Postnatal microcephaly and retinal involvement expand the phenotype of RPL10-related disorder.

Hemizygous missense variants in the RPL10 gene encoding a ribosomal unit are responsible for an X-linked syndrome presenting with intellectual disability (ID), autism spectrum disorder, epilepsy, dysmorphic features, and multiple congenital anomalies. Among 15 individuals with RPL10-related disorder reported so far, only one patient had retinitis pigmentosa and microcephaly was observed in approximately half of the cases. By exome sequencing, three Italian and one Spanish male children, from three independent families, were found to carry the same hemizygous novel missense variant p.(Arg32Leu) in RPL10, inherited by their unaffected mother in all cases. The variant, not reported in gnomAD, is located in the 28S rRNA binding region, affecting an evolutionary conserved residue and predicted to disrupt the salt-bridge between Arg32 and Asp28. In addition to features consistent with RPL10-related disorder, all four boys had retinal degeneration and postnatal microcephaly. Pathogenic variants in genes responsible for inherited retinal degenerations were ruled out in all the probands. A novel missense RPL10 variant was detected in four probands with a recurrent phenotype including ID, dysmorphic features, progressive postnatal microcephaly, and retinal anomalies. The presented individuals suggest that retinopathy and postnatal microcephaly are clinical clues of RPL10-related disorder, and at least the retinal defect might be more specific for the p.(Arg32Leu) RPL10 variant, suggesting a specific genotype/phenotype correlation.

A Time-Resolved Cryo-EM Study of Saccharomyces cerevisiae 80S Ribosome Protein Composition in Response to a Change in Carbon Source.

The role of the ribosome in the regulation of gene expression has come into increased focus. It is proposed that ribosomes are catalytic engines capable of changing their protein composition in response to environmental stimuli. Time-resolved cryo-electron microscopy (cryo-EM) techniques are employed to identify quantitative changes in the protein composition and structure of the Saccharomyces cerevisiae 80S ribosomes after shifting the carbon source from glucose to glycerol. Using cryo-EM combined with the computational classification approach, it is found that a fraction of the yeast cells' 80S ribosomes lack ribosomal proteins at the entrance and exit sites for tRNAs, including uL16(RPL10), eS1(RPS1), uS11(RPS14A/B), and eS26(RPS26A/B). This fraction increased after a change from glucose to glycerol medium. The quantitative structural analysis supports the hypothesis that ribosomes are dynamic complexes that alter their composition in response to changes in growth or environmental conditions.

A QM protein from Bombyx mori negatively regulates prophenoloxidase activation and melanization by interacting with Jun protein.

The QM gene that encodes for the ribosomal protein L10 was firstly identified from human tumour cells as a tumour suppressor. In this study, a QM gene was identified in silkworm Bombyx mori (BmQM) and its immunomodulatory function was explored. BmQM messenger RNA (mRNA) and protein were highly expressed in the silk gland and fat body, and expressed in all stages of silkworm growth. After challenged with four different microorganisms, the expression levels of BmQM mRNA in fat body or haemocytes were significantly upregulated compared with the control. After knock-down of BmQM gene, the expressions of some immune genes (PGRPS6, Gloverin0, Lysozyme and Moricin) were affected, and the transcripts of prophenoloxidase1 and prophenoloxidase2 have different degrees of change. The phenoloxidase activity was significantly reduced when the purified recombinant BmQM protein was injected. Recombinant BmQM protein inhibited systemic melanization and suppressed prophenoloxidase activation stimulated by Micrococcus luteus, but it did not affect phenoloxidase activity. Far-western blotting assays showed that the BmQM protein interacted with silkworm BmJun protein, which negatively regulates AP-1 expression. Our results indicated that BmQM protein could affect some immune gene expression and negatively regulate the prophenoloxidase-activating system, and it may play an important role in regulation of the innate immunity in insects.

A Novel Mutation in RPL10 (Ribosomal Protein L10) Causes X-Linked Intellectual Disability, Cerebellar Hypoplasia, and Spondylo-Epiphyseal Dysplasia.

RPL10 encodes ribosomal protein L10 (uL16), a highly conserved multifunctional component of the large ribosomal subunit, involved in ribosome biogenesis and function. Using X-exome resequencing, we identified a novel missense mutation (c.191C>T; p.(A64V)) in the N-terminal domain of the protein, in a family with two affected cousins presenting with X-linked intellectual disability, cerebellar hypoplasia, and spondylo-epiphyseal dysplasia (SED). We assessed the impact of the mutation on the translational capacity of the cell using yeast as model system. The mutation generates a functional ribosomal protein, able to complement the translational defects of a conditional lethal mutation of yeast rpl10. However, unlike previously reported mutations, this novel RPL10 missense mutation results in an increase in the actively translating ribosome population. Our results expand the mutational and clinical spectrum of RPL10 identifying a new genetic cause of SED and highlight the emerging role of ribosomal proteins in the pathogenesis of neurodevelopmental disorders.

RPL10 mutation segregating in a family with X-linked syndromic Intellectual Disability.

Intellectual disability is a neurodevelopmental disorder of impaired adaptive skills and low intelligence quotient. The overall prevalence is estimated at 2-3% in the general population with extreme clinical and genetic heterogeneity, and it has been associated with possibly causative mutations in more than 700 identified genes. In a recent review, among over 100 X-linked intellectual disability causative genes, eight were reported as "awaiting replication." Exome sequencing in a large family identified a missense mutation in RPL10 highly suggestive of X-linked intellectual disability. Herein, we report on the clinical description of four affected males. All patients presented apparent intellectual disability (4/4), psychomotor delay (4/4) with syndromic features including amniotic fluid excess (3/4), microcephaly (2/4), urogenital anomalies (3/4), cerebellar syndrome (2/4), and facial dysmorphism. In the literature, two mutations were reported in three families with affected males presenting with autism. This report confirms the implication of RPL10 mutations in neurodevelopmental disorders and extends the associated clinical spectrum from autism to syndromic intellectual disability.

Cytoskeletal proteins associate with components of the ribosomal maturation and translation apparatus in Xenopus stage I oocytes.

Actin-based cytoskeleton (CSK) and microtubules may bind to RNAs and related molecules implicated in translation. However, many questions remain to be answered regarding the role of cytoskeletal components in supporting the proteins involved in steps in the maturation and translation processes. Here, we performed co-immunoprecipitation and immunofluorescence to examine the association between spectrins, keratins and tubulin and proteins involved in 60S ribosomal maturation and translation in Xenopus stage I oocytes, including ribosomal rpl10, eukaryotic initiation factor 6 (Eif6), thesaurins A/B, homologs of the eEF1α elongation factor, and P0, the ribosomal stalk protein. We found that rpl10 and eif6 cross-reacted with the actin-based CSK and with tubulin. rpl10 co-localizes with spectrin, particularly in the perinuclear region. eif6 is similarly localized. Given that upon ribosomal maturation, the insertion of rpl10 into the 60S subunit occurs simultaneously with the release of eif6, one can hypothesise that actin-based CSK and microtubules provide the necessary scaffold for the insertion/release of these two molecules and, subsequently, for eif6 transport and binding to the mature 60S subunit. P0 and thesaurins cross-reacted with only spectrin and cytokeratins. Thesaurins aggregated at the oocyte periphery, rendering this a territory favourable site for protein synthesis; the CSK may support the interaction between thesaurins and sites of the translating ribosome. Moreover, given that the assembly of the ribosome stalk, where P0 is located, to the 60S subunit is essential for the release of eif6, it can be hypothesised that the CSK can facilitate the binding of the stalk to the 60S.

Translatome profiling reveals Itih4 as a novel smooth muscle cell-specific gene in atherosclerosis.

AIMS: Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply translating ribosome affinity purification sequencing to profile SMC-specific gene expression directly from tissue. METHODS AND RESULTS: To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, which are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, inter-alpha-trypsin inhibitor-4 (Itih4), Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease through the colocalization of genome-wide association studies, splice quantitative trait loci (QTL), and protein QTL signals. CONCLUSION: We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.

Drivers of de novo Serine/Glycine synthesis in acute leukemia.

Cancer cells hijack metabolic pathways in order to provide themselves with building blocks to support their proliferation and survival. Upregulation and addiction to de novo serine/glycine synthesis is an example of metabolic rewiring in cancer cells whereby serine and glycine are synthesised via a side branch of glycolysis. In this review, we focus on upregulation of endogenous serine/glycine production in acute leukemia, namely T-cell acute leukemia (T-ALL) and acute myeloid leukemia (AML). Several genetic lesions directly driving the serine/glycine addiction in acute leukemia have been established. Additionally, indirect regulation of de novo serine/glycine synthesis is observed in acute leukemia.

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10-R98S). Our data support that RPL10-R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock-in of RPL10-R98S was associated with a 1.7- to 2.5-fold increased production of four transiently expressed recombinant proteins and 1.7-fold for one out of two stably expressed proteins. In CHO-S cells, eGFP reached a 2-fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10-R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10-R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

Mutational analysis of ribosomal proteins in a cohort of pediatric patients with T-cell acute lymphoblastic leukemia reveals Q123R, a novel mutation in RPL10.

T-cell acute lymphoblastic leukemia (T-ALL) is a subtype of ALL involving the malignant expansion of T-cell progenitors. It is driven by a number of different possible genetic lesions, including mutations in genes encoding for ribosomal proteins (RPs). These are structural constituents of ribosomes, ubiquitous effectors of protein synthesis. Albeit the R98S mutation in RPL10, recurring with a higher frequency among RP mutations, has been extensively studied, less is known about the contribution of mutations occurring in other RPs. Alterations affecting translational machinery may not be well tolerated by cells, and there may be a selective pressure that determines the emergence of mutations with a compensatory effect. To explore this hypothesis, we sequenced the exomes of a cohort of 37 pediatric patients affected by T-ALL, and analyzed them to explore the co-occurrence of mutations in genes involved in ribosome biogenesis (including RPs) and translational control, and in known T-ALL driver genes. We found that some of the mutations in these sub-classes of genes tend to cluster together in different patients, indicating that their co-occurrence may confer some kind of advantage to leukemia cells. In addition, our sequencing highlighted the presence of a novel mutation in RPL10, namely the Q123R, which we found associated with a defect in protein synthesis. Our findings indicate that genetic alterations involving ribosome biogenesis and translational control should be carefully considered in the context of precision medicine in T-ALL.

Alternatively Splicing Interactomes Identify Novel Isoform-Specific Partners for NSD2.

Nuclear receptor SET domain protein (NSD2) plays a fundamental role in the pathogenesis of Wolf-Hirschhorn Syndrome (WHS) and is overexpressed in multiple human myelomas, but its protein-protein interaction (PPI) patterns, particularly at the isoform/exon levels, are poorly understood. We explored the subcellular localizations of four representative NSD2 transcripts with immunofluorescence microscopy. Next, we used label-free quantification to perform immunoprecipitation mass spectrometry (IP-MS) analyses of the transcripts. Using the interaction partners for each transcript detected in the IP-MS results, we identified 890 isoform-specific PPI partners (83% are novel). These PPI networks were further divided into four categories of the exon-specific interactome. In these exon-specific PPI partners, two genes, RPL10 and HSPA8, were successfully confirmed by co-immunoprecipitation and Western blotting. RPL10 primarily interacted with Isoforms 1, 3, and 5, and HSPA8 interacted with all four isoforms, respectively. Using our extended NSD2 protein interactions, we constructed an isoform-level PPI landscape for NSD2 to serve as reference interactome data for NSD2 spliceosome-level studies. Furthermore, the RNA splicing processes supported by these isoform partners shed light on the diverse roles NSD2 plays in WHS and myeloma development. We also validated the interactions using Western blotting, RPL10, and the three NSD2 (Isoform 1, 3, and 5). Our results expand gene-level NSD2 PPI networks and provide a basis for the treatment of NSD2-related developmental diseases.

Body-fat sensor triggers ribosome maturation in the steroidogenic gland to initiate sexual maturation in Drosophila.

Fat stores are critical for reproductive success and may govern maturation initiation. Here, we report that signaling and sensing fat sufficiency for sexual maturation commitment requires the lipid carrier apolipophorin in fat cells and Sema1a in the neuroendocrine prothoracic gland (PG). Larvae lacking apolpp or Sema1a fail to initiate maturation despite accruing sufficient fat stores, and they continue gaining weight until death. Mechanistically, sensing peripheral body-fat levels via the apolipophorin/Sema1a axis regulates endocytosis, endoplasmic reticulum remodeling, and ribosomal maturation for the acquisition of the PG cells' high biosynthetic and secretory capacity. Downstream of apolipophorin/Sema1a, leptin-like upd2 triggers the cessation of feeding and initiates sexual maturation. Human Leptin in the insect PG substitutes for upd2, preventing obesity and triggering maturation downstream of Sema1a. These data show how peripheral fat levels regulate the control of the maturation decision-making process via remodeling of endomembranes and ribosomal biogenesis in gland cells.

New possible silver lining for pancreatic cancer therapy: Hydrogen sulfide and its donors.

As one of the most lethal diseases, pancreatic cancer shows a dismal overall prognosis and high resistance to most treatment modalities. Furthermore, pancreatic cancer escapes early detection during the curable period because early symptoms rarely emerge and specific markers for this disease have not been found. Although combinations of new drugs, multimodal therapies, and adjuvants prolong survival, most patients still relapse after surgery and eventually die. Consequently, the search for more effective treatments for pancreatic cancer is highly relevant and justified. As a newly re-discovered mediator of gasotransmission, hydrogen sulfide (H2S) undertakes essential functions, encompassing various signaling complexes that occupy key processes in human biology. Accumulating evidence indicates that H2S exhibits bimodal modulation of cancer development. Thus, endogenous or low levels of exogenous H2S are thought to promote cancer, whereas high doses of exogenous H2S suppress tumor proliferation. Similarly, inhibition of endogenous H2S production also suppresses tumor proliferation. Accordingly, H2S biosynthesis inhibitors and H2S supplementation (H2S donors) are two distinct strategies for the treatment of cancer. Unfortunately, modulation of endogenous H2S on pancreatic cancer has not been studied so far. However, H2S donors and their derivatives have been extensively studied as potential therapeutic agents for pancreatic cancer therapy by inhibiting cell proliferation, inducing apoptosis, arresting cell cycle, and suppressing invasion and migration through exploiting multiple signaling pathways. As far as we know, there is no review of the effects of H2S donors on pancreatic cancer. Based on these concerns, the therapeutic effects of some H2S donors and NO-H2S dual donors on pancreatic cancer were summarized in this paper. Exogenous H2S donors may be promising compounds for pancreatic cancer treatment.

Ribosomal Protein L10: From Function to Dysfunction.

Eukaryotic cytoplasmic ribosomes are highly structured macromolecular complexes made up of four different ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs), which play a central role in the decoding of genetic code for the synthesis of new proteins. Over the past 25 years, studies on yeast and human models have made it possible to identify RPL10 (ribosomal protein L10 gene), which is a constituent of the large subunit of the ribosome, as an important player in the final stages of ribosome biogenesis and in ribosome function. Here, we reviewed the literature to give an overview of the role of RPL10 in physiologic and pathologic processes, including inherited disease and cancer.

Ribosomal Protein RPL10A Contributes to Early Plant Development and Abscisic Acid-Dependent Responses in Arabidopsis.

Plant ribosomal proteins play universal roles in translation, although they are also involved in developmental processes and hormone signaling pathways. Among Arabidopsis RPL10 family members, RPL10A exhibits the highest expression during germination and early development, suggesting that RPL10A is the main contributor to these processes. In this work, we first analyzed RPL10A expression pattern in Arabidopsis thaliana using transgenic RPL10Apro:GUS plants. The gene exhibits a ubiquitous expression pattern throughout the plant, but it is most strongly expressed in undifferentiated tissues. Interestingly, gene expression was also detected in stomatal cells. We then examined protein function during seedling establishment and abscisic acid (ABA) response. Heterozygous rpl10A mutant plants show decreased ABA-sensitivity during seed germination, are impaired in early seedling and root development, and exhibit reduced ABA-inhibition of stomatal aperture under light conditions. Overexpression of RPL10A does not affect the germination and seedling growth, but RPL10A-overexpressing lines are more sensitive to ABA during early plant development and exhibit higher stomatal closure under light condition both with and without ABA treatment than wild type plants. Interestingly, RPL10A expression is induced by ABA. Together, we conclude that RPL10A could act as a positive regulator for ABA-dependent responses in Arabidopsis plants.

A homozygous RPL10L missense mutation associated with male factor infertility and severe oligozoospermia.

OBJECTIVE: To identify the genetic cause of male factor infertility characterized by severe oligozoospermia. DESIGN: Genetic studies. SETTING: Medical university. PATIENT(S): Two infertile brothers with severe oligozoospermia in a consanguineous Han Chinese family, 414 additional patients with oligo-/azoospermia, and 223 fertile (control) subjects. INVENTION(S): None. MAIN OUTCOME MEASURE(S): Genetic analyses using whole-exome and Sanger sequencing were performed for two brothers with severe oligozoospermia. The effects of an identified candidate causative mutation were investigated in silico and in vitro. Whole-exome sequencing screening for the candidate mutation was conducted in 414 patients with oligo-/azoospermia and 223 fertile subjects. RESULT(S): A homozygous missense variant (NM_080746:c.A257C: p.H86P) in RPL10L was identified in the two affected brothers and shown to cosegregate with the severe oligozoospermia phenotype. The mutation was absent in public databases, including the 1000 Genomes Project and the Exome Aggregation Consortium. All queried databases predicted the mutation to be damaging, consistent with the fact that it decreased protein levels in vitro. Subsequent mutation screening identified three additional heterozygous RPL10L mutations in three of 414 subjects with oligo-/azoospermia, but no RPL10L mutations among 223 fertile subjects. CONCLUSION(S): Our findings implicate RPL10L as a novel candidate gene in the pathogenesis of human male factor infertility and severe oligozoospermia.

Ribosomal protein L10 in mitochondria serves as a regulator for ROS level in pancreatic cancer cells.

Tumorigenesis is commonly known as a complicated process, in which reactive oxygen species (ROS) plays a critical role to involve in signal transduction, metabolism, cell proliferation and differentiation. Previously, ribosomal protein L10 (RPL10) was suggested to possess extra-ribosomal functions in pancreatic cancer cells in addition to being proposed as a tumor suppressor or transcription co-regulator. To better understand the relationship between RPL10 and tumorigenic potential in pancreatic cancer cells, chromatin immunoprecipitation sequencing reveals that RPL10 is unlikely to be a transcription factor without a specific binding motif for gene transcription. Additionally, transcriptome analysis indicates that RPL10 could regulate the expression of proteins related to ROS production. Moreover, RPL10 in mitochondria is closely associated with the regulation of ROS level by affecting Complex I activity and the subsequent events. Together, the present study suggests that the regulation of ROS level by mitochondrial RPL10 is one of the major extra-ribosomal functions in pancreatic cancer cells, which could be used as an indicator for the tumorigenesis of pancreatic cancer.

A de novo mutation in RPL10 causes a rare X-linked ribosomopathy characterized by syndromic intellectual disability and epilepsy: A new case and review of the literature.

Intellectual disability (ID) affects 1-2% of the general population and up to 50% of those with ID are estimated to have an underlying genetic cause. Next-generation sequencing provides an efficient means to identify the molecular causes of monogenic forms of ID. Here we present an 18 year old male with severe ID, absent speech, microcephaly, ataxia, dysmorphic facial features, and a refractory, early-onset seizure disorder. Exome sequencing revealed a rare de novo mutation in the X-linked gene RPL10 (c.232A > G, p.K78E). Previous reports of inherited mutations in RPL10 have suggested a role for the gene in neurodevelopment and the individual reported shows marked similarities to three members of a family with the same mutation reported in the literature. The p.K78E substitution appears to be associated with severe microcephaly, seizures, hearing loss, growth retardation, cardiac defects, and dysmorphic facial features. This is the first instance that a de novo mutation in RPL10 has been reported.

Biological Function of Ribosomal Protein L10 on Cell Behavior in Human Epithelial Ovarian Cancer.

Ribosomal protein L10 (RPL10) is one of large ribosomal proteins and plays a role in Wilms' tumor and premature ovarian failure. However, the function of RPL10 in human epithelial ovarian cancer (EOC) remains unknown. The purpose of this study was to examine the expression level and function of RPL10 in EOC. RPL10 protein expression was detected by immunohistochemistry and Western blot. The association RPL10 expression with clinical features was analyzed. Loss-of-function and gain-of-function approaches were applied in cellular assays, including cell viability, migration, invasion, and apoptosis. Our study demonstrated for the first time that RPL10 was upregulated in human EOC compared with normal ovarian tissues. Knockdown of RPL10 inhibited cell viability, migration, and invasion, and increased cell apoptosis. On the contrary, upregulation of RPL10 increased cell viability, migration, invasion, and decreased cell apoptosis. Furthermore, miR-143-3p regulated RPL10 expression. Our data indicate that RPL10 is a potential tissue biomarker of patients with EOC and may be a therapeutic target of ovarian cancer.