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1.
JCO Clin Cancer Inform ; 5: 1085-1095, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34731027

RESUMO

PURPOSE: The ability of next-generation sequencing (NGS) assays to interrogate thousands of genomic loci has revolutionized genetic testing. However, translation to the clinic is impeded by false-negative results that pose a risk to patients. In response, regulatory bodies are calling for reliability measures to be reported alongside NGS results. Existing methods to estimate reliability do not account for sample- and position-specific variability, which can be significant. Here, we report an approach that computes reliability metrics for every genomic position and sample interrogated by an NGS assay. METHODS: Our approach predicts the limit of detection (LOD), the lowest reliably detectable variant fraction, by taking technical factors into account. We initially explored how LOD is affected by input material amount, library conversion rate, sequencing coverage, and sequencing error rate. This revealed that LOD depends heavily on genomic context and sample properties. Using these insights, we developed a computational approach to predict LOD on the basis of a biophysical model of the NGS workflow. We focused on targeted assays for cell-free DNA, but, in principle, this approach applies to any NGS assay. RESULTS: We validated our approach by showing that it accurately predicts LOD and distinguishes reliable from unreliable results when screening 580 lung cancer samples for actionable mutations. Compared with a standard variant calling workflow, our approach avoided most false negatives and improved interassay concordance from 94% to 99%. CONCLUSION: Our approach, which we name LAVA (LOD-aware variant analysis), reports the LOD for every position and sample interrogated by an NGS assay. This enables reliable results to be identified and improves the transparency and safety of genetic tests.


Assuntos
Neoplasias Pulmonares , Nucleotídeos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Mutação , Reprodutibilidade dos Testes
2.
Methods Mol Biol ; 2167: 287-301, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712926

RESUMO

Some long non-coding RNA (lncRNA) genes encode a functional RNA product, whereas others act as DNA elements or via the act of transcription . We describe here a ribozyme-based approach to deplete an endogenous lncRNA in mouse embryonic stem cells, with minimal disruption of its gene. This enables the role of the lncRNA product to be tested.


Assuntos
Células-Tronco Embrionárias/metabolismo , Edição de Genes/métodos , RNA Catalítico/genética , RNA Longo não Codificante/genética , RNA Viral/genética , Animais , Sistemas CRISPR-Cas , Células-Tronco Embrionárias/enzimologia , Camundongos , Conformação de Ácido Nucleico , RNA Catalítico/metabolismo , RNA Longo não Codificante/metabolismo , Recombinação Genética
3.
Genes Dev ; 33(17-18): 1221-1235, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371437

RESUMO

TRIM71/LIN-41, a phylogenetically conserved regulator of development, controls stem cell fates. Mammalian TRIM71 exhibits both RNA-binding and protein ubiquitylation activities, but the functional contribution of either activity and relevant primary targets remain poorly understood. Here, we demonstrate that TRIM71 shapes the transcriptome of mouse embryonic stem cells (mESCs) predominantly through its RNA-binding activity. We reveal that TRIM71 binds targets through 3' untranslated region (UTR) hairpin motifs and that it acts predominantly by target degradation. TRIM71 mutations implicated in etiogenesis of human congenital hydrocephalus impair target silencing. We identify a set of primary targets consistently regulated in various human and mouse cell lines, including MBNL1 (Muscleblind-like protein 1). MBNL1 promotes cell differentiation through regulation of alternative splicing, and we demonstrate that TRIM71 promotes embryonic splicing patterns through MBNL1 repression. Hence, repression of MBNL1-dependent alternative splicing may contribute to TRIM71's function in regulating stem cell fates.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Proteínas de Ligação a RNA/genética , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Células-Tronco Embrionárias , Humanos , Camundongos , Camundongos Knockout , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Domínios Proteicos/genética , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo
4.
Life Sci Alliance ; 1(4): e201800124, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30456373

RESUMO

Eukaryotic genomes produce RNAs lacking protein-coding potential, with enigmatic roles. We integrated three approaches to study large intervening noncoding RNA (lincRNA) gene functions. First, we profiled mouse embryonic stem cells and neural precursor cells at single-cell resolution, revealing lincRNAs expressed in specific cell types, cell subpopulations, or cell cycle stages. Second, we assembled a transcriptome-wide atlas of nuclear lincRNA degradation by identifying targets of the exosome cofactor Mtr4. Third, we developed a reversible depletion system to separate the role of a lincRNA gene from that of its RNA. Our approach distinguished lincRNA loci functioning in trans from those modulating local gene expression. Some genes express stable and/or abundant lincRNAs in single cells, but many prematurely terminate transcription and produce lincRNAs rapidly degraded by the nuclear exosome. This suggests that besides RNA-dependent functions, lincRNA loci act as DNA elements or through transcription. Our integrative approach helps distinguish these mechanisms.

5.
PLoS Genet ; 11(12): e1005735, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26694144

RESUMO

Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression.


Assuntos
Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética , Regiões 3' não Traduzidas , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Cell Stem Cell ; 17(4): 471-85, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26431182

RESUMO

Embryonic stem cell (ESC) culture conditions are important for maintaining long-term self-renewal, and they influence cellular pluripotency state. Here, we report single cell RNA-sequencing of mESCs cultured in three different conditions: serum, 2i, and the alternative ground state a2i. We find that the cellular transcriptomes of cells grown in these conditions are distinct, with 2i being the most similar to blastocyst cells and including a subpopulation resembling the two-cell embryo state. Overall levels of intercellular gene expression heterogeneity are comparable across the three conditions. However, this masks variable expression of pluripotency genes in serum cells and homogeneous expression in 2i and a2i cells. Additionally, genes related to the cell cycle are more variably expressed in the 2i and a2i conditions. Mining of our dataset for correlations in gene expression allowed us to identify additional components of the pluripotency network, including Ptma and Zfp640, illustrating its value as a resource for future discovery.


Assuntos
Células-Tronco Embrionárias Murinas/fisiologia , RNA/genética , Transcriptoma , Animais , Diferenciação Celular/genética , Células Cultivadas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , RNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Célula Única
7.
Mol Cell ; 45(4): 435-6, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22365825

RESUMO

In this issue of Molecular Cell, single-cell analyses by Bumgarner et al. (2012) reveal how two antagonistic long noncoding RNAs at the FLO11 locus define a toggle responsible for morphological heterogeneity in genetically identical populations of budding yeast.

8.
Trends Genet ; 27(10): 422-32, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21741109

RESUMO

Eukaryotic genomes accommodate numerous types of information within diverse DNA and RNA sequence elements. At many loci, these elements overlap and the same sequence is read multiple times during the production, processing, localization, function and turnover of a single transcript. Moreover, two or more transcripts from the same locus might use a common sequence in different ways, to perform distinct biological roles. Recent results show that many transcripts also undergo post-transcriptional cleavage to release specific fragments, which can then function independently. This phenomenon appears remarkably widespread, with even well-documented transcript classes such as messenger RNAs yielding fragments. RNA fragmentation significantly expands the already extraordinary spectrum of transcripts present within eukaryotic cells, and also calls into question how the 'gene' should be defined.


Assuntos
MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/metabolismo , Processamento Alternativo , Animais , DNA/genética , Endorribonucleases/metabolismo , Pleiotropia Genética , Humanos , MicroRNAs/química , MicroRNAs/genética , Dobramento de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA de Transferência/química , RNA de Transferência/genética , Transcrição Gênica
9.
J Mol Biol ; 382(4): 870-83, 2008 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-18691600

RESUMO

The Escherichia coli RNA degradosome is a multienzyme assembly that functions in transcript turnover and maturation of structured RNA precursors. We have developed a procedure to reconstitute the RNA degradosome from recombinant components using modular coexpression vectors. The reconstituted assembly can be purified on a scale that has enabled biochemical and biophysical analyses, and we compare the properties of recombinant and cell-extracted RNA degradosomes. We present evidence that auxiliary protein components can be recruited to the 'superprotomer' core of the assembly through a dynamic equilibrium involving RNA intermediaries. We discuss the implications for the regulation of RNA degradosome function in vivo.


Assuntos
Endorribonucleases , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos , Polirribonucleotídeo Nucleotidiltransferase , RNA Helicases , RNA Bacteriano/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Conformação de Ácido Nucleico , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Polirribonucleotídeo Nucleotidiltransferase/química , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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