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Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs
Published on: June 27, 2013
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Mitotic noncoding RNA processing promotes kinetochore and spindle assembly in Xenopus.
Andrew W Grenfell1, Rebecca Heald2, Magdalena Strzelecka1
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
The Journal of Cell Biology
|July 13, 2016
Summary
Mitotic RNA splicing is crucial for proper spindle assembly and kinetochore formation. Inhibiting RNA processing disrupts centromere protein recruitment and spindle organization, challenging existing mitosis models.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Centromeric transcription and noncoding RNAs are implicated in kinetochore assembly and Aurora B activation.
- The role of mitotic RNA processing in spindle assembly remains largely unexplored.
Purpose of the Study:
- To investigate the contribution of mitotic RNA processing to spindle assembly in Xenopus egg extracts.
- To determine whether RNA splicing or translation inhibition affects spindle formation.
Main Methods:
- Utilized Xenopus egg extracts to study mitotic spindle assembly.
- Inhibited transcription initiation, RNA splicing, and translation to observe effects on spindle formation.
- Analyzed the recruitment of centromere/kinetochore proteins (CENP-A, CENP-C, NDC80) and MCAK levels.
Main Results:
- Inhibition of transcription or RNA splicing, but not translation, caused significant spindle defects.
- Spliceosome inhibition led to accumulation of high molecular weight centromeric transcripts.
- Blocking RNA synthesis or processing resulted in decreased recruitment of key centromere/kinetochore proteins and misregulation of Aurora B kinase activity, evidenced by MCAK accumulation.
Conclusions:
- Co-transcriptional recruitment of RNA processing machinery to nascent mitotic transcripts is vital for kinetochore and spindle assembly.
- The study challenges the notion that RNA processing is globally repressed during mitosis, highlighting its active role.
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