UV-RNA Immunoprecipitation (UV-RIP) Protocol in Neurons.
With the many advances in genome-wide sequencing, it has been discovered that much more of the genome is transcribed into RNA than previously appreciated. These nonprotein-coding RNAs (ncRNAs) come in many different forms, and they have been shown to have a variety of functions within the cell, influencing processes such as gene expression, mRNA splicing, and transport, just as a few examples. As we delve deeper into studying their mechanisms of action, it becomes important to understand how they play these roles, in particular by understanding what proteins these ncRNAs interact with. This protocol describes one technique that can be used to study this, ultra-violet light cross-linking RNA immunoprecipitation (UV-RIP), which uses an antibody to pull down a specific protein of interest and then detects RNA that is bound to it. This technique utilizes UV light to cross-link the cells, which takes advantage of the fact that UV light will only cross-link proteins and nucleic acids that are directly interacting. This approach can provide key mechanistic insight into the function of these newly identified ncRNAs.
Department of Neuroscience, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390-9111, USA.
Katie
K
Schaukowitch
Department of Neuroscience, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390-9111, USA.
Jae-Yeol
JY
Joo
Department of Neuroscience, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390-9111, USA. taekyung.kim@utsouthwestern.edu.
Tae-Kyung
TK
Kim
D000818:Animals; D001665:Binding Sites; D016764:Cell Polarity; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004742:Enhancer Elements, Genetic; D047468:Immunoprecipitation / Q000379:methods*; D051379:Mice; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D011485:Protein Binding; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins
RNA immunoprecipitation; RNA-binding proteins; UV cross-linking; lncRNA