RESUMEN
Long non-coding RNAs (lncRNAs) play key roles in cellular pathways and disease progression, yet their molecular mechanisms remain largely understudied. The lncRNA lncTCF7 has been shown to promote tumor progression by recruiting the SWI/SNF complex to the TCF7 promoter, activating its expression and the WNT signaling pathway. However, how lncTCF7 recruits SWI/SNF remains to be determined, and lncTCF7-specific binding partners are unknown. Using RNA-pulldown and quantitative mass spectrometry, we identified a novel interacting protein partner for lncTCF7, SND1, a multifunctional RNA binding protein that can also function as a transcription co-activator. Knockdown analysis of lncTCF7 and SND1 reveals that they are both required for the expression of TCF7. Chromatin immunoprecipitation assays suggest that both SND1 and lncTCF7 are required for recruiting the SWI/SNF chromatin remodeling complex, and these functions, in tandem, activate the expression of TCF7. Finally, using structural probing and RNA-pulldown of lncTCF7 and its subdomains, we highlight the potential binding region for SND1 in the 3'-end of lncTCF7. Overall, this study highlights the critical roles lncRNAs play in regulating gene expression and provides new insights into the complex network of interactions that underlie this process.
Asunto(s)
Endonucleasas , Unión Proteica , ARN Largo no Codificante , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , Endonucleasas/metabolismo , Endonucleasas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Factores de Transcripción TCF/metabolismo , Factores de Transcripción TCF/genética , Factor 1 de Transcripción de Linfocitos TRESUMEN
Long noncoding RNAs (lncRNAs) are now accepted as key players in diverse cellular functions, yet the structure-function relationships of these novel RNAs remain mostly unknown. Homogenous purification of lncRNAs is a necessary first-step for downstream structural studies. The large size of lncRNAs (often more than 1 kb) presents many unique challenges during the purification process. Here, we detail the purification of lncRNAs, including strategies to identify proper folding conditions of the target lncRNA. Next, we discuss two recently developed RNA structure probing techniques, SHAPE-MaP (SHAPE probing followed by mutational profiling) and DMS-MaP (DMS probing followed by mutational profiling). These techniques couple traditional RNA chemical probing methods with next-generation sequencing and allow high-throughput determination of RNA structures. Using the datasets resulting from these orthogonal probing experiments, we lay out the steps to determine and validate the secondary structure of the target lncRNA. Overall, this chapter details an adaptable protocol that can lead to a better understanding of the structure-function relationships of lncRNAs.
Asunto(s)
ARN Largo no Codificante/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Conformación de Ácido Nucleico , Análisis de Secuencia de ARNRESUMEN
While natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic. The wild-type (WT) interface showed a negligible switch. The overall binding affinity under mild acidic pH for PrG-EHHE outperformed the wild-type PrG (PrG-WT) interaction. The new reagent PrG-EHHE can be revolutionary in IgG purification, since the standard method of using an extreme acidic pH for elution can be circumvented.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Ácido Glutámico/química , Histidina/química , Humanos , Concentración de Iones de Hidrógeno , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/química , Mutación , Unión Proteica , Dominios Proteicos , Ingeniería de Proteínas , Streptococcus/químicaRESUMEN
Long non-coding RNA (lncRNA) biology is a rapidly growing area of study. Thousands of lncRNAs are implicated as key players in cellular pathways and cancer biology. However, the structure-function relationships of these novel biomolecules are not well understood. Recent structural studies suggest that lncRNAs contain modular structural domains, which play a crucial role in their function. Here, we hypothesized that such structural domains exist in lncTCF7, a conserved lncRNA implicated in the development and progression of several cancers. To understand the structure-function relationship of lncTCF7, we characterized its secondary structure using chemical probing methods. Our model revealed structural domains and conserved regions in lncTCF7. One of the modular domains identified here coincides with a known protein-interacting domain. The model reported herein is, to our knowledge, the first structural model of lncTCF7 and thus will serve to direct future studies that will provide fundamental insights into the function of this lncRNA.