Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance

Abstract

Highlights d Strategy for affinity purification of native chromatin fragments d Heterochromatin and euchromatin have overlapping and non-overlapping proteomes d The heterochromatin proteome includes inner nuclear membrane proteins and nucleoporins d Inner-ring nucleoporins are required for clustering and maintenance of heterochromatin

An RNA Degradation Complex Required for Spreading and Epigenetic Inheritance of Heterochromatin

“…The Crb3-TAP purification from wild-type cells pulled down the core subunits reported previously to be part of the rixosome ( Figure 3B, Figure S4D) and its rRNA processing and ribosome biogenesis interaction network (Tables S3 and S4). Importantly, we also recovered the heterochromatin-associated protein Swi6, which was previously shown to associate with the rixosome in an H3K9me-independent manner (Iglesias et al, 2019). When we purified Crb3-TAP from cells carrying the crb3-D198N mutation, we detected a modest reduction in the amount of co-precipitated Swi6 in the mutant ( Figure S4D), which was not explained by a decrease in the overall Swi6 protein levels ( Figure S4E).…”

“…To determine whether the rixosome mutations affect the integrity of the complex or its interactions with other proteins, we affinity purified wild-type and mutant rixosome complexes and analyzed them by quantitative tandem mass tag mass spectrometry (TMT-MS)(Navarrete-Perea et al, 2018). The rixosome co-purifies with native heterochromatin fragments (Iglesias et al, 2019). We therefore used this native purification protocol to test whether the rixosome mutations affect its interaction with native heterochromatin (Iglesias et al, 2019).…”

“…The rixosome co-purifies with native heterochromatin fragments (Iglesias et al, 2019). We therefore used this native purification protocol to test whether the rixosome mutations affect its interaction with native heterochromatin (Iglesias et al, 2019). We tagged the C-terminus of the Crb3 subunit with a tandem affinity purification (TAP) tag (Puig et al, 2001), which did not interfere with the maintenance of silencing at the 10xtetO-ade6+ reporter ( Figure S4A).…”

“…Purifications of Crb3-TAP tagged rixosome complexes were performed by a chromatin purification method described previously (Iglesias et al, 2019) with minor modifications. Cells carrying Crb3-TAP in the wild-type and mutant (grc3-V70M or crb3-D198N) backgrounds, as well as corresponding untagged cells, were grown in rich YES medium to exponential phase (OD600 = 1-1.5) and 600 OD600 units were collected per immunoprecipitation.…”

“…Our screen identified mutations in several pathways, including mutations in three subunits of an RNA processing complex, composed of Rix1, Crb3, Grc3, Mdn1, Las1 and Ipi1 (Castle et al, 2012, Schillewaert et al, 2012, Castle et al, 2013, Gasse et al, 2015, Fromm et al, 2017. This complex, which we propose to name the "rixosome", is conserved from yeast to human, has well-characterized essential functions in ribosomal RNA (rRNA) processing and has been previously shown to localize to heterochromatin in a Swi6-dependent manner (Kitano et al, 2011, Iglesias et al, 2019, but its role in heterochromatin function remains unknown. We show that the rixosome targets heterochromatic RNAs for degradation through the conserved 5'-3' exoribonuclease pathway to clear a path for Clr4-mediated read-write.…”