The cohesin loader NIPBL interacts with pre-ribosomal RNA and treacle to regulate ribosomal RNA synthesis

Abstract

NIPBL is an essential loader of cohesin to mediate sister chromatid cohesion and chromatin loop organization. NIPBL mutations cause Cornelia de Lange Syndrome. How NIPBL's genomic localization is specified is not fully understood. We found that NIPBL localizes to the nucleolus in an RNA-dependent manner and binds directly to ribosomal RNA (rRNA). We identified two RNA binding domains in NIPBL in vitro, both of which are required for efficient rRNA binding in vivo. NIPBL binds to ribosomal DNA (rDNA) in an RNA-stimulated manner, recruits PAF1 and promotes pre-rRNA transcription. Stress that inhibits rRNA synthesis displaces NIPBL from the nucleolus and rDNA. Interestingly, treacle, mutated in Treacher Collins syndrome, tightly binds to and recruits NIPBL to the nucleolus, nucleolar organizer regions, and the stress-induced nucleolar cap. The results reveal that a subpopulation of NIPBL is recruited to the nucleolus through its interaction with RNA and treacle and regulates pre-rRNA transcription.6 Results NIPBL localizes to the Fibrillar Center of the nucleolusThe nucleolus has a "tripartite structure" consisting of the Fibrillar Center (FC), the Dense Fibrillar Component (DFC), and the Granular Component (GC), each with distinct functions (Fig. 1A) (Boulon et al., 2010a). The FC is the site for pre-rRNA transcription and is enriched in RNA polymerase I (Pol I) machinery. Pre-rRNA is processed in the DFC, which contains many RNA processing factors such as snoRNA and fibrillarin. The GC is the site where pre-ribosome assembly takes place, and contains ribosome assembly factors such as B23 (nucleophosmin). Consistent with cohesin binding to the rDNA region (Zeng et al., 2009a), biochemical fractionation analysis indicates that Nipbl and cohesin are present in both nuclear and nucleolar fractions (Fig. 1B). Using an affinity-purified polyclonal antibody directed against the C-terminus of human NIPBL, we observed clear localization of both mouse Nipbl and human NIPBL in the nucleolus (mouse embryonic fibroblasts (MEFs) in Fig. 1C and D; human 293T cells in Supplemental Fig. S1, respectively). Costaining with antibodies specific for B23 (a marker for the GC) and fibrillarin (a marker for the DFC) revealed that Nipbl tightly clusters to the inside of the DFC corresponding to the FC (Fig. 1C and D). Furthermore, nucleolar Nipbl staining signals were reduced significantly in a Nipbl heterozygous mutant MEFs (modeling NIPBL haploinsufficiency in CdLS (Kawauchi et al., 2009;Newkirk et al., 2017)) and Nipbl siRNAdepleted MEFs compared to the wild type and control siRNA-treated MEFs, respectively (Fig. 1E). These results confirmed the specificity of immunostaining and also indicate that Nipbl in the nucleolus is sensitive to partial protein depletion (haploinsufficiency). NIPBL localizes to the nucleolus in an RNA-dependent manner and binds to preribosomal RNARNA is highly enriched and is an important constituent of nucleoli. Interestingly, RNase treatment dispersed Nipbl nucleolar foci while bulk Nipbl remained in the nucl...

Treacle and TOPBP1 control replication stress response in the nucleolus

“…Its direct role in Pol I–dependent transcription is not clear ( Lin and Yeh, 2009 ). Furthermore, an increasing number of studies have reported the role of Treacle in recruitment and/or retention of different nonnucleolar proteins in nucleoli ( Fages et al, 2020 ; Kong et al, 2019 ; Larsen et al, 2014 ; Mooser et al, 2020 ; Rawlinson et al, 2018 ; Velichko et al, 2019 ). It is clear that Treacle can interact with BRCT-containing factors ( Larsen et al, 2014 ; Mooser et al, 2020 ; Velichko et al, 2019 ); however, it is not known whether Nbs1 and TOPBP1 are the only BRCT proteins that interact with Treacle.…”

Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes

“…The possession of more than a single RBD could theoretically allow RBPs to be multifunctional, for instance through the separate regulation of different sets of targets (Kong et al, 2019). Multifunctionality may additionally be established through the condition- or cell type-specific expression of RBPs and their interaction partners, or through dynamic shuttling of RBPs between different subcellular compartments, such as the nucleus and cytosol (Dassi, 2017; Díaz-Muñoz & Turner, 2018; Gray et al, 2015; Yugami et al, 2020).…”

“…The possession of more than a single RBD would theoretically allow RBPs to be multifunctional, for instance through the separate regulation of different sets of targets (Kong et al, 2019).…”

The bromodomain inhibitor JQ1 is a molecular glue targeting centromeres

“…centromeres, rDNA), selected Topological Associated Domain (TAD) boundaries and gene promoters. The top hits for selected loci contain well-characterized locus- specific proteins and proteins that have only recently been identified in association with specific loci, such as NIPBL and RNA polymerase II for rDNA 22, 23 , and GTF2I and BRD4 for TAD boundaries 24, 25 (Fig. 1c and 1d).…”