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To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

Accumulating evidences favor the existence of heterogeneous cellular ribosomes for versatile regulation of gene expression. Validation of distinctive ribosomal protein paralog-specific ribosomes in single cells at the protein level in nearly wild type background is essential for detailed understanding regarding the properties and functions of specialized ribosomes. Here we present results from cleanly purified RPL36 paralog-specific translating ribosomes to demonstrate they represent two distinctive populations characterized by specifically-enriched components. Under amino acid starvation, there is a dramatic reorganization in the composition of the two paralog-specific ribosomes together with reciprocal redistribution of the general translational factors and altered translational preference. pADH1-RPL36A manipulation improves the relative incompetency of the RPL36A gene; alters composition of both RPL36 paralog-specific ribosomes; reprograms genome-wide translational profile; and modifies the cellular ability to adapt to stress conditions while still preserving the paralog-specifically enriched components. Distinct folding is suggested to underlie functional differences between the paralogs.

“Ribosome code” hypothesis proposes ribosomes composed of different ribosomal protein paralogs might govern distinct functions. Identification of the regulatory elements likely involved in controlling ribosomal protein functions and the distinct composition of the correspondingly assembled paralog-specific ribosomes should be informative to this phenomenon. Here we show that modulation of different RPL36 non-coding elements can influence protein functions independently of the protein abundance. This is manifested by distinct growth rate, differential biogenesis of the 60S ribosomal subunits, and characteristic composition of the purified translating ribosomes. Proteomic analysis of highly purified paralog-specific ribosomes from strain co-expressing identical Arabidopsis RPL36B proteins from two different constructs reveals their unique composition as two distinctive classes of ribosomes characterized by specifically-enriched components. This observation is remarkably analogous to the composition of endogenous RPL36 paralog-specific ribosomes. The proposed mechanism underlying functional distinction between ribosomal protein paralogs and the significance of evolving paralog-specific ribosomes are presented.

Current glioma therapies allow in situ delivery of cytotoxic drugs to the tumour; however, gliomas show early recurrence due to their highly proliferative character. Long non‐coding (lnc)RNAs play critical roles in tumorigenesis by controlling cell proliferation and cycling. However, the mechanism of action of lncRNAs in glioma development remains unclear. Here, we report that the lncRNA PLAC2 induces cell cycle arrest by targeting ribosomal protein (RP)L36 in glioma. RPL36 promoted cell proliferation and G1/S cell cycle progression. Mass spectrometry analysis revealed that signal transducer and activator of transcription (STAT)1 interacted with both lncRNA PLAC2 and the RPL36 promoter. We also found that the nucleus PLAC2 bind with STAT1 and interact with RPL36 promoters but the cytoplasmic lncRNA PLAC2 inhibited STAT1 nuclear transfer, thereby decreasing RP36 expression, inhibiting cell proliferation and inducing cell cycle arrest. These results provide evidence for a novel cell cycle regulatory network in glioma comprising the lncRNA PLAC2 along with STAT1 and RPL36 that can serve as a therapeutic target for glioma treatment.

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.

Based on analyses of two-dimensional gel and cDNA microarrays, our laboratory and others have demonstrated that a number of genes show altered expression during the development of cisplatin resistance (CP-r) in human cancer cells, including genes associated with DNA damage repair, proto-oncogenes, apoptosis, stress-response, and transcription factors. To verify these results and find genes that are directly responsible for CP-r, as opposed to those reflecting a secondary response induced by cisplatin treatment or resulting from CP-r, we constructed a retroviral cDNA library in the vector pLNCX2 from KB-CP.5 (KCP.5), a cell line selected in one step after exposure to cisplatin at 0.5 microg/ml. Using a library of cDNAs (1.8 x 10(6) cDNA clones) and an intermittent cisplatin selection system to allow more effective functional cloning, 11 expressed cDNAs were identified in a primary pool of 93,000 transfected cell clones. Metallothionein 2A, a known CP-r gene, was among these 11 genes found in the transfectants after CP selection. Several other genes, including those encoding ribosomal proteins (e.g., RPL36) and heat shock protein (e.g., HSP10), were also found among the cisplatin-selected clones. Transfection of either the RPL36 cDNA or HSP10 cDNA conferred on KB-3-1 cells 2.5- to 3-fold resistance to cisplatin by clonogenic assays. A subsequent transfection also identified RPL36 as a CP-r gene. The finding that a ribosomal protein gene, RPL36, contributes to CP-r should stimulate study of the role of ribosomal proteins in multifactorial mechanisms of cisplatin resistance.

We amplified and sequenced the dihydrofolate reductase (DHFR) gene of the basidiomycete Coprinus cinereus. Downstream of the DHFR coding region, a mitochondrial (mt) ribosomal protein L36 (RPL36) gene was discovered in the opposite orientation to DHFR gene. Putative polyadenylation signals of the two genes overlapped, both containing the 8-bp palindrome 5'-aatatatt-3'. The finding that C. cinereus DHFR gene is closely clustered with a mt protein gene strongly suggests that C. cinereus DHFR is closely related to mt function and evolution. The amino acid sequence of C. cinereus DHFR is most homologous to eukaryotic proteins such as Cryptococcus neoformans and Pneumocystis carinii DHFRs. However, the sequence of C. cinereus mt RPL36 closely resembles RPL36 of bacteria and cyanobacteria such as Synechocystis sp. and Escherichia coli. This result strongly supports the serial endosymbiotic theory of the development of ancestral eukaryotes, and suggests that C. cinereus mt RPL36 gene originated from the ancestral eubacterial genome.

We report the isolation and expression profiles of a single-copy gene from the mycoparasitic fungus Trichoderma hamatum encoding a 60 S cytoplasmic ribosomal protein. The gene, named RPL36, was cloned through its nutrient-mediated expression, using mRNA differential screening. Its predicted ORF, interrupted by two introns, encoded a 105-aa polypeptide. The deduced rpL36 protein showed high overall homologies with other L36-type ribosomal proteins isolated from yeast, rat and human. Analysis of the promoter region of RPL36 revealed the presence of two ribosomal protein gene (RPG) boxes and a T-rich region known to be involved in the regulation of most ribosomal protein genes. Expression of RPL36 was tightly regulated by carbon and nitrogen availability. The mRNA levels of this gene decreased upon exposure of the mycelium to different stresses, whereas the addition of cycloheximide resulted in a super-induction. Levels of RPL36 transcripts also increased during mycoparasitic interaction between T. hamatum and Botrytis cinerea.

Most ribosomal proteins (RPs) are stoichiometrically incorporated into ribosomal subunits and play essential roles in ribosome biogenesis and function. However, a number of RPs appear to have non-ribosomal functions, which involve direct association with pre-mRNA and transcription factors at transcription sites. The consensus is that the RPs found at these sites are off ribosomal subunits, but observation that different RPs are usually found together suggests that ribosomal or ribosomal-like subunits might be present. Notably, it has previously been reported that antibodies against 20 different RPs stain the same Pol II transcription sites in Drosophila polytene chromosomes. Some concerns, however, were raised about the specificity of the antibodies. To investigate further whether RPs are present at transcription sites in Drosophila, we have generated several transgenic flies expressing RPs (RpS2, RpS5a, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, and RpL36) tagged with either green or red fluorescent protein. Imaging of salivary gland cells showed that these proteins are, as expected, abundant in the cytoplasm as well as in the nucleolus. However, these RPs are also apparent in the nucleus in the region occupied by the chromosomes. Indeed, polytene chromosome immunostaining of a representative subset of tagged RPs confirms the association with transcribed loci. Furthermore, characterization of a strain expressing RpL41 functionally tagged at its native genomic locus with YFP, also showed apparent nuclear accumulation and chromosomal association, suggesting that such a nuclear localization pattern might be a shared feature of RPs and is biologically important. We anticipate that the transgenes described here should provide a useful research tool to visualize ribosomal subunits in Drosophila tissues and to study the non-ribosomal functions of RPs.

In Cyanophora paradoxa photosynthetic organelles termed cyanelles perform the functions of chloroplasts in higher plants, while the structural and biochemical characteristics of the cyanelle are essentially cyanobacterial. Our interest in studying the evolutionary relationship between cyanelles and chloroplasts led us to focus on cyanelle-encoded genes of the translational apparatus, specifically genes equivalent to those of the bacterial S10 and spc operons. The structure of a large ribosomal protein gene cluster from cyanelle DNA was characterized and compared with that from plastids and bacteria. Sequences of the following cyanelle genes encompassing 4.8 kb are reported here: 5'-rpl22-rps3-rpl16-rps17-rpl14-rpl5-rps8-rpl6-rpl18- rps5-3'. Cyanelles contain five more ribosomal protein genes than do higher plant chloroplasts and four more genes than Euglena gracilis plastids in the S10/spc region of this gene cluster. The gene encoding rpl36 is absent, in contrast to the case in other plastid DNAs. These genes, including the previously characterized genes rpl3, rpl2 and rps19, are transcribed as a primary transcript of approximately 7500 nucleotides. The occurrence of transcripts smaller than this presumptive primary transcript suggests that it is processed into defined segments. Transcription terminates 3' of rps5 where a 40 bp hairpin with one mismatch (-42.2 kcal) may be folded. Immediately downstream of rps5 an open reading frame, ORF492, is contained on a separate transcript. A comparison of gene content, operon structure and deduced amino acid sequence of the genes in the S10 and spc operons from different organisms supports the notion that cyanelles are intermediary between known plastids and cyanobacteria.