RESUMO
The Src substrate associated in mitosis of 68 kDa (Sam68) is a KH-type RNA binding protein that has been shown to regulate several aspects of RNA metabolism; however, its physiologic role has remained elusive. Herein we report the generation of Sam68-null mice by homologous recombination. Aged Sam68-/- mice preserved their bone mass, in sharp contrast with 12-month-old wild-type littermates in which bone mass was decreased up to approximately 75%. In fact, the bone volume of the 12-month-old Sam68-/- mice was virtually indistinguishable from that of 4-month-old wild-type or Sam68-/- mice. Sam68-/- bone marrow stromal cells had a differentiation advantage for the osteogenic pathway. Moreover, the knockdown of Sam68 using short hairpin RNA in the embryonic mesenchymal multipotential progenitor C3H10T1/2 cells resulted in more pronounced expression of the mature osteoblast marker osteocalcin when differentiation was induced with bone morphogenetic protein-2. Cultures of mouse embryo fibroblasts generated from Sam68+/+ and Sam68-/- littermates were induced to differentiate into adipocytes with culture medium containing pioglitazone and the Sam68-/- mouse embryo fibroblasts shown to have impaired adipocyte differentiation. Furthermore, in vivo it was shown that sections of bone from 12-month-old Sam68-/- mice had few marrow adipocytes compared with their age-matched wild-type littermate controls, which exhibited fatty bone marrow. Our findings identify endogenous Sam68 as a positive regulator of adipocyte differentiation and a negative regulator of osteoblast differentiation, which is consistent with Sam68 being a modulator of bone marrow mesenchymal cell differentiation, and hence bone metabolism, in aged mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ligação a DNA/fisiologia , Osteoporose/genética , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA/fisiologia , Adipócitos/citologia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Osteoblastos/citologia , Osteocalcina/genética , Recombinação GenéticaRESUMO
The Src associated substrate in mitosis of 68kDa, Sam68, is an RNA-binding protein that belongs to the KH domain family of proteins. KH-type RNA binding proteins are known to mediate high affinity RNA binding and regulate RNA metabolism including pre-mRNA splicing, mRNA export and protein translation. The RNA binding specificity of Sam68 as well as its RNA targets are poorly understood. Herein we cross-linked mRNA associated with Sam68 and identified some of the mRNA associated with the Sam68 RNA binding protein complex. By using this strategy, we have identified 23 mRNAs that are associated with the immunoprecipitated endogenous Sam68 protein complex. Five of the identified mRNAs were validated by co-immunoprecipitation assay followed by reverse transcription PCR confirming that we had indeed identified mRNAs associated with the Sam68 protein complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Reagentes de Ligações Cruzadas , Imunoprecipitação , Camundongos , Complexos Multiproteicos , Células NIH 3T3 , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Here we describe a process for the generation of oligonucleotide libraries representative of a given nucleic acid. Starting from at random pool of DNA oligonucleotides, the technique selects only those that hybridize to the nucleic acid template. This selection yields a highly specific library that represents an oligonucleotide image of the chosen template. The novel quality of this approach is the generation of amplifiable oligonucleotide probes that are of unique length and are easily subjected to differential selection. Here we apply this technique to produce different genomic oligonucleotide libraries and show that these genomic oligonucleotide libraries do not cross-hybridize. Differential selection of these genomic oligonucleotide libraries produces oligonucleotides that can be used in the identification, characterzation, and isolation of nucleic acids.
Assuntos
Biblioteca Genômica , Sondas de Oligonucleotídeos/isolamento & purificação , Adenoviridae/genética , Bacteriófago lambda/genética , Southern Blotting , DNA Viral/isolamento & purificação , Métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/isolamento & purificação , Reação em Cadeia da PolimeraseRESUMO
This article summarizes bioanalytical avenues for the determination of siRNA and oligonucleotide therapeutics, with an emphasis on hybridization methods. Aspects of the chemistry and delivery of investigational oligonucleotide therapeutics are considered. The nature of the oligonucleotide under investigation will dictate the best analytical course of action; each method has its advantages and disadvantages, depending upon the oligonucleotide test article and the anticipated toxicokinetic and pharmacokinetic study parameters. Stringent method development and specific validation criteria are essential to attain the best quality results in support of a regulatory filing.