Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins.

The muscleblind-like (Mbnl) family of RNA-binding proteins plays important roles in muscle and eye development and in myotonic dystrophy (DM), in which expanded CUG or CCUG repeats functionally deplete Mbnl proteins. We identified transcriptome-wide functional and biophysical targets of Mbnl proteins in brain, heart, muscle, and myoblasts by using RNA-seq and CLIP-seq approaches. This analysis identified several hundred splicing events whose regulation depended on Mbnl function in a pattern indicating functional interchangeability between Mbnl1 and Mbnl2. A nucleotide resolution RNA map associated repression or activation of exon splicing with Mbnl binding near either 3' splice site or near the downstream 5' splice site, respectively. Transcriptomic analysis of subcellular compartments uncovered a global role for Mbnls in regulating localization of mRNAs in both mouse and Drosophila cells, and Mbnl-dependent translation and protein secretion were observed for a subset of mRNAs with Mbnl-dependent localization. These findings hold several new implications for DM pathogenesis.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

Long-read HiFi sequencing of NUDT15: Phased full-gene haplotyping and pharmacogenomic allele discovery.

To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.

CeFra-seq reveals broad asymmetric mRNA and noncoding RNA distribution profiles in Drosophila and human cells.

Cells are highly asymmetrical, a feature that relies on the sorting of molecular constituents, including proteins, lipids, and nucleic acids, to distinct subcellular locales. The localization of RNA molecules is an important layer of gene regulation required to modulate localized cellular activities, although its global prevalence remains unclear. We combine biochemical cell fractionation with RNA-sequencing (CeFra-seq) analysis to assess the prevalence and conservation of RNA asymmetric distribution on a transcriptome-wide scale in Drosophila and human cells. This approach reveals that the majority (∼80%) of cellular RNA species are asymmetrically distributed, whether considering coding or noncoding transcript populations, in patterns that are broadly conserved evolutionarily. Notably, a large number of Drosophila and human long noncoding RNAs and circular RNAs display enriched levels within specific cytoplasmic compartments, suggesting that these RNAs fulfill extra-nuclear functions. Moreover, fraction-specific mRNA populations exhibit distinctive sequence characteristics. Comparative analysis of mRNA fractionation profiles with that of their encoded proteins reveals a general lack of correlation in subcellular distribution, marked by strong cases of asymmetry. However, coincident distribution profiles are observed for mRNA/protein pairs related to a variety of functional protein modules, suggesting complex regulatory inputs of RNA localization to cellular organization.

Acute hepatic porphyrias: Identification of 46 hydroxymethylbilane synthase, 11 coproporphyrinogen oxidase, and 20 protoporphyrinogen oxidase novel mutations.

The acute hepatic porphyrias (AHPs) are inborn errors of heme biosynthesis, which include three autosomal dominant porphyrias, Acute Intermittent Porphyria (AIP), Hereditary Coproporphyria (HCP), and Variegate Porphyria (VP), and the ultra-rare autosomal recessive porphyria, δ-Aminolevulinic Acid Dehydratase Deficiency Porphyria (ADP). AIP, HCP, VP, and ADP each results from loss-of-function (LOF) mutations in their disease-causing genes: hydroxymethylbilane synthase (HMBS); coproporphyrinogen oxidase (CPOX); protoporphyrinogen oxidase (PPOX), and δ-aminolevulinic acid dehydratase (ALAD), respectively. During the 11-year period from January 1, 2007 through December 31, 2017, the Mount Sinai Porphyrias Diagnostic Laboratory diagnosed 315 unrelated AIP individuals with HMBS mutations, including 46 previously unreported mutations, 29 unrelated HCP individuals with CPOX mutations, including 11 previously unreported mutations, and 54 unrelated VP individuals with PPOX mutations, including 20 previously unreported mutations. Overall, of the 1692 unrelated individuals referred for AHP molecular diagnostic testing, 398 (23.5%) had an AHP mutation. Of the 650 family members of mutation-positive individuals tested for an autosomal dominant AHP, 304 (46.8%) had their respective family mutation. These data expand the molecular genetic heterogeneity of the AHPs and document the usefulness of molecular testing to confirm the positive biochemical findings in symptomatic patients and identify at-risk asymptomatic family members.

CeFra-seq: Systematic mapping of RNA subcellular distribution properties through cell fractionation coupled to deep-sequencing.

The subcellular trafficking of RNA molecules is a conserved feature of eukaryotic cells and plays key functions in diverse processes implicating polarised cellular activities. Large-scale imaging and subcellular transcriptomic studies suggest that regulated RNA localization is a highly prevalent process that appears to be disrupted in several neuromuscular disorders. These features underline the importance and usefulness of implementing procedures to assess global transcriptome subcellular distribution properties. Here, we present a method combining biochemical fractionation of cells and high-throughput RNA sequencing (CeFra-seq) that enables rapid and efficient systematic mapping of RNA cytotopic distributions in cells. The described procedure involves biochemical fractionation to derive extracts of nuclear, cytosolic, endomembrane, cytoplasmic insoluble and extracellular material from cell culture lines. The RNA content of each fraction can then be profiled by deep-sequencing, revealing global subcellular signatures. We provide a detailed protocol for the CeFra-seq procedure along with relevant validation steps and data analysis guidelines to graphically represent RNA spatial distribution features. As a complement to imaging approaches, CeFra-seq represents a powerful and scalable tool to investigate global alterations in RNA trafficking.

Clinical Pharmacogenomic MT-RNR1 Screening for Aminoglycoside-Induced Ototoxicity and the Post-Test Counseling Conundrum.

Aminoglycoside antibiotic exposure can result in ototoxicity and irreversible hearing loss among individuals that harbor the m.1555A>G variant in the mitochondrial 12S rRNA gene, MT-RNR1. Importantly, pre-emptive m.1555A>G screening has been shown to reduce the prevalence of pediatric aminoglycoside-induced ototoxicity; however, professional guidelines to support and guide post-test pharmacogenomic counseling in this context are not currently available. This Perspective highlights key issues with delivering MT-RNR1 results, including longitudinal familial care considerations and communicating m.1555A>G heteroplasmy.

Clinical Pharmacogenetics Implementation Consortium Guideline for the Use of Aminoglycosides Based on MT-RNR1 Genotype.

Aminoglycosides are widely used antibiotics with notable side effects, such as nephrotoxicity, vestibulotoxicity, and sensorineural hearing loss (cochleotoxicity). MT-RNR1 is a gene that encodes the 12s rRNA subunit and is the mitochondrial homologue of the prokaryotic 16s rRNA. Some MT-RNR1 variants (i.e., m.1095T>C; m.1494C>T; m.1555A>G) more closely resemble the bacterial 16s rRNA subunit and result in increased risk of aminoglycoside-induced hearing loss. Use of aminoglycosides should be avoided in individuals with an MT-RNR1 variant associated with an increased risk of aminoglycoside-induced hearing loss unless the high risk of permanent hearing loss is outweighed by the severity of infection and safe or effective alternative therapies are not available. We summarize evidence from the literature supporting this association and provide therapeutic recommendations for the use of aminoglycosides based on MT-RNR1 genotype (updates at https://cpicpgx.org/guidelines/ and www.pharmgkb.org).

Characterization of Reference Materials for TPMT and NUDT15: A GeT-RM Collaborative Project.

Pharmacogenetic testing is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the TPMT and NUDT15 variants included in clinical tests. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Material (GeT-RM) coordination program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 19 DNA samples derived from Coriell cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using a variety of commercially available and laboratory developed tests and/or Sanger sequencing. Of the 12 samples characterized for TPMT, newly identified variants include TPMT∗2, ∗6, ∗12, ∗16, ∗21, ∗24, ∗32, ∗33, and ∗40; for the 7 NUDT15 reference material samples, newly identified variants are NUDT15∗2, ∗3, ∗4, ∗5, ∗6, and ∗9. In addition, a novel haplotype, TPMT∗46, was identified in this study. Preexisting data on an additional 11 Coriell samples, as well as some supplemental testing, were used to create comprehensive reference material panels for TPMT and NUDT15. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

Persistent mRNA localization defects and cell death in ALS neurons caused by transient cellular stress.

Persistent cytoplasmic aggregates containing RNA binding proteins (RBPs) are central to the pathogenesis of late-onset neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). These aggregates share components, molecular mechanisms, and cellular protein quality control pathways with stress-induced RNA granules (SGs). Here, we assess the impact of stress on the global mRNA localization landscape of human pluripotent stem cell-derived motor neurons (PSC-MNs) using subcellular fractionation with RNA sequencing and proteomics. Transient stress disrupts subcellular RNA and protein distributions, alters the RNA binding profile of SG- and ALS-relevant RBPs and recapitulates disease-associated molecular changes such as aberrant splicing of STMN2. Although neurotypical PSC-MNs re-establish a normal subcellular localization landscape upon recovery from stress, cells harboring ALS-linked mutations are intransigent and display a delayed-onset increase in neuronal cell death. Our results highlight subcellular molecular distributions as predictive features and underscore the utility of cellular stress as a paradigm to study ALS-relevant mechanisms.

Development and Analytical Validation of a 29 Gene Clinical Pharmacogenetic Genotyping Panel: Multi-Ethnic Allele and Copy Number Variant Detection.

To develop a novel pharmacogenetic genotyping panel, a multidisciplinary team evaluated available evidence and selected 29 genes implicated in interindividual drug response variability, including 130 sequence variants and additional copy number variants (CNVs). Of the 29 genes, 11 had guidelines published by the Clinical Pharmacogenetics Implementation Consortium. Targeted genotyping and CNV interrogation were accomplished by multiplex single-base extension using the MassARRAY platform (Agena Biosciences) and multiplex ligation-dependent probe amplification (MRC Holland), respectively. Analytical validation of the panel was accomplished by a strategic combination of > 500 independent tests performed on 170 unique reference material DNA samples, which included sequence variant and CNV accuracy, reproducibility, and specimen (blood, saliva, and buccal swab) controls. Among the accuracy controls were 32 samples from the 1000 Genomes Project that were selected based on their enrichment of sequence variants included in the pharmacogenetic panel (VarCover.org). Coupled with publicly available samples from the Genetic Testing Reference Materials Coordination Program (GeT-RM), accuracy validation material was available for the majority (77%) of interrogated sequence variants (100% with average allele frequencies > 0.1%), as well as additional structural alleles with unique copy number signatures (e.g., CYP2D6*5, *13, *36, *68; CYP2B6*29; and CYP2C19*36). Accuracy and reproducibility for both genotyping and copy number were > 99.9%, indicating that the optimized panel platforms were precise and robust. Importantly, multi-ethnic allele frequencies of the interrogated variants indicate that the vast majority of the general population carries at least one of these clinically relevant pharmacogenetic variants, supporting the implementation of this panel for pharmacogenetic research and/or clinical implementation programs.

Correction: DPH1 syndrome: two novel variants and structural and functional analyses of seven missense variants identified in syndromic patients.

Following the publication of the article, it was noted that the last column in Table 1, the total % should have read 5/8 (62.5) for the 'Epilepsy' row, and not 5.7 (71.4). This has now been amended in the HTML and PDF of the original article.

DPH1 syndrome: two novel variants and structural and functional analyses of seven missense variants identified in syndromic patients.

DPH1 variants have been associated with an ultra-rare and severe neurodevelopmental disorder, mainly characterized by variable developmental delay, short stature, dysmorphic features, and sparse hair. We have identified four new patients (from two different families) carrying novel variants in DPH1, enriching the clinical delineation of the DPH1 syndrome. Using a diphtheria toxin ADP-ribosylation assay, we have analyzed the activity of seven identified variants and demonstrated compromised function for five of them [p.(Leu234Pro); p.(Ala411Argfs*91); p.(Leu164Pro); p.(Leu125Pro); and p.(Tyr112Cys)]. We have built a homology model of the human DPH1-DPH2 heterodimer and have performed molecular dynamics simulations to study the effect of these variants on the catalytic sites as well as on the interactions between subunits of the heterodimer. The results show correlation between loss of activity, reduced size of the opening to the catalytic site, and changes in the size of the catalytic site with clinical severity. This is the first report of functional tests of DPH1 variants associated with the DPH1 syndrome. We demonstrate that the in vitro assay for DPH1 protein activity, together with structural modeling, are useful tools for assessing the effect of the variants on DPH1 function and may be used for predicting patient outcomes and prognoses.

Characterization of the 3p12.3-pcen region associated with tumor suppression in a novel ovarian cancer cell line model genetically modified by chromosome 3 fragment transfer.

The genetic analysis of nontumorigenic radiation hybrids generated by transfer of chromosome 3 fragments into the tumorigenic OV-90 ovarian cancer cell line identified the 3p12.3-pcen region as a candidate tumor suppressor gene (TSG) locus. In the present study, polymorphic microsatellite repeat analysis of the hybrids further defined the 3p12.3-pcen interval to a 16.1 Mb common region containing 12 known or hypothetical genes: 3ptel-ROBO2-ROBO1-GBE1-CADM2-VGLL3-CHMP2B-POU1F1-HTR1F-CGGBP1-ZNF654-C3orf38-EPHA3-3pcen. Seven of these genes, ROBO1, GBE1, VGLL3, CHMP2B, CGGBP1, ZNF654, and C3orf38, exhibited gene expression in the hybrids, placing them as top TSG candidates for further analysis. The expression of all but one (VGLL3) of these genes was also detected in the parental OV-90 cell line. Mutations were not identified in a comparative sequence analysis of the predicted protein coding regions of these candidates in OV-90 and donor normal chromosome 3 contig. However, the nondeleterious sequence variants identified in the transcribed regions distinguished parent of origin alleles for ROBO1, VGLL3, CHMP2B, and CGGBP1 and cDNA sequencing of the hybrids revealed biallelic expression of these genes. Interestingly, underexpression of VGLL3 and ZNF654 were observed in malignant ovarian tumor samples as compared with primary cultures of normal ovarian surface epithelial cells or benign ovarian tumors, and this occurred regardless of allelic content of 3p12.3-pcen. The results taken together suggest that dysregulation of VGLL3 and/or ZNF654 expression may have affected pathways important in ovarian tumorigenesis which was offset by the transfer of chromosome 3 fragments in OV-90, a cell line hemizygous for 3p.

Developmentally regulated elimination of damaged nuclei involves a Chk2-dependent mechanism of mRNA nuclear retention.

The faithful execution of embryogenesis relies on the ability of organisms to respond to genotoxic stress and to eliminate defective cells that could otherwise compromise viability. In syncytial-stage Drosophila embryos, nuclei with excessive DNA damage undergo programmed elimination through an as-yet poorly understood process of nuclear fallout at the midblastula transition. We show that this involves a Chk2-dependent mechanism of mRNA nuclear retention that is induced by DNA damage and prevents the translation of specific zygotic mRNAs encoding key mitotic, cytoskeletal, and nuclear proteins required to maintain nuclear viability. For histone messages, we show that nuclear retention involves Chk2-mediated inactivation of the Drosophila stem loop binding protein (SLBP), the levels of which are specifically depleted in damaged nuclei following Chk2 phosphorylation, an event that contributes to nuclear fallout. These results reveal a layer of regulation within the DNA damage surveillance systems that safeguard genome integrity in eukaryotes.

The many functions of mRNA localization during normal development and disease: from pillar to post.

The regulated intracellular trafficking and localized translation of mRNA molecules represents an important and prevalent mechanism of gene regulation. This process plays a key role in modulating asymmetric protein distribution linked to a wide variety of biological processes in different organisms and cell types. In this review, we begin by discussing the diverse biological functions, advantages, and mechanisms of mRNA localization that have been characterized to date. We then review recent technological innovations in RNA imaging and functional genomics methods that will undoubtedly provide powerful new strategies for the elucidation of mRNA trafficking pathways. Finally, we discuss several examples linking human disease pathogenesis to defects in transcript localization, which further underlines the critical importance of this gene regulatory mechanism.

Whole mount RNA fluorescent in situ hybridization of Drosophila embryos.

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Influence of monolayer, spheroid, and tumor growth conditions on chromosome 3 gene expression in tumorigenic epithelial ovarian cancer cell lines.

BACKGROUND: Expression microarray analyses of epithelial ovarian cancer (EOC) cell lines may be exploited to elucidate genetic and epigenetic events important in this disease. A possible variable is the influence of growth conditions on discerning candidates. The present study examined the influence of growth conditions on the expression of chromosome 3 genes in the tumorigenic EOC cell lines, OV-90, TOV-21G and TOV-112D using Affymetrix GeneChip(R) HG-U133A expression microarray analysis. METHODS: Chromosome 3 gene expression profiles (n = 1147 probe sets, representing 735 genes) were extracted from U133A expression microarray analyses of the EOC cell lines OV-90, TOV-21G and TOV-112D that were grown as monolayers, spheroids or nude mouse xenografts and monolayers derived from these tumors. Hierarchical cluster analysis was performed to compare chromosome 3 transcriptome patterns of each growth condition. Differentially expressed genes were identified and characterized by two-way comparative analyses of fold-differences in gene expression between monolayer cultures and each of the other growth conditions, and between the maximum and minimum values of expression of all growth conditions for each EOC cell line. RESULTS: An overall high degree of similarity (> 90%) in gene expression was observed when expression values of alternative growth conditions were compared within each EOC cell line group. Two-way comparative analysis of each EOC cell line grown in an alternative condition relative to the monolayer culture showed that overall less than 15% of probe sets exhibited at least a 3-fold difference in expression profile. Less than 23% of probe sets exhibited greater than 3-fold differences in gene expression in comparisons of the maximum and minimum value of expression of all growth conditions within each EOC cell line group. The majority of these differences were less than 5-fold. There were 17 genes in common which were differentially expressed in all EOC cell lines. However, the patterns of expression of these genes were not necessarily the same for each growth condition when one cell line was compared with another. CONCLUSION: The various alternative in vivo and in vitro growth conditions of tumorigenic EOC cell lines appeared to modestly influence the global chromosome 3 transcriptome supporting the notion that the in vitro cell line models are a viable option for testing gene candidates.