# Pre-rRNAs control mitosis by maintaining chromosomal segregation through protecting SMC2 from AURKA-mediated phosphorylation

## TL;DR

This study reveals that pre-rRNAs prevent chromosomal segregation errors during mitosis by protecting a key protein from harmful phosphorylation.

## Contribution

The novel finding is that pre-rRNAs directly protect SMC2 from AURKA-mediated phosphorylation, ensuring proper chromosomal segregation during mitosis.

## Key findings

- Inhibition of pre-rRNA transcription leads to prolonged mitosis and chromosomal segregation defects.
- AURKA binds and phosphorylates SMC2 at T574 when pre-rRNAs are absent, disrupting chromosomal DNA binding.
- Phosphorylation-deficient SMC2 T574A rescues mitotic defects caused by pre-rRNA depletion.

## Abstract

In interphase, 47S pre-rRNA is transcribed by RNA polymerase I (Pol I) and processed to form intermediate pre-rRNAs and finally produce mature rRNAs in the nucleolus. During mitosis, nucleolus disassembles and pre-rRNAs including 45S, 30S and 32S pre-rRNAs relocate in the peri-chromosomal region (PR). Inhibition of pre-rRNA transcription impairs chromosome dispersion in prometaphase. However, how pre-rRNAs regulate mitosis remains elusive. Here, we unravel a novel mechanism for pre-rRNAs to control mitosis. Inhibition of Pol I prolongs the mitotic process and induces defective chromosomal segregation, resulting in mitotic catastrophe. We isolated chromosome and determined the chromosome-binding proteins by mass-spectrometry. Using quantitative proteomics analysis, immunoprecipitation and immunofluorescent staining, we found that AURKA approaches chromosome when Pol I is inhibited. The AURKA-binding proteins on the chromosome were determined by immunoprecipitation and mass-spectrometry after cells were treated with Act D, BMH-21 or CX5461, respectively, and the chromosomal segregation controlling proteins were selected. When Pol I was inhibited, the binding of AURKA with SMC2, the crucial component of Condensin, is significantly enhanced. Importantly, SMC2 is phosphorylated by AURKA only when Pol I was inhibited. Alignment of SMC2 amino acid sequence with substrates of AURKA shows that SMC2 possesses the consensus R/K/N-R-X-S/T-B, and T574 is the only potential AURKA-catalyzed phosphorylation site. Indeed, SMC2 T574 is phosphorylated by AURKA in cell and in vitro. Thereafter, we generated SMC2 T574-P specific antibody, and confirmed that endogenous SMC2 T574 is phosphorylated by AURKA in mitosis in the absence of pre-rRNAs. Consequently, phosphorylation of SMC2 T574 disrupts the SMC2/SMC4 binding and the binding of SMC2 and SMC4 to chromosomal DNA, leading to chromosomal segregation defect. The phosphorylation deficient Flag-SMC2 T574A reverses the mitotic catastrophe caused by Pol I inhibition. Collectively, we demonstrate that pre-rRNAs protect SMC2 from the AURKA-mediated phosphorylation to maintain normal mitosis.