Towards optimizing diversifying base editors for high-throughput studies of single- nucleotide variants.

Determining the phenotypic effects of single nucleotide variants is critical for understanding the genome and interpreting clinical sequencing results. Base editors, including diversifying base editors that create C>N mutations, are potent tools for installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing.

ChemRAP uncovers specific mRNA translation regulation via RNA 5' phospho-methylation.

5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers".

Transcriptomics and chromatin accessibility in multiple African population samples.

Mapping the functional human genome and impact of genetic variants is often limited to European-descendent population samples. To aid in overcoming this limitation, we measured gene expression using RNA sequencing in lymphoblastoid cell lines (LCLs) from 599 individuals from six African populations to identify novel transcripts including those not represented in the hg38 reference genome. We used whole genomes from the 1000 Genomes Project and 164 Maasai individuals to identify 8,881 expression and 6,949 splicing quantitative trait loci (eQTLs/sQTLs), and 2,611 structural variants associated with gene expression (SV-eQTLs).

Integrative analyses highlight functional regulatory variants associated with neuropsychiatric diseases.

Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types.

Deep learning-assisted genome-wide characterization of massively parallel reporter assays.

Massively parallel reporter assay (MPRA) is a high-throughput method that enables the study of the regulatory activities of tens of thousands of DNA oligonucleotides in a single experiment. While MPRA experiments have grown in popularity, their small sample sizes compared to the scale of the human genome limits our understanding of the regulatory effects they detect. To address this, we develop a deep learning model, MpraNet, to distinguish potential MPRA targets from the background genome.

Multiple causal variants underlie genetic associations in humans.

Associations between genetic variation and traits are often in noncoding regions with strong linkage disequilibrium (LD), where a single causal variant is assumed to underlie the association. We applied a massively parallel reporter assay (MPRA) to functionally evaluate genetic variants in high, local LD for independent cis-expression quantitative trait loci (eQTL). We found that 17.

BCDIN3D RNA methyltransferase stimulates Aldolase C expression and glycolysis through let-7 microRNA in breast cancer cells.

Type II diabetes (T2D) and specific cancers share many risk factors, however, the molecular mechanisms underlying these connections are often not well-understood. BCDIN3D is an RNA modifying enzyme that methylates specific precursor microRNAs and tRNA. In addition to breast cancer, BCDIN3D may also be linked to metabolism, as its gene locus is associated with obesity and T2D.

Post-transcriptional regulation of antiviral gene expression by N6-methyladenosine.

Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (mA) in the regulation of ISGs.

Evaluating the Genomic Parameters Governing rAAV-Mediated Homologous Recombination.

Recombinant adeno-associated virus (rAAV) vectors have the unique ability to promote targeted integration of transgenes via homologous recombination at specified genomic sites, reaching frequencies of 0.1%-1%. We studied genomic parameters that influence targeting efficiencies on a large scale.

Transcriptomic signatures across human tissues identify functional rare genetic variation.

Rare genetic variants are abundant across the human genome, and identifying their function and phenotypic impact is a major challenge. Measuring aberrant gene expression has aided in identifying functional, large-effect rare variants (RVs). Here, we expanded detection of genetically driven transcriptome abnormalities by analyzing gene expression, allele-specific expression, and alternative splicing from multitissue RNA-sequencing data, and demonstrate that each signal informs unique classes of RVs.

Impact of admixture and ancestry on eQTL analysis and GWAS colocalization in GTEx.

Background: Population structure among study subjects may confound genetic association studies, and lack of proper correction can lead to spurious findings. The Genotype-Tissue Expression (GTEx) project largely contains individuals of European ancestry, but the v8 release also includes up to 15% of individuals of non-European ancestry. Assessing ancestry-based adjustments in GTEx improves portability of this research across populations and further characterizes the impact of population structure on GWAS colocalization.

Genetic regulation of gene expression and splicing during a 10-year period of human aging.

Background: Molecular and cellular changes are intrinsic to aging and age-related diseases. Prior cross-sectional studies have investigated the combined effects of age and genetics on gene expression and alternative splicing; however, there has been no long-term, longitudinal characterization of these molecular changes, especially in older age.

Results: We perform RNA sequencing in whole blood from the same individuals at ages 70 and 80 to quantify how gene expression, alternative splicing, and their genetic regulation are altered during this 10-year period of advanced aging at a population and individual level.

BCDIN3D regulates tRNAHis 3' fragment processing.

5' ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5' monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq).

Genetic analyses of human fetal retinal pigment epithelium gene expression suggest ocular disease mechanisms.

The retinal pigment epithelium (RPE) serves vital roles in ocular development and retinal homeostasis but has limited representation in large-scale functional genomics datasets. Understanding how common human genetic variants affect RPE gene expression could elucidate the sources of phenotypic variability in selected monogenic ocular diseases and pinpoint causal genes at genome-wide association study (GWAS) loci. We interrogated the genetics of gene expression of cultured human fetal RPE (fRPE) cells under two metabolic conditions and discovered hundreds of shared or condition-specific expression or splice quantitative trait loci (e/sQTLs).

Pharmacy Refill Data are Poor Predictors of Virologic Treatment Outcomes in Adolescents with HIV in Botswana.

In adults living with HIV, pharmacy refill data are good predictors of virologic failure (VF). The utility of pharmacy refill data for predicting VF in adolescents has not been reported. We evaluated data from 291 adolescents on antiretroviral therapy.

Crosstalk between the RNA Methylation and Histone-Binding Activities of MePCE Regulates P-TEFb Activation on Chromatin.

RNAP II switching from the paused to the productive transcription elongation state is a pivotal regulatory step that requires specific phosphorylations catalyzed by the P-TEFb kinase. Nucleosolic P-TEFb activity is inhibited by its interaction with the ribonuclear protein complex built around the 7SK small nuclear RNA (7SK snRNP). MePCE is the RNA methyltransferase that methylates and stabilizes 7SK in the nucleosol.

Chromatin Regulates Genome Targeting with Cisplatin.

Cisplatin derivatives can form various types of DNA lesions (DNA-Pt) and trigger pleiotropic DNA damage responses. Here, we report a strategy to visualize DNA-Pt with high resolution, taking advantage of a novel azide-containing derivative of cisplatin we named APPA, a cellular pre-extraction protocol and the labeling of DNA-Pt by means of click chemistry in cells. Our investigation revealed that pretreating cells with the histone deacetylase (HDAC) inhibitor SAHA led to detectable clusters of DNA-Pt that colocalized with the ubiquitin ligase RAD18 and the replication protein PCNA.

Click Quantitative Mass Spectrometry Identifies PIWIL3 as a Mechanistic Target of RNA Interference Activator Enoxacin in Cancer Cells.

Enoxacin is a small molecule that stimulates RNA interference (RNAi) and acts as a growth inhibitor selectively in cancer but not in untransformed cells. Here, we used alkenox, a clickable enoxacin surrogate, coupled with quantitative mass spectrometry, to identify PIWIL3 as a mechanistic target of enoxacin. PIWIL3 is an Argonaute protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNAi through piRNAs.

Small RNA Sequencing in Cells and Exosomes Identifies eQTLs and 14q32 as a Region of Active Export.

Exosomes are small extracellular vesicles that carry heterogeneous cargo, including RNA, between cells. Increasing evidence suggests that exosomes are important mediators of intercellular communication and biomarkers of disease. Despite this, the variability of exosomal RNA between individuals has not been well quantified.

MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes.

miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile.

miR-503 represses human cell proliferation and directly targets the oncogene DDHD2 by non-canonical target pairing.

Background: The pathways regulating the transition of mammalian cells from quiescence to proliferation are mediated by multiple miRNAs. Despite significant improvements in our understanding of miRNA targeting, the majority of miRNA regulatory networks are still largely unknown and require experimental validation.

Results: Here we identified miR-503, miR-103, and miR-494 as negative regulators of proliferation in primary human cells.

A Myc-microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes.

The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. However, the interplay between transcription factors and miRNAs in modulating gene regulatory networks involved in human cell proliferation is largely unknown. Here we show that the miRNA miR-22 promotes proliferation in primary human cells, and through a combination of Argonaute-2 immunoprecipitation and reporter assays, we identified multiple novel targets of miR-22, including several cell-cycle arrest genes that mediate the effects of the tumor-suppressor p53.

Measuring stigma among health care and social service providers: The HIV/AIDS Provider Stigma Inventory.

Initial validation of the HIV/AIDS Provider Stigma Inventory (HAPSI), piloted on a sample of 174 nursing students, supported the psychometric qualities of a suite of measures capturing tendencies to stigmatize and discriminate against people living with HIV/AIDS (PLHA). Derived from social psychology and mindfulness theories, separate scales addressing awareness, acceptance, and action were designed to include notions of labeling, stereotyping, outgrouping, and discriminating. These were enhanced to capture differences associated with personal characteristics of PLHA that trigger secondary stigma (e.

Life-sustaining treatment decisions: a social work response to meet needs of health care surrogates.

Surrogates must possess essential patient information prior to legitimately exercising legal and moral obligations to act on patients' medical care preferences. This descriptive, in vivo study examined factors influencing surrogate and proxy decisions (N = 132) following life-sustaining treatment decisions. Patient communication and self-efficacy variables accounted for approximately 38% of the variance in surrogates' perceptions of benefits/barriers associated with decision making.

Factors affecting life-sustaining treatment decisions by health care surrogates and proxies: assessing benefits and barriers.

Terri Schiavo's life and death placed international focus on social workers' need to better understand and serve health care surrogates and proxies making life-sustaining treatment decisions on behalf of medical patients. This study (N = 132) reports initial validation of a new measure, the Acceptance of Treatment Choice Inventory (ATCI), designed to evaluate benefits and barriers considered by health care surrogates and proxies prior to making a life-sustaining treatment decision about CPR, tube-feedings, dialysis, and ventilation. Affirming a two-factor model with acceptable reliability and preliminary construct validity, surrogates sought decision support and desired to represent patients' wishes adequately.