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SummaryRibosomal defects perturb stem cell differentiation, causing diseases called ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discovered three RNA helicases are required for ribosome biogenesis and forDrosophilaoogenesis. Loss of these helicases, which we named Aramis, Athos and Porthos, lead to aberrant stabilization of p53, cell cycle arrest and stalled GSC differentiation. Unexpectedly, Aramis is required for efficient translation of a cohort of mRNAs containing a 5’-Terminal-Oligo-Pyrimidine (TOP)-motif, including mRNAs that encode ribosomal proteins and a conserved p53 inhibitor,NovelNucleolar protein 1 (Non1). The TOP-motif co-regulates the translation of growth-related mRNAs in mammals. As in mammals, the La-related protein co-regulates the translation of TOP-motif containing RNAs duringDrosophilaoogenesis. Thus, a previously unappreciated TOP-motif inDrosophilaresponds to reduced ribosome biogenesis to co-regulate the translation of ribosomal proteins and a p53 repressor, thus coupling ribosome biogenesis to GSC differentiation.

LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5′TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1’s translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 ‘pendular hook’ engages the TOP mRNA 5′-end to repress translation, but only in conditions of mTORC1 inhibition.

The EepR transcription factor positively regulates secondary metabolites and tissue-damaging metalloproteases. To gain insight into mechanisms by which EepR regulates pigment and co-regulated factors, genetic suppressor analysis was performed. Suppressor mutations that restored pigment to the non-pigmented ∆eepR mutant mapped to the hexS ORF. Mutation of hexS also restored haemolysis, swarming motility and protease production to the eepR mutant. HexS is a known direct and negative regulator of secondary metabolites in Serratia marcescens and is a LysR family regulator and an orthologue of LrhA. Here, we demonstrate that HexS directly controls eepR and the serralysin gene prtS. EepR was shown to directly regulate eepR expression but indirectly regulate hexS expression. Together, these data indicate that EepR and HexS oppose each other in controlling stationary phase-associated molecules and enzymes.

Summary The RNA-binding protein La-related protein 1 (LARP1) plays a central role in ribosome biosynthesis. Its C-terminal DM15 region binds the 7-methylguanosine (m7G) cap and 5′ terminal oligopyrimidine (TOP) motif characteristic of transcripts encoding ribosomal proteins and translation factors. Under the control of mammalian target of rapamycin complex 1 (mTORC1), LARP1 regulates translation of these transcripts. Characterizing the dynamics of DM15-TOP recognition is essential to understanding this fundamental biological process. We use molecular dynamics simulations, biophysical assays, and X-ray crystallography to reveal the mechanism of DM15 binding to TOP transcripts. Residues C-terminal to the m7G-binding site play important roles in cap recognition. Furthermore, we show that the unusually static pocket that recognizes the +1 cytosine characteristic of TOP transcripts drives binding specificity. Finally, we demonstrate that the DM15 pockets involved in TOP-specific m7GpppC-motif recognition are likely druggable. Collectively, these studies suggest unique opportunities for further pharmacological development.

The mammalian target of rapamycin complex 1 (mTORC1) controls critical cellular functions such as protein synthesis, lipid metabolism, protein turnover and ribosome biogenesis through the phosphorylation of multiple substrates. In this study, we examined the phosphorylation of a recently identified target of mTORC1: La-related protein 1 (LARP1), a member of the LARP superfamily. Previously, we and others have shown that LARP1 plays an important role in repressing TOP mRNA translation downstream of mTORC1. LARP1 binds the 7-methylguanosine triphosphate (m7Gppp) cap moiety and the adjacent 5’terminal oligopyrimidine (5’TOP) motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 plays a critical role in the control of TOP mRNA translationviaLARP1 but the precise mechanism by which this occurs is incompletely understood. The data described herein help to elucidate this process. Specifically, it show that: (i) mTORC1 interacts with LARP1, but not other LARP superfamily members,viathe C-terminal region that comprises the DM15 domain, (ii) mTORC1 pathway controls the phosphorylation of multiple (up to 26) serine and threonine residues on LARP1in vivo, (iii) mTORC1 regulates the binding of LARP1 to TOP mRNAs and (iv) phosphorylation of S689 by mTORC1 is particularly important for the association of the DM15 domain of LARP1 with the 5’UTR of RPS6 TOP mRNA. These data reveal LARP1 as a major substrate of mTORC1.

The ribosome is an essential unit of all living organisms that commands protein synthesis, ultimately fuelling cell growth (accumulation of cell mass) and cell proliferation (increase in cell number). The eukaryotic ribosome consists of 4 ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs). Despite its fundamental role in every living organism, our present understanding of how higher eukaryotes produce the various ribosome components is incomplete. Uncovering the mechanisms utilized by human cells to generate functional ribosomes will likely have far-reaching implications in human disease. Recent biochemical and structural studies revealed La-related protein 1 (LARP1) as a key new player in RP production. LARP1 is an RNA-binding protein that belongs to the LARP superfamily; it controls the translation and stability of the mRNAs that encode RPs and translation factors, which are characterized by a 5′ terminal oligopyrimidine (5′TOP) motif and are thus known as TOP mRNAs. The activity of LARP1 is regulated by the mammalian target of rapamycin complex 1 (mTORC1): a eukaryotic protein kinase complex that integrates nutrient sensing with mRNA translation, particularly that of TOP mRNAs. In this review, we provide an overview of the role of LARP1 in the control of ribosome production in multicellular eukaryotes. This article is categorized under: Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Processing > Capping and 5′ End Modifications.

Viral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, using in vitro and cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the family Reoviridae encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the family Reoviridae diverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the family Reoviridae assort and replicate their genomes.

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5′TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5′ UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5′TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001

Generation of many useful microbe-derived secondary metabolites, including the red pigment prodigiosin of the bacterium Serratia marcescens, is inhibited by glucose. In a previous report, a genetic approach was used to determine that glucose dehydrogenase activity (GDH) is required for inhibiting prodigiosin production and transcription of the prodigiosin biosynthetic operon (pigA-N). However, the transcription factor(s) that regulate this process were not characterized. Here we tested the hypothesis that HexS, a LysR-family transcription factor similar to LrhA of Escherichia coli, is required for inhibition of prodigiosin by growth in glucose. We observed that mutation of the hexS gene in S. marcescens allowed the precocious production of prodigiosin in glucose-rich medium conditions that completely inhibited prodigiosin production by the wild type. Unlike previously described mutants able to generate prodigiosin in glucoserich medium, hexS mutants exhibited GDH activity and medium acidification similar to the wild type. Glucose inhibittion of pigA expression was shown to be dependent upon HexS, suggesting that HexS is a key transcription factor in secondary metabolite regulation in response to medium pH. These data give insight into the prodigiosin regulatory pathway and could be used to enhance the production of secondary metabolites.

RNA-binding proteins (RBPs) are increasingly identified as post-transcriptional drivers of cancer progression. The RBP LARP1 is an mRNA stability regulator, and elevated expression of the protein in hepatocellular and lung cancers is correlated with adverse prognosis. LARP1 associates with an mRNA interactome that is enriched for oncogenic transcripts. Here we explore the role of LARP1 in epithelial ovarian cancer, a disease characterized by the rapid acquisition of resistance to chemotherapy through the induction of pro-survival signalling. We show, using ovarian cell lines and xenografts, that LARP1 is required for cancer cell survival and chemotherapy resistance. LARP1 promotes tumour formation in vivo and maintains cancer stem cell-like populations. Using transcriptomic analysis following LARP1 knockdown, cross-referenced against the LARP1 interactome, we identify BCL2 and BIK as LARP1 mRNA targets. We demonstrate that, through an interaction with the 3′ untranslated regions (3′ UTRs) of BCL2 and BIK, LARP1 stabilizes BCL2 but destabilizes BIK with the net effect of resisting apoptosis. Together, our data indicate that by differentially regulating the stability of a selection of mRNAs, LARP1 promotes ovarian cancer progression and chemotherapy resistance.

Serratia marcescensgenerates secondary metabolites and secreted enzymes, and it causes hospital infections and community-acquired ocular infections. Previous studies identified cyclic AMP (cAMP) receptor protein (CRP) as an indirect inhibitor of antimicrobial secondary metabolites. Here, we identified a putative two-component regulator that suppressedcrpmutant phenotypes. Evidence supports that the putative response regulatoreepRwas directly transcriptionally inhibited by cAMP-CRP. EepR and the putative sensor kinase EepS were necessary for the biosynthesis of secondary metabolites, including prodigiosin- and serratamolide-dependent phenotypes, swarming motility, and hemolysis. Recombinant EepR bound to the prodigiosin and serratamolide promotersin vitro. Together, these data introduce a novel regulator of secondary metabolites that directly connects the broadly conserved metabolism regulator CRP with biosynthetic genes that may contribute to competition with other microbes.IMPORTANCEThis study identifies a new transcription factor that is directly controlled by a broadly conserved transcription factor, CRP. CRP is well studied in its role to help bacteria respond to the amount of nutrients in their environment. The new transcription factor EepR is essential for the bacteriumSerratia marcescensto produce two biologically active compounds, prodigiosin and serratamolide. These two compounds are antimicrobial and may allowS. marcescensto compete for limited nutrients with other microorganisms. Results from this study tie together the CRP environmental nutrient sensor with a new regulator of antimicrobial compounds. Beyond microbial ecology, prodigiosin and serratamolide have therapeutic potential; therefore, understanding their regulation is important for both applied and basic science.

Purpose The Serratia marcescens bacterium causes vision-threatening keratitis, life-threatening hospital acquired infections and contaminates contact lens cases. The goal of this study was to identify a hemolytic factor produced by keratitis isolates and laboratory strains of S. marcescens. Methods Hemolysis was measured using sheep and mouse erythrocytes. S. marcescens was mutated with a mariner transposon. Hemolysin defective mutants were isolated on blood agar plates. Transposon insertion sites were mapped by marker rescue and sequencing. Complementation analysis was performed with plasmids. Serratamolide was extracted with ethyl acetate and purified by preparative HPLC and verified as pure with high-resolution mass spectroscopy (HR-MS) and 1H NMR analysis. Cytotoxicity to human airway and ocular epithelial cells was performed using Alamar Blue viability dye. The presence of swrW in ocular isolates was determined using PCR. Results Mutation of the swrW gene conferred a hemolysis defect that was complemented by the wild-type swrW gene on a plasmid. The SwrW protein catalyzes production of the cyclic lipopeptide serratamolide. Purified serratamolide was hemolytic to mammalian erythrocytes, and cytotoxic to human airway and ocular cells in vitro. The swrW gene was found in the majority of contact-lens associated keratitis isolates. Conclusion Serratamolide is a novel hemolysin produced by S. marcescens ocular isolates and may contribute to the ability of contact lens associated bacteria to cause infections.

Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that d -glucono-1,5-lactone and d -gluconic acid, but not d -gluconate, were able to inhibit prodigiosin production. These data support a model in which the oxidation of d -glucose by quinoprotein GDH initiates a reduction in pH that inhibits prodigiosin production through transcriptional control of the prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites.

Serratia marcescens is a common contaminant of contact lens cases and lenses. Hemolytic factors of S. marcescens contribute to the virulence of this opportunistic bacterial pathogen. We took advantage of an observed hyper-hemolytic phenotype of crp mutants to investigate mechanisms of hemolysis. A genetic screen revealed that swrW is necessary for the hyper-hemolysis phenotype of crp mutants. The swrW gene is required for biosynthesis of the biosurfactant serratamolide, previously shown to be a broad-spectrum antibiotic and to contribute to swarming motility. Multicopy expression of swrW or mutation of the hexS transcription factor gene, a known inhibitor of swrW expression, led to an increase in hemolysis. Surfactant zones and expression from an swrW-transcriptional reporter were elevated in a crp mutant compared to the wild type. Purified serratamolide was hemolytic to sheep and murine red blood cells and cytotoxic to human airway and corneal limbal epithelial cells in vitro. The swrW gene was found in the majority of contact lens isolates tested. Genetic and biochemical analysis implicate the biosurfactant serratamolide as a hemolysin. This novel hemolysin may contribute to irritation and infections associated with contact lens use.

A case of endophthalmitis following uneventful phacoemulsification and posterior chamber intraocular lens (IOL) implantation in a 77-year-old diabetic man was culture-positive for Enterococcus faecalis . After successful treatment with intravitreal, topical, and systemic antibiotic agents, the infection seemed to clear and the patient achieved a corrected visual acuity of 20/25. Four months after the initial presentation, the patient again developed signs and symptoms of endophthalmitis, with regrowth of E faecalis . The antibiotic therapy was repeated. One month later, the IOL was removed surgically and found to harbor a biofilm of the strain demonstrated by DNA analysis. The microbiologic and DNA analyses support that a biofilm on an IOL could be a vector for a cause of recurrent endophthalmitis. Intraocular lens exchange in cases of postoperative endophthalmitis caused by E faecalis may be considered to decrease the risk for recurrent infection. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.