Protein products of non-stop mRNA disrupt nucleolar homeostasis

SummaryMutations that cause ribosome stalling or impair the cell’s protective response to stalling have been demonstrated to cause neurodegeneration, yet the mechanisms underlying these pathologies remain poorly understood. Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the Ribosome-associated Quality Control (RQC) E3 ligase LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tail of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress. Changes in nucleolar morphology are consistent with a simple physical model in which LTN1 impairment enhances the inter-molecular interactions of nucleolar components. Our work elucidates how failure to degrade the protein products of stalled translation may affect distal cellular processes and will inform studies on the pathology of neurodegenerative disease.

Sorting the trash: Micronucleophagy gets selective

During micronucleophagy, the nucleolus is targeted by autophagic degradation, but although nucleolar proteins are recycled, ribosomal DNA is spared. Mostofa et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706164) reveal that the separation of these two nucleolar components is mediated by the CLIP and cohibin complexes and is vital for cell survival during starvation.

CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy

Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear–vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA–nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins.

The chromosome peripheral proteins play an active role in chromosome dynamics

The chromosome periphery is a chromosomal structure that covers the surface of mitotic chromosomes. The structure and function of the chromosome periphery has been poorly understood since its first description in 1882. It has, however, been proposed to be an insulator or barrier to protect chromosomes from subcellular substances and to act as a carrier of nuclear and nucleolar components to direct their equal distribution to daughter cells because most chromosome peripheral proteins (CPPs) are derived from the nucleolus or nucleus. Until now, more than 30 CPPs were identified in mammalians. Recent immunostaining analyses of CPPs have revealed that the chromosome periphery covers the centromeric region of mitotic chromosomes in addition to telomeres and regions between two sister chromatids. Knockdown analyses of CPPs using RNAi have revealed functions in chromosome dynamics, including cohesion of sister chromatids, kinetochore-microtubule attachments, spindle assembly and chromosome segregation. Because most CPPs are involved in various subcellular events in the nucleolus or nuclear at interphase, a temporal and spatial-specific knockdown method of CPPs in the chromosome periphery will be useful to understand the function of chromosome periphery in cell division.

Compartmentation of the Nucleolar Processing Proteins in the Granular Component Is a CK2-driven Process

To analyze the compartmentation of nucleolar protein complexes, the mechanisms controlling targeting of nucleolar processing proteins onto rRNA transcription sites has been investigated. We studied the reversible disconnection of transcripts and processing proteins using digitonin-permeabilized cells in assays capable of promoting nucleolar reorganization. The assays show that the dynamics of nucleolar reformation is ATP/GTP-dependent, sensitive to temperature, and CK2-driven. We further demonstrate the role of CK2 on the rRNA-processing protein B23. Mutation of the major CK2 site on B23 induces reorganization of nucleolar components that separate from each other. This was confirmed in assays using extracts containing B23 mutated in the CK2-binding sites. We propose that phosphorylation controls the compartmentation of the rRNA-processing proteins and that CK2 is involved in this process.

Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition

Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.