanish-language rap video on HIV concepts and examined its acceptability and feasibility as a learning tool among ALHIV in Lima, Peru. Twenty-eight ALHIV receiving care at an urban pediatric hospital and ten stakeholders engaged in the care of adolescents watched the video. Adolescents completed a pre- and post-video questionnaire. We conducted focus groups with ALHIV and in-depth interviews with stakeholders and analyzed transcripts to identify themes. ALHIV described concepts of CD4 cell count and viral load as they were portrayed. Participants reported the video was relatable, accessible, and provided hope that ALHIV could lead healthy lives and advocated for future videos to address topics such as transmission and sexual health. Questionnaires indicated some improvement in viral load knowledge. An HIV health literacy music video intervention was feasible to implement and accepted by ALHIV and their healthcare providers. Communicating HIV knowledge via music videos may be promising; further study is needed to optimize implementation.

RESUMEN: Los resultados clínicos entre los adolescentes que viven con el VIH (AVVIH) podrían mejorarse mediante intervenciones dirigidas a abordar la limitada alfabetización sanitaria. Desarrollamos un video de rap en español sobre los conceptos del VIH y examinamos su aceptabilidad y viabilidad como herramienta de aprendizaje entre los AVVIH en Lima, Perú. Veintiocho AVVIH que reciben atención en un hospital pediátrico urbano y diez interesados involucrados en la atención de adolescentes vieron el video. Los adolescentes completaron un cuestionario previo y posterior al video. Realizamos grupos focales con AVVIH y entrevistas a profundidad a los interesados y analizamos las transcripciones para identificar los temas. Los AVVIH describieron conceptos de recuento de células CD4 y carga viral tal como se retrataron. Los participantes informaron que el video era identificable, accesible y brindaba la esperanza de que los AVVIH pudiera llevar una vida saludable y abogaron por videos futuros para abordar temas como la transmisión y la salud sexual. Los cuestionarios indicaron cierta mejora en el conocimiento de la carga viral. Una intervención de video musical para educación en salud sobre el VIH fue factible de implementar y fue aceptada por los AVVIH y sus proveedores de atención médica. La comunicación de conocimientos sobre el VIH a través de videos musicales puede ser prometedora; se necesitan más estudios para optimizar la implementación.

Women in radiation: a brief discussion of the intersectionality of race and gender.

As we progress towards more diverse, equitable, and inclusive communities, workplaces, and organizations in radiation protection (RP), we begin to consider the experiences of our fellow radiation protectionists in parallel with our own. This is to identify, and ideally take down, barriers to pursuing a fulfilling career and positive professional relationships. Recognition and active consideration of differential experiences and needs will help promote both personal and community level success. Over the past few years there have been concerted efforts within the RP community to consider and promote the participation of women. An example outcome of this effort is that we are seeing more women assuming prominent leadership positions in our community. As we celebrate the progress made towards equity, specifically with respect to women, it is important to consider that other factors such as race and gender identity can present their own barriers. Those at the intersection of these or other identities often find themselves in the so-called 'double bind' of more pronounced, or stacking, barriers to success. This paper briefly highlights the need for considering the intersectionality of race and gender in the promotion of equity for all women.

Accumulation of radio-iron and plutonium, alone and in combination, inPseudomonas putidagrown in liquid cultures.

The impact of low doses of ionising radiation on biological and environmental systems have been historically difficult to study. Modern biological tools have provided new methods for studying these mechanisms but applying these tools to a dose-response relationship may require refinement of dosimetric techniques that incorporate a detailed understand of radionuclide accumulation in biological cells, particularly when assessing the impact of low doses of ionising radiation. In this workPseudomonas putida (KT2440) grown in liquid culture was exposed to low dose rates (10-20 mGy d-1) of239Pu and55Fe, both alone and in combination, for a period of 20 days, and the accumulation of239Pu and55Fe in cell pellets was analysed via liquid scintillation counting. The study also considered of cells grown with239Pu and stable Fe (primarily56Fe). In addition to the analysis of cell pellet and media samples, this work includes analysis of the radiological content of ribonucleic acid extraction samples to examine uptake of radionuclides. Results indicate that239Pu inhibited the uptake of55Fe, and that the presence of stable and radioactive isotopes of Fe in cultures may promote pathways for Fe accumulation that are used by239Pu. The work herein provides foundational insight into future dosimetric models for our work with environmental bacteria.

Dissolution and Vertical Transport of Uranium from Stable Mineral Forms by Plants as Influenced by the Co-occurrence of Uranium with Phosphorus.

Plants could mobilize (dissolution followed by vertical transport) uranium (U) from mineral forms that are otherwise stable. However, the variability of this plant-mediated mobilization of U as a function of the presence of various essential plant nutrients contained in these minerals remains unknown. A series of column experiments were conducted using Andropogon virginicus to quantify the vertical transport of U from stable mineral forms as influenced by the chemical and physical coexistence of U with the essential nutrient, phosphorus (P). The presence of plants significantly increased the vertical migration of U only when U was precipitated with P (UO2HPO4·4H2O; chernikovite) but not from UO2 (uraninite) that lacks any essential plant nutrient. The U dissolution was further increased when chernikovite co-occurred with a sparingly available form of P (FePO4) under P-limited growing conditions. Similarly, A. virginicus accumulated the highest amount of U from chernikovite (0.05 mg/g) in the presence of FePO4 compared to that of uraninite (no-P) and chernikovite supplemented with KH2PO4. These results signify an increased plant-mediated dissolution, uptake, and leaching of radioactive contaminants in soils that are nutrient deficient, a key factor that should be considered in management at legacy contamination sites.

Phosphorus Stress-Induced Changes in Plant Root Exudation Could Potentially Facilitate Uranium Mobilization from Stable Mineral Forms.

Apparent deficiency of soil mineral nutrients often triggers specific physio-morphological changes in plants, and some of these changes could also inadvertently increase the ability of plants to mobilize radionuclides from stable mineral forms. This work, through a series of sand-culture, hydroponics, and batch-equilibration experiments, investigated the differential ability of root exudates of Andropogon virginicus grown under conditions with variable phosphorus (P) availability (KH2PO4, FePO4, Ca3(PO4)2, and no P) to solubilize uranium (U) from the uranyl phosphate mineral Chernikovite. The mineral form of P, and hence the bioavailability of P, affected the overall composition of the root exudates. The lower bioavailable forms of P (FePO4 and Ca3(PO4)2), but not the complete absence of P, resulted in a higher abundance of root metabolites with chelating capacity at 72 hrs after treatment application. In treatments with lower P-bioavailability, the physiological amino acid concentration inside of the roots increased, whereas the concentration of organic acids in the roots decreased due to the active exudation. In batch dissolution experiments, the organic acids, but not amino acids, increase the dissolution U from Chernikovite. The root exudate matrix of plants exposed to low available forms of P induced a >60% increase in U dissolution from Chernikovite due to 5-16 times greater abundance of organic acids in these treatments. However, this was ca. 70% of the theoretical dissolution achievable by this exudate matrix. These results highlight the potential of using active management of soil P as an effective tool to alter the plant-mediated mobilization of U in contaminated soil.

Induced transcription and stability of CELF2 mRNA drives widespread alternative splicing during T-cell signaling.

Studies in several cell types have highlighted dramatic and diverse changes in mRNA processing that occur upon cellular stimulation. However, the mechanisms and pathways that lead to regulated changes in mRNA processing remain poorly understood. Here we demonstrate that expression of the splicing factor CELF2 (CUGBP, Elav-like family member 2) is regulated in response to T-cell signaling through combined increases in transcription and mRNA stability. Transcriptional induction occurs within 6 h of stimulation and is dependent on activation of NF-κB. Subsequently, there is an increase in the stability of the CELF2 mRNA that correlates with a change in CELF2 3'UTR length and contributes to the total signal-induced enhancement of CELF2 expression. Importantly, we uncover dozens of splicing events in cultured T cells whose changes upon stimulation are dependent on CELF2 expression, and provide evidence that CELF2 controls a similar proportion of splicing events during human thymic T-cell development. Taken together, these findings expand the physiologic impact of CELF2 beyond that previously documented in developing neuronal and muscle cells to T-cell development and function, identify unappreciated instances of alternative splicing in the human thymus, and uncover novel mechanisms for CELF2 regulation that may broadly impact CELF2 expression across diverse cell types.

Reflectance-Based Vegetation Index Assessment of Four Plant Species Exposed to Lithium Chloride.

This study considers whether a relationship exists between response to lithium (Li) exposure and select vegetation indices (VI) determined from reflectance spectra in each of four plant species: Arabidopsis thaliana, Helianthus annuus (sunflower), Brassica napus (rape), and Zea mays (corn). Reflectance spectra were collected every week for three weeks using an ASD FieldSpec Pro spectroradiometer with both a contact probe (CP) and a field of view probe (FOV) for plants treated twice weekly in a laboratory setting with 0 mM (control) or 15 mM of lithium chloride (LiCl) solution. Plants were harvested each week after spectra collection for determination of relevant physical endpoints such as relative water content and chlorophyll content. Mixed effects analyses were conducted on selected endpoints and vegetation indices (VI) to determine the significance of the effects of treatment level and length of treatment as well as to determine which VI would be appropriate predictors of treatment-dependent endpoints. Of the species considered, A. thaliana exhibited the most significant effects and corresponding shifts in reflectance spectra. Depending on the species and endpoint, the most relevant VIs in this study were NDVI, PSND, YI, R1676/R1933, R750/R550, and R950/R750.