# The nuclear poly(A)-binding protein Pab2/PABPN1 promotes heterochromatin assembly through the formation of Pab2 nuclear condensates

## TL;DR

This study shows that the protein Pab2 helps form heterochromatin by creating nuclear condensates, which are important for genome stability in fission yeast.

## Contribution

The study reveals a novel role of Pab2 in heterochromatin assembly through phase separation and RNA-binding interactions.

## Key findings

- Pab2 forms nuclear condensates via its RNA-recognition motif and intrinsically disordered domain.
- Pab2 is essential for H3K9 methylation at centromeres, a marker of heterochromatin.
- Pab2 interacts with Clr4 and Mit1, key heterochromatin factors, through Red5 and Rmn1.

## Abstract

The assembly of constitutive heterochromatin is a prerequisite for maintaining genome stability. However, the mechanism of heterochromatin formation has yet to be completely understood. Here, we demonstrate a crucial role of the nuclear poly(A)-binding protein (PABP) Pab2/PABPN1 in promoting constitutive heterochromatin formation in the fission yeast Schizosaccharomyces japonicus. Histone H3 Lys 9 di- and tri-methylation, hallmarks of heterochromatin, are significantly reduced at centromeres in the absence of Pab2. Pab2 forms nuclear condensates through its RNA-recognition motif (RRM) and the intrinsically disordered domain (IDR), both of which bind to centromeric non-coding RNAs. Intriguingly, two key heterochromatin factors, the histone H3 Lys9 methyltransferase Clr4 and the Mi2-type chromatin remodeler Mit1, associate with centromeres in a Pab2-dependent manner. Pab2 interacts with two putative RNA-binding proteins, the ZC3H3 ortholog Red5 and the RBM26·27 ortholog Rmn1, both essential for heterochromatin formation. Deletion of the Pab2 N-terminal region, which disrupts this interaction, largely abolishes Pab2 function, underscoring the importance of this complex. Pab2 also associates and colocalizes with Ppn1 (a PPP1R10 ortholog), a component of the cleavage and polyadenylation specificity factor (CPSF) complex, and ppn1 mutations disrupt constitutive heterochromatin. Notably, both Ppn1 and Rmn1 are able to interact with Clr4. Our findings reveal that Pab2 plays a pivotal role in heterochromatin assembly by forming nuclear condensates through its RRM/IDR, and Pab2 condensates facilitate the recruitment of Clr4 and Mit1 to centromeres, potentially through its binding proteins, Ppn1 and Rmn1. This study provides new insights into the mechanisms underlying heterochromatin formation and highlights the importance of RNA-binding proteins and phase separation in this process.

Constitutive heterochromatin is crucial for genome stability, yet its assembly mechanisms are not fully understood. In this study, we investigate the role of the nuclear poly(A)-binding protein Pab2 (human PABPN1 ortholog) in heterochromatin formation in the fission yeast Schizosaccharomyces japonicus. We show that Pab2 is essential for constitutive heterochromatin assembly, as evidenced by reduced H3K9 methylation, a hallmark of heterochromatin, in its absence. Pab2 forms nuclear condensates through its RNA-recognition motif and intrinsically disordered region, both binding to transcripts derived from centromeres; the Pab2 condensates are crucial for Pab2 functions. In addition, the stable association of the H3 Lys 9 methyltransferase Clr4 and the chromatin remodeler Mit1 with centromeres depends on Pab2. In addition, Pab2 interacts with two putative RNA-binding proteins Red5 and Rmn1, essential for heterochromatin formation, and theses interactions are required for its function. Moreover, Pab2 associates with Ppn1, a cleavage and polyadenylation specificity factor (CPSF) complex component, and Ppn1 contributes to heterochromatin integrity. Notably, Ppn1 and Rmn1 interact with the H3 Lys 9 methyltransferase Clr4. Our findings reveal that Pab2 promotes heterochromatin assembly by forming condensates that facilitate Clr4 and Mit1 recruitment to centromeres, potentially through Ppn1 and Rmn1. This highlights the critical role of RNA-binding proteins and phase separation in heterochromatin formation.