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1.  The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant 
BMC Biology  2013;11:123.
Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1.
The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs.
These data represent the first genome-wide analysis of translation in a eukaryote defective in the large ribosomal subunit. RPL24 and eIF3h play similar but non-identical roles in eukaryotic translation. The data also shed light on the fine structure of the regulon of ribosomal protein mRNAs.
PMCID: PMC3901033  PMID: 24377433
Translation state; Ribosome occupancy; RPL24; PABP; Regulon; Arabidopsis
2.  Post-transcriptional regulation in the myo1Δ mutant of Saccharomyces cerevisiae 
BMC Genomics  2010;11:690.
Saccharomyces cerevisiae myosin type II-deficient (myo1Δ) strains remain viable and divide, despite the absence of a cytokinetic ring, by activation of the PKC1-dependent cell wall integrity pathway (CWIP). Since the myo1Δ transcriptional fingerprint is a subset of the CWIP fingerprint, the myo1Δ strain may provide a simplified paradigm for cell wall stress survival.
To explore the post-transcriptional regulation of the myo1Δ stress response, 1,301 differentially regulated ribosome-bound mRNAs were identified by microarray analysis of which 204 were co-regulated by transcription and translation. Four categories of mRNA were significantly affected - protein biosynthesis, metabolism, carbohydrate metabolism, and unknown functions. Nine genes of the 20 CWIP fingerprint genes were post-transcriptionally regulated. Down and up regulation of selected ribosomal protein and cell wall biosynthesis mRNAs was validated by their distribution in polysomes from wild type and myo1Δ strains. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2α-P) and a reduction in the steady state levels of the translation initiation factor eIF4Gp in myo1Δ strains. Deletion of GCN2 in myo1Δ abolished eIF2αp phosphorylation, and showed a severe growth defect. The presence of P-bodies in myo1Δ strains suggests that the process of mRNA sequestration is active, however, the three representative down regulated RP mRNAs, RPS8A, RPL3 and RPL7B were present at equivalent levels in Dcp2p-mCh-positive immunoprecipitated fractions from myo1Δ and wild type cells. These same RP mRNAs were also selectively co-precipitated with eIF2α-P in myo1Δ strains.
Quantitative analysis of ribosome-associated mRNAs and their polyribosome distributions suggests selective regulation of mRNA translation efficiency in myo1Δ strains. Inhibition of translation initiation factor eIF2α (eIF2α-P) in these strains was by Gcn2p-dependent phosphorylation. The increase in the levels of eIF2α-P; the genetic interaction between GCN2 and MYO1; and the reduced levels of eIF4Gp suggest that other signaling pathways, in addition to the CWIP, may be important for myo1Δ strain survival. Selective co-immunoprecipitation of RP mRNAs with eIF2α-P in myo1Δ strains suggests a novel mode of translational regulation. These results indicate that post-transcriptional control is important in the myo1Δ stress response and possibly other stresses in yeast.
PMCID: PMC3017085  PMID: 21126371
3.  RPL24: a potential therapeutic target whose depletion or acetylation inhibits polysome assembly and cancer cell growth 
Oncotarget  2014;5(13):5165-5176.
Partial loss of large ribosomal subunit protein 24 (RPL24) function is known to protect mice against Akt or Myc-driven cancers, in part via translational inhibition of a subset of cap(eIF4E)-dependently translated mRNAs. The role of RPL24 in human malignancies is unknown. By analyzing a public dataset of matched human breast cancers and normal mammary tissue, we found that breast cancers express significantly more RPL24 than matched normal breast samples. Depletion of RPL24 in breast cancer cells by >70% reduced cell viability by 80% and decreased protein expression of the eIF4E-dependently translated proteins cyclin D1 (75%), survivin (46%) and NBS1 (30%) without altering GAPDH or beta-tubulin levels. RPL24 knockdown also reduced 80S subunit levels relative to 40S and 60S levels. These effects on expression of eIF4E-dependent proteins and ribosome assembly were mimicked by 2-24 h treatment with the pan-HDACi, trichostatin A (TSA), which induced acetylation of 15 different polysome-associated proteins including RPL24. Furthermore, HDAC6-selective inhibition or HDAC6 knockdown induced ribosomal protein acetylation. Via mass spectrometry, we found that 60S-associated, but not, polysome-associated, RPL24 undergoes HDACi-induced acetylation on K27. Thus, RPL24 K27 acetylation may play a role in ribosome assembly. These findings point toward a novel acetylation-dependent polysome assembly mechanism regulating tumorigenesis.
PMCID: PMC4148130  PMID: 24970821
RPL24; eIF6; ribosome assembly; translation; HDACs; breast cancer
4.  Dynamics of mRNA and polysomal abundance in early 3T3-L1 adipogenesis 
BMC Genomics  2014;15(1):381.
Adipogenesis is a complex process, in which immature pre-adipocytes change morphology, micro-anatomy and physiology to become mature adipocytes. These store and accumulate fat and release diverse hormones. Massive changes in protein content and protein composition of the transforming cell take place within a short time-frame.
In a previous study we analyzed changes in the abundance of free and polysomal, i.e. ribosome bound, RNAs in the first hours of adipogenesis in the murine cell line 3T3-L1. Here we analyze changes of mRNA levels and their potential contribution to the changing protein pool by determination of mRNA levels and ribosome binding to mRNAs in 3T3-L1 cells stimulated for adipogenesis. We grouped mRNA species into categories with respect to up- or down-regulated transcription and translation and analyzed the groups regarding specific functionalities based on Gene Ontology (GO).
A shift towards up-regulation of gene expression in early adipogenesis was detected. Genes up-regulated at the transcriptional (TC:up) and translational (TL:up) level (TC:up/TL:up) are very likely involved in control and logistics of translation. Many of them are known to contain a TOP motif. In the TC:up/TL:unchanged group we detected most of the metal binding proteins and metal transporters. In the TC:unchanged/TL:up group several factors of the olfactory receptor family were identified, while in TC:unchanged/TL:down methylation and repair genes are represented. In the TC:down/TL:up group we detected many signaling factors. The TC:down/TL:unchanged group mainly consists of regulatory factors.
Within the first hours of adipogenesis, changes in transcriptional and translational regulation take place. Notably, genes with a specific biological or molecular function tend to cluster in groups according to their transcriptional and translational regulation.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-381) contains supplementary material, which is available to authorized users.
PMCID: PMC4039748  PMID: 24886538
Adipogenesis; Transcription; Translation; 3T3-L1 pre-adipocytes; Gene ontology; Gene enrichment; Differential expression; TOP motif
5.  An investigation of ribosomal protein L10 gene in autism spectrum disorders 
BMC Medical Genetics  2009;10:7.
Autism spectrum disorders (ASD) are severe neurodevelopmental disorders with the male:female ratio of 4:1, implying the contribution of X chromosome genetic factors to the susceptibility of ASD. The ribosomal protein L10 (RPL10) gene, located on chromosome Xq28, codes for a key protein in assembling large ribosomal subunit and protein synthesis. Two non-synonymous mutations of RPL10, L206M and H213Q, were identified in four boys with ASD. Moreover, functional studies of mutant RPL10 in yeast exhibited aberrant ribosomal profiles. These results provided a novel aspect of disease mechanisms for autism – aberrant processes of ribosome biosynthesis and translation. To confirm these initial findings, we re-sequenced RPL10 exons and quantified mRNA transcript level of RPL10 in our samples.
141 individuals with ASD were recruited in this study. All RPL10 exons and flanking junctions were sequenced. Furthermore, mRNA transcript level of RPL10 was quantified in B lymphoblastoid cell lines (BLCL) of 48 patients and 27 controls using the method of SYBR Green quantitative PCR. Two sets of primer pairs were used to quantify the mRNA expression level of RPL10: RPL10-A and RPL10-B.
No non-synonymous mutations were detected in our cohort. Male controls showed similar transcript level of RPL10 compared with female controls (RPL10-A, U = 81, P = 0.7; RPL10-B, U = 61.5, P = 0.2). We did not observe any significant difference in RPL10 transcript levels between cases and controls (RPL10-A, U = 531, P = 0.2; RPL10-B, U = 607.5, P = 0.7).
Our results suggest that RPL10 has no major effect on the susceptibility to ASD.
PMCID: PMC2645381  PMID: 19166581
6.  Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced 
Nucleic Acids Research  2010;39(7):2701-2716.
Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expression was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal component. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated ‘rpL22-like short’) that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like.
PMCID: PMC3074143  PMID: 21138957
7.  Organization and Transcriptional Analysis of a Six-Gene Cluster around the rplK-rplA Operon of Corynebacterium glutamicum Encoding the Ribosomal Proteins L11 and L1 
A cluster of six genes, tRNATrp-secE-nusG-rplK-rplA-pkwR, was cloned and sequenced from a Corynebacterium glutamicum cosmid library and shown to be contiguous in the C. glutamicum genome. These genes encode a tryptophanyl tRNA, the protein translocase component SecE, the antiterminator protein NusG, and the ribosomal proteins L11 and L1 in addition to PkwR, a putative regulatory protein of the LacI-GalR family. S1 nuclease mapping analysis revealed that nusG and rplK are expressed as separate transcriptional units and rplK and rplA are cotranscribed as a single mRNA. A 19-nucleotide inverted repeat that appears to correspond to a transcriptional terminator was located in the 3′ region downstream from nusG. Northern analysis with different probes confirmed the S1 mapping results and showed that the secE-rplA four-gene region gives rise to four transcripts. secE was transcribed as a 0.5-kb monocistronic mRNA, nusG formed two transcripts of 1.4 and 1.0 kb from different initiation sites, and the two ribosomal protein genes rplK and rplA were cotranscribed as a single mRNA of 1.6 kb. A consensus L1 protein binding sequence was identified in the leader region of the rplK-rplA transcript, suggesting that expression of the rplK-rplA cluster was regulated by autogenous regulation exerted by the L1 protein at the translation level. The promoters of the nusG and rplK-rplA genes were subcloned in a novel corynebacterial promoter-probe vector and shown to confer strong expression of the reporter gene.
PMCID: PMC92853  PMID: 11319098
8.  GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1991;11(6):3203-3216.
The GCD2 protein is a translational repressor of GCN4, the transcriptional activator of multiple amino acid biosynthetic genes in Saccharomyces cerevisiae. We present evidence that GCD2 has a general function in the initiation of protein synthesis in addition to its gene-specific role in translational control of GCN4 expression. Two temperature-sensitive lethal gcd2 mutations result in sensitivity to inhibitors of protein synthesis at the permissive temperature, and the gcd2-503 mutation leads to reduced incorporation of labeled leucine into total protein following a shift to the restrictive temperature of 36 degrees C. The gcd2-503 mutation also results in polysome runoff, accumulation of inactive 80S ribosomal couples, and accumulation of at least one of the subunits of the general translation initiation factor 2 (eIF-2 alpha) in 43S-48S particles following a shift to the restrictive temperature. The gcd2-502 mutation causes accumulation of 40S subunits in polysomes, known as halfmers, that are indicative of reduced 40S-60S subunit joining at the initiation codon. These phenotypes suggest that GCD2 functions in the translation initiation pathway at a step following the binding of eIF-2.GTP.Met-tRNA(iMet) to 40S ribosomal subunits. consistent with this hypothesis, we found that inhibiting 40S-60S subunit joining by deleting one copy (RPL16B) of the duplicated gene encoding the 60S ribosomal protein L16 qualitatively mimics the phenotype of gcd2 mutations in causing derepression of GCN4 expression under nonstarvation conditions. However, deletion of RPL16B also prevents efficient derepression of GCN4 under starvation conditions, indicating that lowering the concentration of 60S subunits and reducing GCD2 function affect translation initiation at GCN4 in different ways. This distinction is in accord with a recently proposed model for GCN4 translational control in which ribosomal reinitiation at short upstream open reading frames in the leader of GCN4 mRNA is suppressed under amino acid starvation conditions to allow for increased reinitiation at the GCN4 start codon.
PMCID: PMC360173  PMID: 2038326
9.  Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1990;10(10):5235-5243.
In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.
PMCID: PMC361207  PMID: 2204809
10.  Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control 
Combining translating ribosome affinity purification with RNA-seq for cell-specific profiling of translating RNAs in developing flowers.Cell type comparisons of cell type-specific hormone responses, promoter motifs, coexpressed cognate binding factor candidates, and splicing isoforms.Widespread post-transcriptional regulation at both the intron splicing and translational stages.A new class of noncoding RNAs associated with polysomes.
What constitutes a differentiated cell type? How much do cell types differ in their transcription of genes? The development and functions of tissues rely on constant interactions among distinct and nonequivalent cell types. Answering these questions will require quantitative information on transcriptomes, proteomes, protein–protein interactions, protein–nucleic acid interactions, and metabolomes at cellular resolution. The systems approaches emerging in biology promise to explain properties of biological systems based on genome-wide measurements of expression, interaction, regulation, and metabolism. To facilitate a systems approach, it is essential first to capture such components in a global manner, ideally at cellular resolution.
Recently, microarray analysis of transcriptomes has been extended to a cellular level of resolution by using laser microdissection or fluorescence-activated sorting (for review, see Nelson et al, 2008). These methods have been limited by stresses associated with cellular separation and isolation procedures, and biases associated with mandatory RNA amplification steps. A newly developed method, translating ribosome affinity purification (TRAP; Zanetti et al, 2005; Heiman et al, 2008; Mustroph et al, 2009), circumvents these problems by epitopetagging a ribosomal protein in specific cellular domains to selectively purify polysomes. We combined TRAP with deep sequencing, which we term TRAP-seq, to provide cell-level spatiotemporal maps for Arabidopsis early floral development at single-base resolution.
Flower development in Arabidopsis has been studied extensively and is one of the best understood aspects of plant development (for review, see Krizek and Fletcher, 2005). Genetic analysis of homeotic mutants established the ABC model, in which three classes of regulatory genes, A, B and C, work in a combinatorial manner to confer organ identities of four whorls (Coen and Meyerowitz, 1991). Each class of regulatory gene is expressed in a specific and evolutionarily conserved domain, and the action of the class A, B and C genes is necessary for specification of organ identity (Figure 1A).
Using TRAP-seq, we purified cell-specific translating mRNA populations, which we and others call the translatome, from the A, B and C domains of early developing flowers, in which floral patterning and the specification of floral organs is established. To achieve temporal specificity, we used a floral induction system to facilitate collection of early stage flowers (Wellmer et al, 2006). The combination of TRAP-seq with domain-specific promoters and this floral induction system enabled fine spatiotemporal isolation of translating mRNA in specific cellular domains, and at specific developmental stages.
Multiple lines of evidence confirmed the specificity of this approach, including detecting the expression in expected domains but not in other domains for well-studied flower marker genes and known physiological functions (Figures 1B–D and 2A–C). Furthermore, we provide numerous examples from flower development in which a spatiotemporal map of rigorously comparable cell-specific translatomes makes possible new views of the properties of cell domains not evident in data obtained from whole organs or tissues, including patterns of transcription and cis-regulation, new physiological differences among cell domains and between flower stages, putative hormone-active centers, and splicing events specific for flower domains (Figure 2A–D). Such findings may provide new targets for reverse genetics studies and may aid in the formulation and validation of interaction and pathway networks.
Beside cellular heterogeneity, the transcriptome is regulated at several steps through the life of mRNA molecules, which are not directly available through traditional transcriptome profiling of total mRNA abundance. By comparing the translatome and transcriptome, we integratively profiled two key posttranscriptional control points, intron splicing and translation state. From our translatome-wide profiling, we (i) confirmed that both posttranscriptional regulation control points were used by a large portion of the transcriptome; (ii) identified a number of cis-acting features within the coding or noncoding sequences that correlate with splicing or translation state; and (iii) revealed correlation between each regulation mechanism and gene function. Our transcriptome-wide surveys have highlighted target genes transcripts of which are probably under extensive posttranscriptional regulation during flower development.
Finally, we reported the finding of a large number of polysome-associated ncRNAs. About one-third of all annotated ncRNA in the Arabidopsis genome were observed co-purified with polysomes. Coding capacity analysis confirmed that most of them are real ncRNA without conserved ORFs. The group of polysome-associated ncRNA reported in this study is a potential new addition to the expanding riboregulator catalog; they could have roles in translational regulation during early flower development.
Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.
PMCID: PMC2990639  PMID: 20924354
Arabidopsis; flower; intron; transcriptome; translation
11.  RPL41, a Small Ribosomal Peptide Deregulated in Tumors, Is Essential for Mitosis and Centrosome Integrity1 
Neoplasia (New York, N.Y.)  2010;12(3):284-293.
Ribosomal large subunit protein RPL41 is a basic (positively charged) peptide consisting of only 25 amino acids. An antisense-based functional screening revealed that the down-regulation of RPL41 led to an anchorage-independent growth of NIH3T3 cells in soft agar plates. RPL41 depletion with gene-specific small interfering RNA also resulted in malignant transformation of NIH3T3 cells including increased tumor growth in mice. RPL41 deletion was detected in 59% of tumor cell lines by fluorescence in situ hybridization analyses and RPL41 down-regulation in 75% of primary breast cancers by real-time quantitative reverse transcription-polymerase chain reaction. These studies suggest a tumor suppression role for RPL41. By mass spectrometry, RPL41 was associated with several cytoskeleton components including tubulin β, γ, and myosin IIA, which was confirmed by Western blot analysis on both cellular lysis and individually in vitro-expressed proteins. RPL41 also bound directly to polymerized tubulins. Cells overexpressing a GFP-RPL41 were resistant to nocodazole-induced microtubule depolymerization. A synthetic RPL41 induced cellular α-tubulin acetylation and G2/M cell cycle arrest. These results indicate a stabilizing role of RPL41 on microtubule. Microtubule spindles are essential for chromosome segregation during mitosis. Cells with RPL41 knock-down showed abnormal spindles, frequent failure of cytokinesis, and formation of polynuclear cells. In interphase cells, RPL41-depleted cells had premature splitting of centrosome. Our results provide evidence that RPL41 is a microtubule-associated protein essential for functional spindles and for the integrity of centrosome and that the abnormal mitosis and disrupted centrosome associated with the RPL41 down-regulation may be related to malignant transformation.
PMCID: PMC2838445  PMID: 20234822
12.  eIF4EBP3L Acts as a Gatekeeper of TORC1 In Activity-Dependent Muscle Growth by Specifically Regulating Mef2ca Translational Initiation 
PLoS Biology  2013;11(10):e1001679.
Muscle activity promotes muscle growth through the TOR-4EBP pathway by controlling the translation of specific mRNAs, including Mef2ca, a muscle transcription factor required for normal growth.
Muscle fiber size is activity-dependent and clinically important in ageing, bed-rest, and cachexia, where muscle weakening leads to disability, prolonged recovery times, and increased costs. Inactivity causes muscle wasting by triggering protein degradation and may simultaneously prevent protein synthesis. During development, muscle tissue grows by several mechanisms, including hypertrophy of existing fibers. As in other tissues, the TOR pathway plays a key role in promoting muscle protein synthesis by inhibition of eIF4EBPs (eukaryotic Initiation Factor 4E Binding Proteins), regulators of the translational initiation. Here, we tested the role of TOR-eIF4EBP in a novel zebrafish muscle inactivity model. Inactivity triggered up-regulation of eIF4EBP3L (a zebrafish homolog of eIF4EBP3) and diminished myosin and actin content, myofibrilogenesis, and fiber growth. The changes were accompanied by preferential reduction of the muscle transcription factor Mef2c, relative to Myod and Vinculin. Polysomal fractionation showed that Mef2c decrease was due to reduced translation of mef2ca mRNA. Loss of Mef2ca function reduced normal muscle growth and diminished the reduction in growth caused by inactivity. We identify eIF4EBP3L as a key regulator of Mef2c translation and protein level following inactivity; blocking eIF4EBP3L function increased Mef2ca translation. Such blockade also prevented the decline in mef2ca translation and level of Mef2c and slow myosin heavy chain proteins caused by inactivity. Conversely, overexpression of active eIF4EBP3L mimicked inactivity by decreasing the proportion of mef2ca mRNA in polysomes, the levels of Mef2c and slow myosin heavy chain, and myofibril content. Inhibiting the TOR pathway without the increase in eIF4EBP3L had a lesser effect on myofibrilogenesis and muscle size. These findings identify eIF4EBP3L as a key TOR-dependent regulator of muscle fiber size in response to activity. We suggest that by selectively inhibiting translational initiation of mef2ca and other mRNAs, eIF4EBP3L reprograms the translational profile of muscle, enabling it to adjust to new environmental conditions.
Author Summary
Most genes are transcribed into mRNA and then translated into proteins that function in various cellular processes. Initiation of mRNA translation is thus a fundamental control point in gene expression. Working in a zebrafish model, we have found that muscle activity (or inactivity) can differentially regulate the translation of specific mRNAs and thereby control the growth of skeletal muscle. Emerging evidence suggests that control of translational initiation of particular mRNAs by an intracellular signaling pathway acting through TORC1 is a major regulator of cell growth and function. We show here that muscle activity both activates the TORC1 pathway and suppresses the expression of a downstream TORC1 target—the translational inhibitor eIF4EBP3L. This removes a brake on translation of certain mRNAs. Conversely, we show that muscle inactivity can up-regulate this translational inhibitor, thereby causing reduced translation of these mRNAs. One of the mRNAs targeted in this manner by eIF4EBP3L is Mef2ca, which encodes a transcription factor that promotes assembly of muscle contractile apparatus. Our work thus reveals a mechanism by which muscle growth can be differentially influenced depending on the context of muscle activity (or lack thereof). If this pathway operates in people, it may help explain how exercise regulates muscle growth and performance.
PMCID: PMC3797031  PMID: 24143132
13.  Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids. 
Molecular and Cellular Biology  1989;9(9):3679-3684.
P1798 murine lymphosarcoma cells cease to proliferate upon exposure to 10(-7) M dexamethasone and exhibit a dramatic inhibition of rRNA and ribosomal protein synthesis (O. Meyuhas, E. Thompson, Jr., and R. P. Perry, Mol. Cell Biol. 7:2691-2699, 1987). These workers demonstrated that ribosomal protein synthesis is regulated primarily at the level of translation, since dexamethasone did not alter mRNA levels but shifted the mRNAs from active polysomes into inactive messenger ribonucleoproteins. We have examined the effects of dexamethasone on the biosynthesis of initiation factor proteins in the same cell line. The relative protein synthesis rates of eIF-4A and eIF-2 alpha were inhibited by about 70% by the hormone, a reduction comparable to that for ribosomal proteins. The mRNA levels of eIF-4A, eIF-4D, and eIF-2 alpha also were reduced by 60 to 70%, indicating that synthesis rates are proportional to mRNA concentrations. Analysis of polysome profiles showed that the average number of ribosomes per initiation factor polysome was only slightly reduced by dexamethasone, and little or no mRNA was present in messenger ribonucleoproteins. The results indicate that initiation factor gene expression is coordinately regulated with ribosomal protein synthesis but is controlled primarily by modulating mRNA levels rather than mRNA efficiency.
PMCID: PMC362428  PMID: 2779563
14.  Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24 
BMC Plant Biology  2010;10:193.
Within the scanning model of translation initiation, reinitiation is a non-canonical mechanism that operates on mRNAs harboring upstream open reading frames. The h subunit of eukaryotic initiation factor 3 (eIF3) boosts translation reinitiation on the uORF-containing mRNA coding for the Arabidopsis bZip transcription factor, AtbZip11, among others. The RPL24B protein of the large ribosomal subunit, which is encoded by SHORT VALVE1, likewise fosters translation of uORF-containing mRNAs, for example mRNAs for auxin response transcription factors (ARFs).
Here we tested the hypothesis that RPL24B and eIF3h affect translation reinitiation in a similar fashion. First, like eif3h mutants, rpl24b mutants under-translate the AtbZip11 mRNA, and the detailed spectrum of translational defects in rpl24b is remarkably similar to that of eif3h. Second, eif3h mutants display defects in auxin mediated organogenesis and gene expression, similar to rpl24b. Like AtbZip11, the uORF-containing ARF mRNAs are indeed undertranslated in eif3h mutant seedlings.
We conclude that, similar to eIF3h, RPL24B bolsters the reinitiation competence of uORF-translating ribosomes. Coordination between eIF3 and the large ribosomal subunit helps to fine-tune translation of uORF-containing mRNAs and, in turn, to orchestrate plant development.
PMCID: PMC3020687  PMID: 20799971
15.  CK2 Is Responsible for Phosphorylation of Human La Protein Serine-366 and Can Modulate rpL37 5′-Terminal Oligopyrimidine mRNA Metabolism 
Molecular and Cellular Biology  2004;24(21):9580-9591.
La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3′ UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5′ regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S366) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5′ untranslated regions comprised of terminal oligopyrimidine (5′TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S366 represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S366 phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5′TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A366 than with La S366, concomitant with La A366-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S366 in vivo, that this limits 5′TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery.
PMCID: PMC522270  PMID: 15485924
16.  Genome-wide Analysis of Transcript Abundance and Translation in Arabidopsis Seedlings Subjected to Oxygen Deprivation 
Annals of Botany  2005;96(4):647-660.
• Background and Aims DNA microarrays allow comprehensive estimation of total cellular mRNA levels but are also amenable to studies of other mRNA populations, such as mRNAs in translation complexes (polysomes). The aim of this study was to evaluate the role of translational regulation in response to oxygen deprivation (hypoxia).
• Methods Alterations in total cellular and large polysome (≥ five ribosomes per mRNA) mRNA levels were monitored in response to 12 h of hypoxia stress in seedlings of Arabidopsis thaliana with a full-genome oligonucleotide microarray.
• Key Results Comparison of two mRNA populations revealed considerable modulation of mRNA accumulation and diversity in translation in response to hypoxia. Consistent with the global decrease in protein synthesis, hypoxia reduced the average proportion of individual mRNA species in large polysome complexes from 56·1 % to 32·1 %. A significant decrease in the association with translational complexes was observed for 77 % of the mRNAs, including a subset of known hypoxia-induced gene transcripts. The examination of mRNA levels of nine genes in polysomes fractionated through sucrose density gradients corroborated the microarray data. Gene cluster analysis was used to identify mRNAs that displayed co-ordinated regulation. Fewer than half of the highly induced mRNAs circumvented the global depression of translation. Moreover, a large number of mRNAs displayed a significant decrease in polysome association without a concomitant decrease in steady-state accumulation. The abundant mRNAs that encode the ribosomal proteins behaved in this manner. By contrast, a small group of abiotic and biotic stress-induced mRNAs showed a significant increase in polysome association, without a change in abundance. Evaluation of quantitative features of mRNA sequences demonstrated that a low GC nucleotide content of the 5′-untranslated region provides a selective advantage for translation under hypoxia.
• Conclusions Alterations in transcript abundance and translation contribute to the differential regulation of gene expression in response to oxygen deprivation.
PMCID: PMC4247032  PMID: 16081496
Hypoxia; DNA microarray; polysome; translational control; mRNA sequence features; Arabidopsis thaliana
17.  c-myc Repression of TSC2 Contributes to Control of Translation Initiation and Myc-Induced Transformation 
Cancer research  2007;67(23):11209-11217.
The c-myc oncogene plays a key role in cellular growth control, and translation initiation factors are among the transcriptional targets of Myc. Here, we describe a defect in translation initiation control in myc-null cells due to alterations in the mammalian target of rapamycin (mTOR) pathway. Myc loss increased sensitivity to dominant inhibition of eukaryotic translation initiation factor 4E function. Polysomal profiles of myc−/− cells revealed decreased translation initiation rates, which were accompanied by decreased 40S/60S ribosomal subunit ratios. Because the 40S small ribosomal subunit contains the key regulatory ribosomal protein S6 (rpS6), we considered that myc loss might affect expression of components of the mTOR signaling pathway that regulate rpS6 function. Among mTOR signaling components, Myc directly affected transcription of tuberous sclerosis 2 (TSC2), as shown by quantitative mRNA analysis and by Myc binding to its promoter in chromatin immunoprecipitation assays. Importantly, Myc acted as a strong and direct repressor for TSC2 expression because its loss increased TSC2 mRNA in myc-null and in HL60 shRNA experiments, activation of a mycER construct in myc−/− cells suppressed TSC2 induction in a myc box II–dependent manner, and mycER activation recruited Myc to the TSC2 promoter. The biological significance of the effect of Myc on TSC2 expression was shown by markedly reduced TSC2 mRNA levels in myc-transformed cells, stimulation of S6 kinase activity in myc-null cells by TSC2 siRNA, and decreased Myc-induced soft agar colony formation following retroviral transduction of TSC2. Together, these findings show that regulation of TSC2 can contribute to the effects of Myc on cell proliferation and neoplastic growth.
PMCID: PMC3022657  PMID: 18056446
18.  Translational profiling in childhood acute lymphoblastic leukemia: no evidence for glucocorticoid regulation of mRNA translation 
BMC Genomics  2013;14(1):844.
Glucocorticoids (GCs) are natural stress induced steroid hormones causing cell cycle arrest and cell death in lymphoid tissues. Therefore they are the central component in the treatment of lymphoid malignancies, in particular childhood acute lymphoblastic leukemia (chALL). GCs act mainly via regulating gene transcription, which has been intensively studied by us and others. GC control of mRNA translation has also been reported but has never been assessed systematically. In this study we investigate the effect of GCs on mRNA translation on a genome-wide scale.
Childhood T- (CCRF-CEM) and precursor B-ALL (NALM6) cells were exposed to GCs and subjected to “translational profiling”, a technique combining sucrose-gradient fractionation followed by Affymetrix Exon microarray analysis of mRNA from different fractions, to assess the translational efficiency of the expressed genes. Analysis of GC regulation in ribosome-bound fractions versus transcriptional regulation revealed no significant differences, i.e., GC did not entail a significant shift between ribosomal bound and unbound mRNAs.
In the present study we analyzed for the first time possible effects of GC on the translational efficiency of expressed genes in two chALL model systems employing whole genome polysome profiling. Our results did not reveal significant differences in translational efficiency of expressed genes thereby arguing against a potential widespread regulatory effect of GCs on translation at least in the investigated in vitro systems.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-14-844) contains supplementary material, which is available to authorized users.
PMCID: PMC4046653  PMID: 24289529
Polysome profiling; Translatome; Gene regulation; Glucocorticoids; Acute lymphoblastic leukemia
19.  Measurement of the Rates of Synthesis of Three Components of Ribosomes of Mycobacterium fortuitum: A Theoretical Approach to qRT-PCR Experimentation 
PLoS ONE  2010;5(7):e11575.
Except for the ribosomal protein L12 (rplL), ribosomal proteins are present as one copy per ribosome; L12 (rplL) is unusual because it is present as four copies per ribosome. Thus, the strategies used by Mycobacterium fortuitum to regulate ribosomal protein synthesis were investigated, including evaluations of the rates of chain elongations of 16S rRNA, rplL and ribosomal protein S12 (rpsL).
RNA was isolated from cell cultures and cDNA was prepared. The numbers of cDNA copies of 16S rRNA, precursor-16S rRNA and transcripts of rpsL and rplL were quantified by qRT-PCR and then related to the rates of 16S rRNA, rpsL and rplL chain elongations by means of a mathematical framework for coupled transcription/translation.
Principal Findings
The rates of synthesis of 16S rRNA, rpsL and rplL respectively were found to be approximately 50×103 nucleotides h−1, 1.6×103 amino acid residues h−1 and 3.4×103 amino acid residues h−1. The number of transcripts of rplL was approximately twice that of rpsL. These data account for the presence of one copy of rpsL and four copies of rplL per ribosome, and reveal that the rate of M. fortuitum ribosome synthesis was closer to that of M. tuberculosis than to E. coli. Except for rplJ, the elongation rate obtained for rpsL was inferred to be appropriate for all other proteins present as one copy per ribosome.
The results obtained provide the basis for a comprehensive view of the kinetics of ribosome synthesis, and of the ways that bacterial cells utilize genes encoding ribosomal proteins. The methodology also applies to proteins involved in transcription, energy generation and to bacterial proteins in general. The method proposed for measuring the fidelity of cDNA preparations is intrinsically much more sensitive than procedures that measure the integrity of 16S rRNA.
PMCID: PMC2904383  PMID: 20644643
20.  SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes 
Journal of Bacteriology  1999;181(10):3136-3143.
Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.
PMCID: PMC93769  PMID: 10322015
21.  Downregulation of RPL6 by siRNA Inhibits Proliferation and Cell Cycle Progression of Human Gastric Cancer Cell Lines 
PLoS ONE  2011;6(10):e26401.
Our previous study revealed that human ribosomal protein L6 (RPL6) was up-regulated in multidrug-resistant gastric cancer cells and over-expression of RPL6 could protect gastric cancer from drug-induced apoptosis. It was further demonstrated that up-regulation of RPL6 accelerated growth and enhanced in vitro colony forming ability of GES cells while down-regulation of RPL6 exhibited the opposite results. The present study was designed to investigate the potential role of RPL6 in therapy of gastric cancer for clinic. The expression of RPL6 and cyclin E in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemisty. It was found that RPL6 and cyclin E were expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa and the two were correlative in gastric cancer. Survival time of postoperative patients was analyzed by Kaplan- Meier analysis and it was found that patients with RPL6 positive expression showed shorter survival time than patients that with RPL6 negative expression. RPL6 was then genetically down-regulated in gastric cancer SGC7901 and AGS cell lines by siRNA. It was demonstrated that down-regulation of RPL6 reduced colony forming ability of gastric cancer cells in vitro and reduced cell growth in vivo. Moreover, down-regulation of RPL6 could suppress G1 to S phase transition in these cells. Further, we evidenced that RPL6 siRNA down-regulated cyclin E expression in SGC7901 and AGS cells. Taken together, these data suggested that RPL6 was over-expressed in human gastric tissues and caused poor prognosis. Down-regulation of RPL6 could suppress cell growth and cell cycle progression at least through down-regulating cyclin E and which might be used as a novel approach to gastric cancer therapy.
PMCID: PMC3197136  PMID: 22043320
22.  RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to the nucleoplasm of Drosophila meiotic spermatocytes 
Nucleus  2013;4(3):241-258.
Duplicated ribosomal protein (Rp) gene families often encode highly similar or identical proteins with redundant or unique roles. Eukaryotic-specific paralogues RpL22e and RpL22e-like-PA are structurally divergent within the N terminus and differentially expressed, suggesting tissue-specific functions. We previously identified RpL22e-like-PA as a testis Rp. Strikingly, RpL22e is detected in immunoblots at its expected molecular mass (m) of 33 kD and at increasing m of ~43–55 kD, suggesting RpL22e post-translational modification (PTM). Numerous PTMs, including N-terminal SUMOylation, are predicted computationally. Based on S2 cell co-immunoprecipitations, bacterial-based SUMOylation assays and in vivo germline-specific RNAi depletion of SUMO, we conclude that RpL22e is a SUMO substrate. Testis-specific PTMs are evident, including a phosphorylated version of SUMOylated RpL22e identified by in vitro phosphatase experiments. In ribosomal profiles from S2 cells, only unconjugated RpL22e co-sediments with active ribosomes, supporting an extra-translational role for SUMOylated RpL22e. Ectopic expression of an RpL22e N-terminal deletion (lacking SUMO motifs) shows that truncated RpL22e co-sediments with polysomes, implying that RpL22e SUMOylation is dispensable for ribosome biogenesis and function. In mitotic germ cells, both paralogues localize within the cytoplasm and nucleolus. However, within meiotic cells, phase contrast microscopy and co-immunohistochemical analysis with nucleolar markers nucleostemin1 and fibrillarin reveals diffuse nucleoplasmic, but not nucleolar RpL22e localization that transitions to a punctate pattern as meiotic cells mature, suggesting an RpL22e role outside of translation. Germline-specific knockdown of SUMO shows that RpL22e nucleoplasmic distribution is sensitive to SUMO levels, as immunostaining becomes more dispersed. Overall, these data suggest distinct male germline roles for RpL22e and RpL22e-like-PA.
PMCID: PMC3720754  PMID: 23778934
RpL22e; RpL22e-like-PA; Drosophila; ribosomal protein paralogues; duplicated ribosomal proteins; SUMOylation; phosphorylation; post-translational modification; male germline
23.  Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies 
BMC Molecular Biology  2010;11:22.
Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium.
The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions.
The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.
PMCID: PMC2907514  PMID: 20331858
24.  Autogenous regulation of the RNA polymerase beta subunit of Escherichia coli occurs at the translational level in vivo. 
Journal of Bacteriology  1989;171(11):6234-6242.
A series of transcriptional and translational fusions of the gene for the beta subunit of RNA polymerase (rpoB) to the lacZ reporter gene have been constructed on lambda vectors. Both transcriptional and translational fusions carry the upstream rplKAJL ribosomal protein gene region, which contains the two strong promoters rplKp and rplJp responsible for the transcription of rpoBC. Monolysogens carrying either the transcriptional translational fusion were assayed for beta-galactosidase, providing a measure of the transcription or of both transcription and translation of rpoB, respectively. Translational fusion monolysogens which also carried a multicopy plasmid containing the beta and beta' genes (rpoBC) under the control of a regulatable promoter, exhibited a substantial decrease in the beta-galactosidase levels upon overproduction of beta and beta'. No significant effect was seen in comparable experiments with the transcriptional fusions. These results argue that in vivo, the synthesis of the RNA polymerase beta subunit is autogenously regulated by a translational mechanism. Furthermore, experiments with the overexpressing plasmids confirm the requirement for a portion of the rplL-rpoB intercistronic region in the vicinity of the RNaseIII processing site for the efficient translation of the beta subunit mRNA.
PMCID: PMC210494  PMID: 2681158
25.  Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3 
PLoS Genetics  2014;10(3):e1004205.
Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.
Author Summary
Recent progress has provided us with detailed knowledge of the structure and function of eukaryotic ribosomes. However, our understanding of the intricate processes of pre-ribosome assembly and the transition to translation-competent ribosomal subunits remains incomplete. The early and intermediate steps of ribosome assembly occur successively in the nucleolus and nucleoplasm. The pre-ribosomal subunits are then exported to the cytoplasm where final maturation steps, notably including D site cleavage of the 20S pre-rRNA to mature 18S rRNA, confer subunit joining and translation competence. Recent evidence indicates that pre-40S subunits are subject to a quality control step involving the GTP-dependent translation initiation factor eIF5B/Fun12, in the context of 80S-like ribosomes. Here, we demonstrate the involvement of 60S subunits in promoting 20S pre-rRNA cleavage. In particular, we show that a specific point mutation in the 60S subunit ribosomal protein L3 (rpl3[W255C]) leads to the accumulation of pre-40S particles that contain the 20S pre-rRNA but are translation-competent. Notably, this mutation prevents the stimulation of the GTPase activity of eIF5B/Fun12, which is also required for site D cleavage. We conclude that L3 plays an important role in regulating the function of eIF5B/Fun12 during 3′ end processing of 18S rRNA at site D, in the context of 80S ribosomes that have not yet engaged in translation.
PMCID: PMC3945201  PMID: 24603549

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