RESUMEN
The lncRNA human Hox transcript antisense intergenic RNA (hHOTAIR) regulates gene expression by recruiting chromatin modifiers. The prevailing model suggests that hHOTAIR recruits hnRNPB1 to facilitate intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target gene transcripts. This B1-mediated RNA-RNA interaction modulates the structure of hHOTAIR, attenuates its inhibitory effect on polycomb repression complex 2, and enhances its methyl transferase activity. However, the molecular details by which the nuclear hnRNPB1 protein assembles on the lncRNA HOTAIR have not yet been described. Here, we investigate the molecular interactions between hnRNPB1 and Helix-12 (hHOTAIR). We show that the low-complexity domain segment (LCD) of hnRNPB1 interacts with a strong affinity for Helix-12. Our studies revealed that unbound Helix-12 folds into a specific base-pairing pattern and contains an internal loop that, as determined by thermal melting and NMR studies, exhibits hydrogen bonding between strands and forms the recognition site for the LCD segment. In addition, mutation studies show that the secondary structure of Helix-12 makes an important contribution by acting as a landing pad for hnRNPB1. The secondary structure of Helix-12 is involved in specific interactions with different domains of hnRNPB1. Finally, we show that the LCD unwinds Helix-12 locally, indicating its importance in the hHOTAIR restructuring mechanism.
Asunto(s)
ARN Largo no Codificante , Humanos , Complejo Represivo Polycomb 2 , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
The thermodynamic landscape of the CRISPR/Cas9 system plays a crucial role in understanding and optimizing the performance of this revolutionary genome-editing technology. In this research, we utilized isothermal titration calorimetry and microscale thermophoresis techniques to thoroughly investigate the thermodynamic properties governing CRISPR/Cas9 interactions. Our findings revealed that the binding between sgRNA and Cas9 is primarily governed by entropy, which compensates for an unfavorable enthalpy change. Conversely, the interaction between the CRISPR RNP complex and the target DNA is characterized by a favorable enthalpy change, offsetting an unfavorable entropy change. Notably, both interactions displayed negative heat capacity changes, indicative of potential hydration, ionization, or structural rearrangements. However, we noted that the involvement of water molecules and counterions in the interactions is minimal, suggesting that structural rearrangements play a significant role in influencing the binding thermodynamics. These results offer a nuanced understanding of the energetic contributions and structural dynamics underlying CRISPR-mediated gene editing. Such insights are invaluable for optimizing the efficiency and specificity of CRISPR-based genome editing applications, ultimately advancing our ability to precisely manipulate genetic material in various organisms for research, therapeutic, and biotechnological purposes.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Termodinámica , Edición Génica/métodos , ADN/química , ADN/metabolismo , ADN/genética , Calorimetría , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/química , ARN Guía de Sistemas CRISPR-Cas/química , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
ABBREVIATIONS: AF2: AlphaFold2; AF2-Mult: AlphaFold2 multimer; ATG: autophagy-related; CTD: C-terminal domain; ECTD: extreme C-terminal domain; FR: flexible region; MD: molecular dynamics; NTD: N-terminal domain; pLDDT: predicted local distance difference test; UBL: ubiquitin-like.