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The spliceosome undergoes extensive rearrangements as it assembles onto precursor messenger RNAs. In the earliest assembly step, U1snRNA identifies the 5' splice site. However, U1snRNA leaves the spliceosome relatively early in assembly, and 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components during the rearrangements that build the catalytic site. Using a forward genetic screen in Caenorhabditis elegans, we have identified suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new suppressor alleles from this screen, two in PRP8 and one in SNRNP200/BRR2. mRNASeq studies of these suppressor strains indicate that they also affect specific native alternative 5'ss, especially for suppressor PRP8 D1549N. A strong suppressor at the unstructured N-terminus of SNRNP200, N18K, indicates a novel role for this region. By examining distinct changes in the splicing of native genes, examining double mutants between suppressors, comparing these new suppressors to previously identified splicing suppressors from yeast, and mapping conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions at multiple stages in spliceosome assembly responsible for maintaining the initial 5'ss identified by U1snRNA for entry into the catalytic core., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5' GU and ending with 3' AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and "custody" of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5' splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5' splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5' splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5' splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process., Competing Interests: The authors have declared that no competing interests exist.

A safe and healthy natural and built environment is fundamental to children's health and represents a significant determinant of community well-being. We aimed to identify and prioritize environmental health concerns within resource-poor neighborhoods in the Dominican Republic using free-listing and semi-structured focus groups composed of parents and caregivers in the perirural community of Consuelo, Dominican Republic. Transcripts were coded and relevant themes identified using qualitative content analysis. Demographic data and information regarding trash disposal practices were also collected. Participants described common health concerns, including respiratory infections, asthma, vector-borne illnesses, and diarrheal diseases and linked them to environmental hazards in their communities, such as air quality and sanitation. Interventional priorities that emerged included reduction of trash accumulation and trash burning as well as improvement of sanitation facilities., Competing Interests: The authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

To date, there appear to be no studies investigating the practice settings of all Medical Toxicology (MT) diplomates. The MT Assessment of Practice Performance Taskforce queried all MT diplomates about their current practice settings relative to the number of patients seen, the most common diagnoses, and the percent of time spent in their roles as medical toxicologists (MTs) and in their primary specialty. One hundred twenty-seven surveys were completed (44% response rate). Seventy-nine percent of respondents were affiliated with poison centers. Eighty-eight percent of participants were clinically active and reported seeing or consulting on behalf of at least ten patients over a 2-year period. Acetaminophen toxicity was the most common diagnosis encountered by respondents. Other common diagnoses included antidepressant toxicity, antipsychotic toxicity, mental status alteration, metal/environmental toxicity, envenomation, and pesticide toxicity. While respondents were likely to spend more time in direct patient care in their primary specialty, compared to consulting on behalf of patients, they were more likely to consult on behalf of patients in their role as MTs. Respondents spent more time in research, education, and population health in their role as an MT than in their primary specialty. Administrative activities were more commonly reported in association with the respondents' primary specialty than in their role as MTs. Most MTs encounter certain diagnoses with significant frequency and see a substantial number of patients within these categories. The majority spends more time on direct patient care in their primary specialty but is actively engaged in MT education, research, population health, and administration. A longitudinal assessment of MT practice patterns could inform MT curricular development and practice performance evaluation.

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.