Point centromere activity requires an optimal level of centromeric noncoding RNA.

Area of Science:

• Epigenetics
• Molecular Biology
• Yeast Genetics

Background:

• Budding yeast possesses simple point centromeres.
• Centromeres are essential for accurate chromosome segregation.
• The role of noncoding RNAs at centromeres is not fully understood.

Purpose of the Study:

• To investigate the expression and function of long noncoding RNAs (cenRNAs) at budding yeast centromeres.
• To determine the regulatory mechanisms controlling cenRNA expression.
• To elucidate the role of cenRNAs in centromere epigenetic regulation and function.

Main Methods:

• Analysis of cenRNA expression in budding yeast.
• Genetic manipulation (deletion mutants) of centromeric factors (Cbf1, H2A.Z).
• RNA interference (RNAi) to knock down cenRNAs.
• Minichromosome loss assays.
• Western blotting for protein level analysis.
• Chromatin immunoprecipitation for protein localization.

Main Results:

• All budding yeast centromeres express long noncoding RNAs (cenRNAs), particularly during S phase and coinciding with CENP-A^Cse4 loading.
• CENP-A^Cse4 loading and cenRNA expression are dependent on DNA replication.
• Centromeric transcription is repressed by Cbf1 and H2A.Z^Htz1; their deletion up-regulates cenRNAs, increases minichromosome loss, and causes aneuploidy.
• Deletion of Cbf1 and H2A.Z^Htz1 leads to down-regulation of key centromeric proteins and reduced chromatin localization of CENP-A^Cse4 and associated proteins.
• cenRNAs function *in trans* to regulate centromere activity, as evidenced by RNAi knockdown effects.
• cenRNA knockdown partially rescues minichromosome loss in deletion mutants, indicating dose-dependent regulation.

Conclusions:

• Long noncoding RNAs (cenRNAs) are integral components of budding yeast centromeres, expressed during S phase and linked to CENP-A^Cse4 loading.
• The epigenetic control of point centromere function is tightly regulated by cenRNA levels, influenced by factors like Cbf1 and H2A.Z^Htz1.
• cenRNAs play a critical role in maintaining centromere integrity and ensuring proper chromosome segregation, functioning both *cis* and *trans*.