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1.
Int J Mol Sci ; 23(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36499391

RESUMO

The SH-SY5Y neuroblastoma cells are a widely used in vitro model approximating neurons for testing the target engagement of therapeutics designed for neurodegenerative diseases and pain disorders. However, their potential as a model for receptor-mediated delivery and uptake of novel modalities, such as antibody-drug conjugates, remains understudied. Investigation of the SH-SY5Y cell surfaceome will aid in greater in vitro to in vivo correlation of delivery and uptake, thereby accelerating drug discovery. So far, the majority of studies have focused on total cell proteomics from undifferentiated and differentiated SH-SY5Y cells. While some studies have investigated the expression of specific proteins in neuroblastoma tissue, a global approach for comparison of neuroblastoma cell surfaceome to the brain and dorsal root ganglion (DRG) neurons remains uninvestigated. Furthermore, an isoform-specific evaluation of cell surface proteins expressed on neuroblastoma cells remains unexplored. In this study, we define a bioinformatic workflow for the identification of high-confidence surface proteins expressed on brain and DRG neurons using tissue proteomic and transcriptomic data. We then delineate the SH-SY5Y cell surfaceome by surface proteomics and show that it significantly overlaps with the human brain and DRG neuronal surface proteome. We find that, for 32% of common surface proteins, SH-SY5Y-specific major isoforms are alternatively spliced, maintaining their protein-coding ability, and are predicted to localize to the cell surface. Validation of these isoforms using surface proteomics confirms a SH-SY5Y-specific alternative NRCAM (neuron-glia related cell adhesion molecule) isoform, which is absent in typical brain neurons, but present in neuroblastomas, making it a receptor of interest for neuroblastoma-specific therapeutics.


Assuntos
Neuroblastoma , Humanos , Neuroblastoma/terapia , Neuroblastoma/tratamento farmacológico , Linhagem Celular Tumoral , Proteômica , Neurônios/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Membrana/metabolismo
2.
Proteomics ; 22(17): e2200002, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35678367

RESUMO

Cell surface proteins form a major fraction of the druggable proteome and can be used for tissue-specific delivery of oligonucleotide/cell-based therapeutics. Surface protein isoforms are regulated by alternative splicing, which drives subcellular localization and transmembrane (TM) topology thereby shaping cell type specific signatures. Current advances in multiomic approaches have developed interest in discovery of tissue-specific alternatively spliced or novel surface protein isoforms. However, there exists a need for bioinformatic approaches for rapidly benchmarking the large number of isoforms identified by these approaches. To address this gap, we have developed, surfaltr, an R package which takes user input isoforms, pairs them with the known principal isoform of the gene, predicts TM topologies, and generates a customizable graphical output. Further, surfaltr facilitates prioritization of topologically diverse isoform pairs through incorporation of three different ranking metrics and through protein alignment functions. Here, we demonstrate the utility of our R package by evaluating the mouse retina-specific novel surface protein isoforms identified in Ray et al. 2020. surfaltr is freely available through Bioconductor (https://bioconductor.org/packages/surfaltr) and the vignette provides extensive instructions for implementation.


Assuntos
Benchmarking , Proteínas de Membrana , Software , Animais , Biologia Computacional , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética
3.
PLoS Genet ; 16(6): e1008830, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502192

RESUMO

Many post-transcriptional mechanisms operate via mRNA 3'UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3'UTR introns (3'UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3'UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3'UI in zebrafish, mouse and humans. foxo3b is one such proximal 3'UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3'UIs may serve gene regulatory function in vertebrates.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Códon de Terminação , Conjuntos de Dados como Assunto , Embrião não Mamífero , Éxons/genética , Redes Reguladoras de Genes/genética , Homozigoto , Humanos , Íntrons/genética , Camundongos , Músculo Esquelético/inervação , Mutagênese , Mutação , Crescimento Neuronal/genética , Proteínas Nucleares/genética , Terminação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Alinhamento de Sequência , Regulação para Cima , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
4.
J Vis Exp ; (149)2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31355789

RESUMO

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT's advantages and applications and discuss some of its limitations.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Células HEK293 , Humanos , Reação em Cadeia da Polimerase
5.
Methods Mol Biol ; 1680: 1-28, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29030838

RESUMO

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.


Assuntos
Proteínas Argonautas/metabolismo , Clonagem Molecular , Sequenciamento de Nucleotídeos em Larga Escala , Pequeno RNA não Traduzido/genética , Biologia Computacional/métodos , Interpretação Estatística de Dados , Biblioteca Gênica , Pequeno RNA não Traduzido/isolamento & purificação , Pequeno RNA não Traduzido/metabolismo , Análise de Sequência de RNA
6.
Artigo em Inglês | MEDLINE | ID: mdl-28008720

RESUMO

During messenger RNA (mRNA) biogenesis and processing in the nucleus, many proteins are imprinted on mRNAs assembling them into messenger ribonucleoproteins (mRNPs). Some of these proteins remain stably bound within mRNPs and have a long-lasting impact on their fate. One of the best-studied examples is the exon junction complex (EJC), a multiprotein complex deposited primarily 24 nucleotides upstream of exon-exon junctions as a consequence of pre-mRNA splicing. The EJC maintains a stable, sequence-independent, hold on the mRNA until its removal during translation in the cytoplasm. Acting as a molecular shepherd, the EJC travels with mRNA across the cellular landscape coupling pre-mRNA splicing to downstream, posttranscriptional processes such as mRNA export, mRNA localization, translation, and nonsense-mediated mRNA decay (NMD). In this review, we discuss our current understanding of the EJC's functions during these processes, and expound its newly discovered functions (e.g., pre-mRNA splicing). Another focal point is the recently unveiled in vivo EJC interactome, which has shed new light on the EJC's location on the spliced RNAs and its intimate relationship with other mRNP components. We summarize new strides being made in connecting the EJC's molecular function with phenotypes, informed by studies of human disorders and model organisms. The progress toward understanding EJC functions has revealed, in its wake, even more questions, which are discussed throughout. WIREs RNA 2017, 8:e1411. doi: 10.1002/wrna.1411 For further resources related to this article, please visit the WIREs website.


Assuntos
Éxons/genética , Splicing de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Humanos , RNA Mensageiro/genética , Ribonucleoproteínas/genética
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