Kinase PIM1 promotes prostate cancer cell growth via c-Myc-RPS7-driven ribosomal stress.

Ribosomal stress is known to increase cancer risk; however, the molecular mechanism underlying its various effects on cancer remains unclear. To decipher this puzzle, we investigated the upstream signaling pathway that might be involved in promoting ribosomal stress that leads to tumor progression. Our results suggested that inhibition of kinase PIM1 attenuated PC3 cell growth and motility following the condensed cellular body and decreased protein translation in PIM1-inhibited cells. In addition, PIM1 was found to be a component of the small 40S ribosomal subunit and could regulate the expression of ribosomal small subunit protein 7 (RPS7). Our investigation also revealed that PIM1 enhanced the protein stability of c-Myc. Furthermore, a functional E-box motif was found upstream of the transcription start site in RPS7, and RPS7 has been proven to be a transcriptional target of c-Myc. Additionally, knocking down RPS7 dramatically reduced cell growth in vitro and in vivo, whereas enhancing RPS7 expression reversed the condensed cellular body and decreased protein translation resulted from PIM1 inhibition. Finally, biochemical recurrence-free survival and overall survival analysis indicated that the concomitant upregulation of PIM1 and RPS7 correlated with the worst prognosis of prostate cancer (PCa). Overall, our results demonstrated that kinase PIM1 promotes cell growth through c-Myc-RPS7-induced ribosomal stress in PCa. These findings substantially expanded our understanding on the molecular mechanism of PIM1-promoted abnormal ribosomal biosynthesis in tumorigenesis and tumor progression in PCa. Therapies that target molecules involved in PIM1-RPS7-induced ribosomal stress could provide a promising approach to treating PCa.

Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution.

Hepatitis C virus (HCV), a widespread human pathogen, is dependent on a highly structured 5'-untranslated region of its mRNA, referred to as internal ribosome entry site (IRES), for the translation of all of its proteins. The HCV IRES initiates translation by directly binding to the small ribosomal subunit (40S), circumventing the need for many eukaryotic translation initiation factors required for mRNA scanning. Here we present the cryo-EM structure of the human 40S ribosomal subunit in complex with the HCV IRES at 3.9 Å resolution, determined by focused refinement of an 80S ribosome-HCV IRES complex. The structure reveals the molecular details of the interactions between the IRES and the 40S, showing that expansion segment 7 (ES7) of the 18S rRNA acts as a central anchor point for the HCV IRES. The structural data rationalizes previous biochemical and genetic evidence regarding the initiation mechanism of the HCV and other related IRESs.

mRNAs that specifically interact with eukaryotic ribosomal subunits.

The accuracy of start codon selection is determined by the translation initiation process. In prokaryotes the initiation step on most mRNAs relies on recruitment of the small ribosomal subunit onto the initiation codon by base pairing between the mRNA and the 16S rRNA. Eukaryotes have evolved a complex molecular machinery involving at least 11 initiation factors, and mRNAs do not directly recruit the small ribosomal subunit. Instead the initiation complex is recruited to the 5' end of the mRNA through a complex protein network including eIF4E that interacts with the 5' cap structure and poly-A binding protein that interacts with the 3'end. However, some viral and cellular mRNAs are able to escape this pathway by internal recruitment of one or several components of the translation machinery. Here we review those eukaryotic mRNAs that have been reported to directly recruit the 40S ribosomal subunit internally. In the well characterized cases of viral IRESes, a specific RNA structure is involved in this process, and in addition to recruitment of the ribosome, the mRNA also manipulates the ribosome structure to stimulate the first translocation step. We also review recently described IRES/ribosome interactions in cases where the molecular mechanism leading to translation initiation has yet to be described. Finally we evaluate the possibility that mRNA may recruit the 40S ribosomal subunit through base pairing with the 18S rRNA.

Neuronal BC RNAs cooperate with eIF4B to mediate activity-dependent translational control.

In neurons, translational regulation of gene expression has been implicated in the activity-dependent management of synapto-dendritic protein repertoires. However, the fundamentals of stimulus-modulated translational control in neurons remain poorly understood. Here we describe a mechanism in which regulatory brain cytoplasmic (BC) RNAs cooperate with eukaryotic initiation factor 4B (eIF4B) to control translation in a manner that is responsive to neuronal activity. eIF4B is required for the translation of mRNAs with structured 5' untranslated regions (UTRs), exemplified here by neuronal protein kinase Mζ (PKMζ) mRNA. Upon neuronal stimulation, synapto-dendritic eIF4B is dephosphorylated at serine 406 in a rapid process that is mediated by protein phosphatase 2A. Such dephosphorylation causes a significant decrease in the binding affinity between eIF4B and BC RNA translational repressors, enabling the factor to engage the 40S small ribosomal subunit for translation initiation. BC RNA translational control, mediated via eIF4B phosphorylation status, couples neuronal activity to translational output, and thus provides a mechanistic basis for long-term plastic changes in nerve cells.

Quality control mechanisms during ribosome maturation.

Protein synthesis on ribosomes is carefully quality-controlled to ensure the faithful transmission of genetic information from mRNA to protein. Many of these mechanisms rely on communication between distant sites on the ribosomes, and thus on the integrity of the ribosome structure. Furthermore, haploinsufficiency of ribosomal proteins, which increases the chances of forming incompletely assembled ribosomes, can predispose to cancer. Finally, release of inactive ribosomes into the translating pool will lead to their degradation together with the degradation of the bound mRNA. Together, these findings suggest that quality control mechanisms must be in place to survey nascent ribosomes and ensure their functionality. This review gives an account of these mechanisms as currently known.

A zebrafish model of dyskeratosis congenita reveals hematopoietic stem cell formation failure resulting from ribosomal protein-mediated p53 stabilization.

Dyskeratosis congenita (DC) is a bone marrow failure disorder characterized by shortened telomeres, defective stem cell maintenance, and highly heterogeneous phenotypes affecting predominantly tissues that require high rates of turnover. Here we present a mutant zebrafish line with decreased expression of nop10, one of the known H/ACA RNP complex genes with mutations linked to DC. We demonstrate that this nop10 loss results in 18S rRNA processing defects and collapse of the small ribosomal subunit, coupled to stabilization of the p53 tumor suppressor protein through small ribosomal proteins binding to Mdm2. These mutants also display a hematopoietic stem cell deficiency that is reversible on loss of p53 function. However, we detect no changes in telomere length in nop10 mutants. Our data support a model of DC whereupon in early development mutations involved in the H/ACA complex contribute to bone marrow failure through p53 deregulation and loss of initial stem cell numbers while their role in telomere maintenance does not contribute to DC until later in life.

eIF3a cooperates with sequences 5' of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA.

Yeast initiation factor eIF3 (eukaryotic initiation factor 3) has been implicated in multiple steps of translation initiation. Previously, we showed that the N-terminal domain (NTD) of eIF3a interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. Here, we demonstrate that a partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of eIF3 and associated eIFs to native preinitiation complexes in vivo. Strikingly, it also severely blocks the induction of GCN4 translation that occurs via reinitiation. Detailed examination unveiled a novel reinitiation defect resulting from an inability of 40S ribosomes to resume scanning after terminating at the first upstream ORF (uORF1). Genetic analysis reveals a functional interaction between the eIF3a-NTD and sequences 5' of uORF1 that is critically required to enhance reinitiation. We further demonstrate that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation efficiency after uORF1 declines with its increasing length. Together, our results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with its 5' enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and enable resumption of scanning for reinitiation downstream.

Mutation of ribosomal protein RPS24 in Diamond-Blackfan anemia results in a ribosome biogenesis disorder.

Diamond-Blackfan anemia (DBA) is a rare congenital disease affecting erythroid precursor differentiation. DBA is emerging as a paradigm for a new class of pathologies potentially linked to disorders in ribosome biogenesis. Three genes encoding ribosomal proteins have been associated to DBA: after RPS19, mutations in genes RPS24 and RPS17 were recently identified in a fraction of the patients. Here, we show that cells from patients carrying mutations in RPS24 have defective pre-rRNA maturation, as in the case of RPS19 mutations. However, in contrast to RPS19 involvement in the maturation of the internal transcribed spacer 1, RPS24 is required for processing of the 5' external transcribed spacer. Remarkably, epistasis experiments with small interfering RNAs indicate that the functions of RPS19 and RPS24 in pre-rRNA processing are connected. Resolution of the crystal structure of RPS24e from the archeon Pyroccocus abyssi reveals domains of RPS24 potentially involved in interactions with pre-ribosomes. Based on these data, we discuss the impact of RPS24 mutations and speculate that RPS19 and RPS24 cooperate at a particular stage of ribosome biogenesis connected to a cell cycle checkpoint, thus affecting differentiation of erythroid precursors as well as developmental processes.