Paralogous ribosomal protein l32-1 and l32-2 in fission yeast may function distinctively in cellular proliferation and quiescence by changing the ratio of rpl32 paralogs.
ABSTRACT: Fission yeast cells express Rpl32-2 highly while Rpl32-1 lowly in log phase; in contrast, expression of Rpl32-1 raises and reaches a peak level while Rpl32-2 is downregulated to a low basic level when cells enter into stationary phase. Overexpression of Rpl32-1 inhibits cell growth while overexpression of Rpl32-2 does not. Deleting rpl32-2 impairs cell growth more severely than deleting rpl32-1 does. Cell growth impaired by deleting either paralog can be rescued completely by reintroducing rpl32-2, but only partly by rpl32-1. Overexpression of Rpl32-1 inhibits cell division, yielding 4c DNA and multiple septa, while overexpressed Rpl32-2 promotes it. Transcriptomics analysis proved that Rpl32 paralogs regulate expression of a subset of genes related with cell division and stress response in a distinctive way. This functional difference of the two paralogs is due to their difference of 95(th) amino acid residue. The significance of a competitive inhibition between Rpl32 paralogs on their expression is discussed.
Project description:Lung cancer is the leading cause of cancer death worldwide, and the overall survival rate of advanced lung cancer patients is unsatisfactory. Ribosomal proteins (RPs) play important roles in carcinogenesis. However, the role of RPL32 in lung cancer has not been demonstrated. Here, we report that RPL32 is aberrantly, highly expressed in lung cancer tissues and that the overexpression of RPL32 is correlated with the poor prognosis of these patients. RPL32 silencing significantly inhibited the proliferation of lung cancer cells, with an observed p53 accumulation and cell-cycle arrest. Mechanistically, knockdown of RPL32 resulted in ribosomal stress and affected rRNA maturation. RPL5 and RPL11 sensed stress and translocated from the nucleus to the nucleoplasm, where they bound to murine double minute 2 (MDM2), an important p53 E3 ubiquitin ligase, which resulted in p53 accumulation and inhibition of cancer cell proliferation. As lung cancer cells usually express high levels of Toll-like receptor 9 (TLR9), we conjugated RPL32 small interfering RNA (siRNA) to the TLR9 ligand CpG to generate CpG-RPL32 siRNA, which could stabilize and guide RPL32 siRNA to lung cancer cells. Excitingly, CpG-RPL32 siRNA displayed strong anticancer abilities in lung cancer xenografts. Therefore, RPL32 is expected to be a potential target for lung cancer treatment.
Project description:We cultured the cells of S. pmbe in EMM2, and harvested them in the OD600 of 0.5 and 10 respectively. We used microarrays to detail the the global program of gene expression of SP-Q01 and SP-Q01 carrying vevtor pESP3X to overexpress either ribosomal protein L32 paralog in the OD600 of 0.5 and 10 respectively. We constructed the ribosomal protein L32 paralog overexpression strains to mimic the expression pattern of RPL32 paralogs in wild-type cells of S.pombe. We cultured the cells to different growth phase, and used microarray to identify the genes were regulated distinctively by RPL32 paralogs.
Project description:BACKGROUND: For accuracy of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), normalisation with suitable reference genes is required. To date, no reference genes have been validated for expression studies of bronchoalveolar (BAL) cells. The aims of this study were to identify gene(s) with stable mRNA expression in BAL cells irrespective of gender, smoking, BAL cellular composition, lung pathology, treatment; and to assess the influence of reference genes on target gene expression data. RESULTS: The mRNA expression of ten housekeeping genes (ACTB, ARF1, CANX, G6PD, GAPDH, GPS1, GNB2L1, PSMB2, PSMD2, RPL32) was investigated by qRT-PCR in BAL cells from 71 subjects across a spectrum of lung diseases. The analyses were validated in an independent BAL cohort from 63 sarcoidosis patients and 17 control subjects. A second derivative method was used to calculate expression values (CTt); an equivalence test, applets BestKeeper, geNorm and NormFinder were applied to investigate gene expression stability. Of the investigated genes, PSMB2 (CTt +/- SD, 23.66 +/- 0.86) and RPL32 (18.65 +/- 0.92) were the most stable; both were constantly expressed in BAL samples from parallel investigated cohorts irrespective of evaluated variables. Finally, to demonstrate effect of traditional (ACTB/GAPDH) and novel (PSMB2/RPL32) reference genes as denominators, expression of two cytokines known associated with sarcoidosis was investigated in sarcoid BAL cells. While normalization with PSMB2/RPL32 resulted in elevated IFNG mRNA expression (p = 0.004); no change was observed using GAPDH/ACTB (p > 0.05). CCL2 mRNA up-regulation was observed only when PSMB2/RPL32 were used as denominators (p < 0.03). CONCLUSION: PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.
Project description:Plastids originated from cyanobacteria and the majority of the ancestral genes were lost or functionally transferred to the nucleus after endosymbiosis. Comparative genomic investigations have shown that gene transfer from plastids to the nucleus is an ongoing evolutionary process but molecular evidence for recent functional gene transfers among seed plants have only been documented for the four genes accD, infA, rpl22, and rpl32.The complete plastid genome of Thalictrum coreanum, the first from the subfamily Thalictroideae (Ranunculaceae), was sequenced and revealed the losses of two genes, infA and rpl32. The functional transfer of these two genes to the nucleus in Thalictrum was verified by examination of nuclear transcriptomes. A survey of the phylogenetic distribution of the rpl32 loss was performed using 17 species of Thalictrum and representatives of related genera in the subfamily Thalictroideae. The plastid-encoded rpl32 gene is likely nonfunctional in members of the subfamily Thalictroideae (Aquilegia, Enemion, Isopyrum, Leptopyrum, Paraquilegia, and Semiaquilegia) including 17 Thalictrum species due to the presence of indels that disrupt the reading frame. A nuclear-encoded rpl32 with high sequence identity was identified in both Thalictrum and Aquilegia. The phylogenetic distribution of this gene loss/transfer and the high level of sequence similarity in transit peptides suggest a single transfer of the plastid-encoded rpl32 to the nucleus in the ancestor of the subfamily Thalictroideae approximately 20-32 Mya.The genome sequence of Thalictrum coreanum provides valuable information for improving the understanding of the evolution of plastid genomes within Ranunculaceae and across angiosperms. Thalictrum is unusual among the three sequenced Ranunculaceae plastid genomes in the loss of two genes infA and rpl32, which have been functionally transferred to the nucleus. In the case of rpl32 this represents the third documented independent transfer from the plastid to the nucleus with the other two transfers occurring in the unrelated angiosperm families Rhizophoraceae and Salicaceae. Furthermore, the transfer of rpl32 provides additional molecular evidence for the monophyly of the subfamily Thalictroideae.
Project description:Toxin-antitoxin (TA) gene cassettes are widely distributed across bacteria, archaea and bacteriophage. The chromosome of the alpha-proteobacterium, Caulobacter crescentus, encodes eight ParE/RelE-superfamily toxins that are organized into operons with their cognate antitoxins. A systematic genetic analysis of these parDE and relBE TA operons demonstrates that seven encode functional toxins. The one exception highlights an example of a non-functional toxin pseudogene. Chromosomally encoded ParD and RelB proteins function as antitoxins, inhibiting their adjacently encoded ParE and RelE toxins. However, these antitoxins do not functionally complement each other, even when overexpressed. Transcription of these paralogous TA systems is differentially regulated under distinct environmental conditions. These data support a model in which multiple TA paralogs encoded by a single bacterial chromosome form independent functional units with insulated protein-protein interactions. Further characterization of the parDE(1) system at the single-cell level reveals that ParE(1) toxin functions to inhibit cell division but not cell growth; residues at the C-terminus of ParE(1) are critical for its stability and toxicity. While continuous ParE(1) overexpression results in a substantial loss in cell viability at the population level, a fraction of cells escape toxicity, providing evidence that ParE(1) toxicity is not uniform within clonal cell populations.
Project description:BACKGROUND: The regulation of ribosomal proteins in plants under stress conditions has not been well studied. Although a few reports have shown stress-specific post-transcriptional and translational mechanisms involved in downregulation of ribosomal proteins yet stress-responsive transcriptional regulation of ribosomal proteins is largely unknown in plants. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, transcriptional regulation of genes encoding rice 60S ribosomal protein L32 (rpL32) in response to salt stress has been studied. Northern and RT-PCR analyses showed a significant downregulation of rpL32 transcripts under abiotic stress conditions in rice. Of the four rpL32 genes in rice genome, the gene on chromosome 8 (rpL32_8.1) showed a higher degree of stress-responsive downregulation in salt sensitive rice variety than in tolerant one and its expression reverted to its original level upon withdrawal of stress. The nuclear run-on and promoter:reporter assays revealed that the downregulation of this gene is transcriptional and originates within the promoter region. Using in vivo footprinting and electrophoretic mobility shift assay (EMSA), cis-elements in the promoter of rpL32_8.1 showing reduced binding to proteins in shoots of salt stressed rice seedlings were identified. CONCLUSIONS: The present work is one of the few reports on study of stress downregulated genes. The data revealed that rpL32 gene is transcriptionally downregulated under abiotic stress in rice and that this transcriptional downregulation is associated with the removal of transcription factors from specific promoter elements.
Project description:Deleting ribosomal protein genes caused haploid cells of S. pombe flocculated in full nutrition YEPD medium. We deleted rpl32-1 and rpl32-2 gene respectively and these two mutant cells cultured in YEPD medium flocculated. We used microarrays to detail the global programme of gene expression between non flocculated rpl32-1 deleted and rpl32-2 deleted, compared with the non flocculated cells SPQ-01, and identified distinct classes of up-regulated genes during this process. Yeast cells cultured in YEPD medium in the log phase were selected for RNA extraction and hybridization on Affymetrix microarrays. The rpl32-1 deleted cells were conformed as SD1 and rpl32-2 deleted cells were conformed as SD2. Control cells SPQ-01 were conformed as WT.
Project description:Ebp1, an ErbB3 receptor-binding protein, inhibits the proliferation and induces the differentiation of human cancer cells. Ebp1 binds nuclear Akt and prevents DNA fragmentation by inhibiting caspase-activated DNase. Here, we show that Ebp1 possesses two different isoforms, p48 and p42, which differentially mediate PC12 cell survival and differentiation. The longer-form p48 localizes in both the cytoplasm and the nucleus and suppresses apoptosis, whereas the shorter-form p42 predominantly resides in the cytoplasm and promotes cell differentiation. EGF strongly stimulates p42 to bind ErbB3, and the association depends on PKC-mediated phosphorylation of Ebp1. By contrast, p48 does not bind to ErbB3 regardless of EGF treatment. Overexpression of p48 provokes cell proliferation, which is inhibited by p42. Moreover, nerve growth factor elicits extensive sprouting in p42 stably transfected PC12 cells, whereas p48 cells reveal modest neurite outgrowth. Although mitogen-activated protein kinase cascade remains similar in both cells, Akt is more active in p48 cells than in p42 cells. Thus, Ebp1 might regulate cell survival and differentiation through two distinctive p48 and p42 isoforms.
Project description:Deletion of ribosomal protein L32 genes resulted in a nonsexual flocculation of fission yeast. Nonsexual flocculation also occurred when two other ribosomal protein genes, rpl21-2 and rpl9-2, were deleted. However, deletion of two nonribosomal protein genes, mpg and fbp, did not cause flocculation. Overall transcript levels of rpl32 in rpl32-1? and rpl32-2? cells were reduced by 35.9% and 46.9%, respectively, and overall ribosome levels in rpl32-1? and rpl32-2? cells dropped 31.1% and 27.8%, respectively, compared to wild-type cells. Interestingly, ribosome protein expression levels and ribosome levels were also reduced greatly in sexually flocculating diploid YHL6381/WT (h?/h?) cells compared to a mixture of YHL6381 (h?) and WT (h?) nonflocculating haploid cells. Transcriptome analysis indicated that the reduction of ribosomal levels in sexual flocculating cells was caused by more-extensive suppression of ribosomal biosynthesis gene expression, while the reduction of ribosomal levels caused by deleting ribosomal protein genes in nonsexual flocculating cells was due to an imbalance between ribosomal proteins. We propose that once the reduction of ribosomal levels is below a certain threshold value, flocculation is triggered.
Project description:The signals that control skeletogenesis are incompletely understood. Here we show that deleting Kindlin-2 in Prx1-expressing mesenchymal progenitors in mice causes neonatal lethality, chondrodysplasia and loss of the skull vault. Kindlin-2 ablation reduces chondrocyte density by decreasing cell proliferation and increasing apoptosis, and disrupts column formation, thus impairing the formation of the primary ossification center and causing severe limb shortening. Remarkably, Kindlin-2 localizes to not only focal adhesions, but also to the nuclei of chondrocytes. Loss of Kindlin-2 reduces, while the overexpression of Kindlin-2 increases, Sox9 expression. Furthermore, the overexpression of Sox9 restores the defects in chondrogenic differentiation induced by Kindlin-2 deletion in vitro. In addition, Kindlin-2 ablation inhibits TGF-?1-induced Smad2 phosphorylation and chondrocyte differentiation. Finally, deleting Kindlin-2 in chondrocytes directly impairs chondrocyte functions, resulting in progressive dwarfism and kyphosis in mice. These studies uncover a previously unrecognized function for Kindlin-2 and a mechanism for regulation of the chondrocyte differentiation programme and chondrogenesis.