title: Identification and Characterization of Mitotic Spindle-Localized Transcripts.
abstract: RNAs associate with the mitotic spindle in a variety of organisms, where they can spatially regulate protein production, ensure their proper segregation during cell division, or perform translation-independent roles in spindle formation. The identification of spindle-associated RNAs is an important first step in understanding the biological consequences of this phenomenon. In this chapter, we describe a method to use Xenopus laevis egg extracts to assemble and isolate mitotic spindles and to identify the spindle-associated RNAs. The method described here can be used in combination with immunodepletions, the addition of inhibitors, or other perturbations to investigate factors that affect RNA localization to the spindle. Finally, we describe a method to assess the consequences of ablating RNA in the extract on spindle formation.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, 02114, USA.|Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA.
authors.forename: Amy B
authors.initials: AB
authors.lastname: Emerman
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, 02114, USA.|Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA.
authors.forename: Ashwini
authors.initials: A
authors.lastname: Jambhekar
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, 02114, USA. blower@molbio.mgh.harvard.edu.|Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA. blower@molbio.mgh.harvard.edu.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Blower
mesh_terms: D000818:Animals; D002457:Cell Extracts; D019295:Computational Biology / Q000379:methods; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D034443:RNA Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D014158:Transcription, Genetic; D014404:Tubulin / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts; D012333:RNA, Messenger; D014404:Tubulin; D012313:RNA; D012259:Ribonuclease, Pancreatic
keywords: Mitotic Spindle; RNA localization; RNaseA; Xenopus laevis