RESUMO
The interactions between various RNA-binding proteins (RBPs) and the RNA transcripts they bind strongly influence posttranscriptional control of gene expression in vertebrates. The hundreds of vertebrate RBPs that have been identified within the genome, often with multiple RNA recognition motifs, are capable of recognizing specific target RNA sequences mediating the maturation, movement, and translational state of their RNA cargoes. To identify the cargoes associated with a specific RBP, we have developed a technique called antibody-positioned RNA amplification (APRA), which positions an oligonucleotide with a degenerate priming sequence in proximity to the RNAs sequestered by a specific RBP. The conjugation of the priming oligonucleotide to the antibody by itself does not interfere with the antibody's intrinsic affinity for the target RBP epitope, thus enabling RNA targets to be reverse-transcribed and amplified via a T7 bacteriophage RNA polymerase promoter sequence located upstream of the degenerate priming sequence in the oligonucleotide. By identifying the mRNA transcripts associated with the RBP in situ, we may be able to ascertain the significance of their temporal expression and physiological activities within the vast transcriptional networks regulating functional responses to stimuli.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Mensageiro/análise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Animais , Anticorpos/metabolismo , Primers do DNA/imunologia , Primers do DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transcrição Reversa , Transcrição Gênica , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
RNA precursors give rise to mRNA after splicing of intronic sequences traditionally thought to occur in the nucleus. Here, we show that intron sequences are retained in a number of dendritically-targeted mRNAs, by using microarray and Illumina sequencing of isolated dendritic mRNA as well as in situ hybridization. Many of the retained introns contain ID elements, a class of SINE retrotransposon. A portion of these SINEs confers dendritic targeting to exogenous and endogenous transcripts showing the necessity of ID-mediated mechanisms for the targeting of different transcripts to dendrites. ID elements are capable of selectively altering the distribution of endogenous proteins, providing a link between intronic SINEs and protein function. As such, the ID element represents a common dendritic targeting element found across multiple RNAs. Retention of intronic sequence is a more general phenomenon than previously thought and plays a functional role in the biology of the neuron, partly mediated by co-opted repetitive sequences.
Assuntos
Citoplasma/genética , Dendritos/genética , Íntrons/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Animais , Células Cultivadas , Citoplasma/metabolismo , Dendritos/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Hibridização In Situ , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
We report that the stress axis-regulated exon (STREX)-containing calcium-activated big potassium (BKCa) channel splice variant expression and physiology are regulated in part by cytoplasmic splicing and intron retention. NextGen sequencing of the mRNA complement of pooled hippocampal dendrite samples found intron 17a (i17a), the intron immediately preceding STREX, in the BKCa mRNA. Further molecular analyses of i17a revealed that the majority of i17a-containing BKCa channel mRNAs associate with STREX. i17a siRNA treatment followed by STREX protein immunocytochemistry demonstrated both reduced levels and altered subcellular distribution of STREX-containing BKCa channel protein. Selective reduction of i17a-BKCa or STREX-BKCa mRNAs induced similar changes in the burst firing properties of hippocampal neurons. Collectively, these data show that STREX splice variant regulation via cytoplasmic splicing and intron retention helps generate STREX-dependent BKCa current diversity in hippocampal neurons.
Assuntos
Processamento Alternativo/genética , Íntrons/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Animais , Dendritos , Hipocampo/citologia , Neurônios , RNA Mensageiro , RatosRESUMO
Few cell types are more adapted for cell-cell signaling than neurons. Their responsiveness lies in the formation of highly specialized compartments composed of unique repertoires of selectively distributed protein complexes generated, in part, by the local translation of mRNAs and regulated by their RNA-binding proteins. Utilizing the selective distribution of these neuronal proteins and the underlying mechanisms that generate the differential patterns of expression as central facets of drug design promises to enhance the therapeutic ratio of a drug. It is in this context that we discuss the unique arrangement of mRNAs, RNA-binding proteins and the protein macromolecular complexes at the dendrite, which is the postsynaptic site of synaptic transmission. Recent advances in identifying the function of dendritic components of the mechanisms of protein and RNA transport, non-nuclear RNA splicing and localized translation underscore their importance as targets of neuropharmacology.
Assuntos
Comunicação Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurofarmacologia , Frações Subcelulares/efeitos dos fármacos , Animais , Sistemas de Liberação de Medicamentos , HumanosRESUMO
High single-channel conductance K+ channels, which respond jointly to membrane depolarization and micromolar concentrations of intracellular Ca2+ ions, arise from extensive cell-specific alternative splicing of pore-forming alpha-subunit mRNAs. Here, we report the discovery of an endogenous BK(Ca) channel alpha-subunit intron-containing mRNA in the cytoplasm of hippocampal neurons. This partially processed mRNA, which comprises approximately 10% of the total BK(Ca) channel alpha-subunit mRNAs, is distributed in a gradient throughout the somatodendritic space. We selectively reduced endogenous cytoplasmic levels of this intron-containing transcript by RNA interference without altering levels of the mature splice forms of the BK(Ca) channel mRNAs. In doing so, we could demonstrate that changes in a unique BK(Ca) channel alpha-subunit intron-containing splice variant mRNA can greatly impact the distribution of the BK(Ca) channel protein to dendritic spines and intrinsic firing properties of hippocampal neurons. These data suggest a new regulatory mechanism for modulating the membrane properties and ion channel gradients of hippocampal neurons.
Assuntos
Hipocampo/fisiologia , Íntrons , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Neurônios/fisiologia , RNA Mensageiro/genética , Potenciais de Ação , Animais , Células Cultivadas , Dendritos , Hipocampo/citologia , Hipocampo/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Neurônios/metabolismo , RNA Interferente Pequeno , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/metabolismoRESUMO
To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3' and 5' UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.
Assuntos
RNA/química , Ribonucleoproteínas/química , Aminoácidos/química , Animais , Sequência de Bases , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , Primers do DNA/química , Corantes Fluorescentes/farmacologia , Hipocampo/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , Neurônios/metabolismo , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Ratos , Ribonucleoproteínas/metabolismo , Fatores de Tempo , Raios UltravioletaRESUMO
The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. We have developed an approach, antibody-positioned RNA amplification (APRA), to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, we have observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP.