Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5′ end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5′ end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg–eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E–eIF4G, eIF4E bound VPg–luciferase RNA conjugates, and these VPg–RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg–RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.

Dyskeratosis congenita (DC) is a rare genetic syndrome that gives rise to a variety of disorders in affected individuals. Remarkably, all causative gene mutations identified to date share a link to telomere/telomerase biology. We found that the most prevalent dyskerin mutation in DC (A353V) did not affect formation of the NAF1-dyskerin-NOP10-NHP2 tetramer that normally assembles with nascent H/ACA RNAs in vivo. However, the A353V mutation slightly reduced pre-RNP assembly with the H/ACA-like domain of human telomerase RNA (hTR). In contrast, NHP2 mutations V126M and Y139H impaired association with NOP10, leading to major pre-RNP assembly defects with all H/ACA RNAs tested, including the H/ACA domain of hTR. Mutation R34W in NOP10 caused no apparent defect in protein tetramer formation, but it severely affected pre-RNP assembly with the H/ACA domain of hTR and a subset of H/ACA RNAs. Surprisingly, H/ACA sno/scaRNAs that encode miRNAs were not affected by the mutation R34W, and they were able to form pre-RNPs with NOP10-R34W. This indicates structural differences between H/ACA RNPs that encode miRNAs and those that do not. Altogether, our results suggest that, in addition to major defects in the telomere/telomerase pathways, some of the disorders occurring in DC may be caused by alteration of most H/ACA RNPs, or by only a subset of them.

To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome maintenance. Nol12 is found in different subcellular compartments-nucleoli, where it associates with ribosomal RNA and is required for efficient separation of large and small subunit precursors at site 2; the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles; as well as GW/P-bodies in the cytoplasm. Loss of Nol12 results in the inability of cells to recover from DNA stress and a rapid p53-independent ATR-Chk1-mediated apoptotic response. Nol12 co-localizes with DNA repair proteins in vivo including Dhx9, as well as with TOPBP1 at sites of replication stalls, suggesting a role for Nol12 in the resolution of DNA stress and maintenance of genome integrity. Identification of a complex Nol12 interactome, which includes NONO, Dhx9, DNA-PK and Stau1, further supports the protein's diverse functions in RNA metabolism and DNA maintenance, establishing Nol12 as a multifunctional RBP essential for genome integrity.