RESUMO
Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.
Assuntos
Sistemas CRISPR-Cas , Reparo de DNA por Recombinação , Humanos , Edição de Genes/métodos , Reparo do DNA , Reparo do DNA por Junção de ExtremidadesRESUMO
We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.
Assuntos
Saccharomyces cerevisiae , Spliceossomos , Humanos , Íntrons/genética , Splicing de RNA , Saccharomyces cerevisiae/genética , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Eukaryotic protein synthesis generally initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but the quantitative importance of this context in different species is unclear. We tested this concept in two pathogenic Cryptococcus yeast species by genome-wide mapping of translation and of mRNA 5' and 3' ends. We observed thousands of AUG-initiated upstream open reading frames (uORFs) that are a major contributor to translation repression. uORF use depends on the Kozak sequence context of its start codon, and uORFs with strong contexts promote nonsense-mediated mRNA decay. Transcript leaders in Cryptococcus and other fungi are substantially longer and more AUG-dense than in Saccharomyces. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Analysis of other fungal species shows that such dual-localization is also predicted to be common in the ascomycete mould, Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that quantitatively programs both the expression and the structures of proteins in diverse fungi.
Assuntos
Códon de Iniciação/química , Cryptococcus/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Iniciação Traducional da Cadeia Peptídica , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Mapeamento Cromossômico , Códon de Iniciação/metabolismo , Cryptococcus/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Fases de Leitura Aberta , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Especificidade da EspécieRESUMO
The human pathogenic yeast Cryptococcus neoformans silences transposable elements using endo-siRNAs and an Argonaute, Ago1. Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1, and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is independent of histone H3 lysine 9 methylation. We describe here an insertional mutagenesis screen for factors required to suppress the mobilization of the C. neoformans HARBINGER family DNA transposon HAR1 Validation experiments uncovered five novel genes (RDE1-5) required for HAR1 suppression and global production of suppressive endo-siRNAs. The RDE genes do not impact transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover. RDE3 encodes a non-Dicer RNase III related to S. cerevisiae Rnt1, RDE4 encodes a predicted terminal nucleotidyltransferase, while RDE5 has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies suggest that Rde3 and Rde5 are physically associated. RDE1 encodes a G-patch protein homologous to the S. cerevisiae Sqs1/Pfa1, a nucleolar protein that directly activates the essential helicase Prp43 during rRNA biogenesis. Rde1 copurifies Rde2, another novel protein obtained in the screen, as well as Ago1, a homolog of Prp43, and numerous predicted nucleolar proteins. We also describe the isolation of conditional alleles of PRP43, which are defective in RNAi. This work reveals unanticipated requirements for a non-Dicer RNase III and presumptive nucleolar factors for endo-siRNA biogenesis and transposon mobilization suppression in C. neoformans.
Assuntos
Cryptococcus neoformans/genética , Elementos de DNA Transponíveis , Interferência de RNA , Ribonuclease III/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Criptococose/microbiologia , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica , Humanos , Mutagênese Insercional , Mutação , RNA Interferente Pequeno/genéticaRESUMO
Tools to understand how the spliceosome functions in vivo have lagged behind advances in the structural biology of the spliceosome. Here, methods are described to globally profile spliceosome-bound pre-mRNA, intermediates, and spliced mRNA at nucleotide resolution. These tools are applied to three yeast species that span 600 million years of evolution. The sensitivity of the approach enables the detection of canonical and non-canonical events, including interrupted, recursive, and nested splicing. This application of statistical modeling uncovers independent roles for the size and position of the intron and the number of introns per transcript in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal the ATP-dependent discard of numerous endogenous substrates after spliceosome assembly in vivo and connect this discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology used to investigate an RNP central to eukaryotic gene expression.
Assuntos
Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Teorema de Bayes , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Imunoprecipitação , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telomerase/genética , Telomerase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , GMP Cíclico/análogos & derivados , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , GMP Cíclico/química , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Conformação de Ácido Nucleico , Riboswitch/genética , Sistemas do Segundo Mensageiro , Transdução de SinaisRESUMO
The platelet serotonergic system has potential biomarker utility for major depressive disorder (MDD). In the present study, platelet expression of 5-HT1A receptors and serotonin transporter (SERT) proteins, and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were quantified in 53 patients with MDD and 22 unaffected controls. All were drug-free, non-smokers and had no other psychiatric and cardiovascular comorbidity. The severity of depression symptoms was evaluated using the 17-item Hamilton Depression Rating Scale (HAMD-17) and the Self-rating Depression Scale (SDS). Patients with MDD had significantly higher expression of platelet 5-HT1A receptors but significantly lower contents of platelet 5-HT, platelet-poor plasma (PPP) 5-HT and PPP 5-HIAA compared to healthy controls, and this was correlated with the severity of depression. SERT expression did not differ between the two groups. Correlation analysis confirmed a strong, inverse relationship between the 5-HT1A receptor expression and the 5-HT and 5-HIAA levels. Thus overexpression of platelet 5-HT1A receptors and reduced 5-HT tone may function as a peripheral marker of depression.
Assuntos
Plaquetas/metabolismo , Transtorno Depressivo Maior/sangue , Receptor 5-HT1A de Serotonina/sangue , Adulto , Análise de Variância , Feminino , Humanos , Ácido Hidroxi-Indolacético/sangue , Masculino , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , Serotonina/sangue , Estatística como Assunto , Adulto JovemRESUMO
The vitamin folate is required for methionine homeostasis in all organisms. In addition to its role in protein synthesis, methionine is the precursor to S-adenosyl-methionine (SAM), which is used in myriad cellular methylation reactions, including all histone methylation reactions. Here, we demonstrate that folate and methionine deficiency led to reduced methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. The effect of nutritional deficiency on H3K79 methylation was less pronounced, but was exacerbated in S. cerevisiae carrying a hypomorphic allele of Dot1, the enzyme responsible for H3K79 methylation. This result suggested a hierarchy of epigenetic modifications in terms of their susceptibility to nutritional limitations. Folate deficiency caused changes in gene transcription that mirrored the effect of complete loss of H3K4 methylation. Histone methylation was also found to respond to nutritional deficiency in the fission yeast Schizosaccharomyces pombe and in human cells in culture.
Assuntos
Epigênese Genética , Ácido Fólico/metabolismo , Metionina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Feminino , Antagonistas do Ácido Fólico/efeitos adversos , Antagonistas do Ácido Fólico/uso terapêutico , Deficiência de Ácido Fólico/complicações , Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Recém-Nascido , Células K562 , Metilação , Defeitos do Tubo Neural/etiologia , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Gravidez , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidade da EspécieRESUMO
Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.