Large-scale functional screen identifies genetic variants with splicing effects in modern and archaic humans.

Humans coexisted and interbred with other hominins which later became extinct. These archaic hominins are known to us only through fossil records and for two cases, genome sequences. Here, we engineer Neanderthal and Denisovan sequences into thousands of artificial genes to reconstruct the pre-mRNA processing patterns of these extinct populations. Of the 5,169 alleles tested in this massively parallel splicing reporter assay (MaPSy), we report 962 exonic splicing mutations that correspond to differences in exon recognition between extant and extinct hominins. Using MaPSy splicing variants, predicted splicing variants, and splicing quantitative trait loci, we show that splice-disrupting variants experienced greater purifying selection in anatomically modern humans than that in Neanderthals. Adaptively introgressed variants were enriched for moderate-effect splicing variants, consistent with positive selection for alternative spliced alleles following introgression. As particularly compelling examples, we characterized a unique tissue-specific alternative splicing variant at the adaptively introgressed innate immunity gene TLR1, as well as a unique Neanderthal introgressed alternative splicing variant in the gene HSPG2 that encodes perlecan. We further identified potentially pathogenic splicing variants found only in Neanderthals and Denisovans in genes related to sperm maturation and immunity. Finally, we found splicing variants that may contribute to variation among modern humans in total bilirubin, balding, hemoglobin levels, and lung capacity. Our findings provide unique insights into natural selection acting on splicing in human evolution and demonstrate how functional assays can be used to identify candidate causal variants underlying differences in gene regulation and phenotype.

Reprogramming RNA processing: an emerging therapeutic landscape.

The production of a mature mRNA requires coordination of multiple processing steps, which ultimately control its content, localization, and stability. These steps include some of the largest macromolecular machines in the cell, which were, until recently, considered undruggable due to their biological complexity. Building from an expanded understanding of the underlying mechanisms that drive these processes, a new wave of therapeutics is seeking to target RNA processing. With a focus on impacting gene regulation at the RNA level, such modalities offer potential for sequence-specific resolution in drug design. Here, we review our current understanding of RNA-processing events and their role in gene regulation, with a focus on the therapeutic opportunities that have emerged within this landscape.

L-bodies are RNA-protein condensates driving RNA localization in Xenopus oocytes.

Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.

Intronic RNA: Ad'junk' mediator of post-transcriptional gene regulation.

RNA splicing, the process through which intervening segments of noncoding RNA (introns) are excised from pre-mRNAs to allow for the formation of a mature mRNA product, has long been appreciated for its capacity to add complexity to eukaryotic proteomes. However, evidence suggests that the utility of this process extends beyond protein output and provides cells with a dynamic tool for gene regulation. In this review, we aim to highlight the role that intronic RNA plays in mediating specific splicing outcomes in pre-mRNA processing, as well as explore an emerging class of stable intronic sequences that have been observed to act in gene expression control. Building from underlying flexibility in both sequence and structure, intronic RNA provides mechanisms for post-transcriptional gene regulation that are amenable to the tissue and condition specific needs of eukaryotic cells. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

Fluorescence In Situ Hybridization of Cryosectioned Xenopus Oocytes.

Xenopus laevis oocytes are widely used to study mechanisms of RNA function and biogenesis. While the large size of Xenopus oocytes is amenable to both biochemical and imaging approaches, the relative opacity of the yolk-rich cytoplasm has limited high-resolution imaging of endogenous RNAs. Here, we present a protocol that combines multi-probe fluorescence in situ hybridization with cryosectioning to provide a highly sensitive means of imaging endogenous oocyte RNAs.

Subepidermal moisture differentiates erythema and stage I pressure ulcers in nursing home residents.

The objective of this descriptive cohort study was to examine the relationship between subepidermal moisture (SEM) and visual assessment of early pressure ulcers (PUs) in 31 nursing home (NH) residents residing in two NHs. Concurrent visual assessments and SEM were obtained at the sacrum, right and left trochanters, buttocks, and ischium weekly for 20 weeks. Visual assessment was rated as normal, erythema, stage I PU, or stage II+ PU. SEM, measured with a dermal phase meter where higher readings indicate greater SEM (range: 0-999 dermal phase units [DPU]), was modeled as a predictor of concurrent visual assessment of skin damage and erythema and stage I PUs at the sacrum 1 week later with covariate PU risk. Participants had a mean age of 84.1 years, were 83% female, 72% non-Hispanic white. SEM was lowest for normal skin (104 DPU, SD 114), higher for erythema (185 DPU, SD 138), stage I PUs (264 DPU, SD 208), and highest for stage II+ PUs (727 DPU, SD 287) across all sites (all p<0.01). SEM was responsive to visual assessment changes, differentiated between erythema and stage I PU, and higher SEM predicted greater likelihood of erythema/stage I PU at the sacrum the next week (odds ratio=1.32 for every 100 DPU increase, p=0.03). SEM was associated with concurrent skin damage and future (1 week later) development of sacral erythema/stage I PUs. SEM differentiates between erythema and stage I PUs. SEM may assist in predicting early PU damage, allowing for earlier intervention to prevent PUs.

Subepidermal moisture predicts erythema and stage 1 pressure ulcers in nursing home residents: a pilot study.

OBJECTIVES: To examine the relationship between a measure of subepidermal moisture (SEM) and visual skin assessment (VSA) of erythema and Stage 1 pressure ulcers (PUs) performed a week later in nursing home (NH) residents. DESIGN: Descriptive, cohort study. SETTING: Two NHs. PARTICIPANTS: Thirty-five residents. METHODS: Concurrent VSAs and SEM readings were obtained at the sacrum, right and left trochanters, buttocks, and ischial tuberosities weekly for 52 weeks. SEM was measured using a handheld dermal phase meter, with higher readings indicating greater SEM (range 0-999 dermal phase units [DPUs]). VSA was rated as normal, erythema/Stage 1 PU, or Stage 2+PU. SEM was modeled as a predictor of VSA of erythema and PUs 1 week later (controlling for clustering), with concurrent moisture, Braden Scale PU risk status, anatomic site, and ethnicity as covariates. RESULTS: Participants had a mean age of 84.7, 83% were female, and 80% were non-Hispanic white. SEM measures were lowest for normal skin (97+/-122 DPU), higher for erythema/Stage 1 PUs (192+/-188 DPU), and highest for Stage 2+PUs (569+/-320 DPU) across all sites (all P<.001). SEM was responsive to changes in VSA, and higher SEM predicted greater likelihood of erythema/Stage 1 PU the next week (odds ratio=1.26 for every 100-DPU increase in SEM, P=.04). CONCLUSION: SEM measures are associated with concurrent erythema and PUs and future (1 week later) development of erythema/Stage 1 PUs. SEM may assist in predicting early PU damage, allowing for earlier intervention to prevent skin damage.