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.

While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein–rRNA interactions, providing intriguing insights into the temporal order of protein–rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.

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.

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A) + RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by proteinprotein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.

Dyskeratosis congenita (DC) is an inherited disorder that implicates defects in the biology of telomeres, which are maintained by telomerase, a ribonucleoprotein with reverse transcriptase activity. Like all H/ACA RNAs, the H/ACA domain of nascent human telomerase RNA (hTR) forms a pre-RNP with H/ACA proteins NAF1, dyskerin, NOP10, and NHP2 in vivo. To assess the pre-RNP assembly of hTR mutants that poorly accumulate in vivo, we developed an in vitro system that uses components of human origin. Pre-RNPs were reconstituted with synthetic 32 P-labeled RNAs and 35 S-labeled proteins produced in rabbit reticulocyte lysate, and immunoprecipitations were carried out to analyze RNP formation. We show that human NAF1 cannot bind directly to the H/ACA domain of hTR, and requires the core trimer dyskerin-NOP10-NHP2 to be efficiently incorporated into the pre-RNP. This order of assembly seems common to H/ACA RNAs since it was observed with snoRNA ACA36 and scaRNA U92, which are predicted to guide pseudouridylation of 18S rRNA and U2 snRNA, respectively. However, the processing H/ACA snoRNA U17 did not conform to this rule, as NAF1 alone was able to bind it. We also provide the first evidence that DC-related mutations of hTR C408G and D378-451 severely impair pre-RNP assembly. Integrity of boxes H and ACA of hTR are also crucial for pre-RNP assembly, while the CAB box is dispensable. Our results offer new insights into the defects caused by some mutations located in the H/ACA domain of hTR.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.