Knockdown of ribosomal protein S3 protects human cells from genotoxic stress

Human ribosomal protein S3 (hS3) has a high apparent binding affinity for the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG). The hS3 ribosomal protein has also been found to inhibit the base excision repair (BER) enzyme hOGG1 from liberating 8-oxoG residing in a 5'-end-labeled oligonucleotide. To understand the in vivo involvement of hS3 in BER, we have turned to RNA interference to generate knockdown of hS3 in cells exposed to DNA damaging agents. Here we show that a 40% knockdown of hS3 resulted in as much as a seven-fold increase in the 24h survival-rate of HEK293 cells exposed to hydrogen peroxide. Significant protection to the alkylating agent methyl methanesulfonate (MMS) was also observed. Protection to the chemotherapeutic alkylating agent Thio-TEPA was only revealed at longer exposure times where the agent became more toxic to untransfected human cells. Overall, these results raise the possibility that hS3 interferes with the repair of the DNA lesions produced by genotoxic agents that potentially could play a role in the onset of cancer and other pathological states such as aging.     

Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation

Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5′–3′ mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5′–3′ exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants.     

Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana.

We have isolated a genomic clone containing Arabidopsis thaliana 5S ribosomal RNA (rRNA)-encoding genes (rDNA) by screening an A. thaliana library with a 5S rDNA probe from flax. The clone isolated contains seven repeat units of 497 bp, plus 11 kb of flanking genomic sequence at one border. Sequencing of individual subcloned repeat units shows that the sequence of the 5S rRNA coding region is very similar to that reported for other flowering plants. Four A. thaliana ecotypes were found to contain approx. 1000 copies of 5S rDNA per haploid genome. Southern-blot analysis of genomic DNA indicates that 5S rDNA occurs in long tandem arrays, and shows the presence of numerous restriction-site polymorphisms among the six ecotypes studied.     

Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana

In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron–sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.     

Ovule abortion in Arabidopsis triggered by stress

Environmental stresses frequently decrease plant fertility. In Arabidopsis, the effect of salt stress on reproduction was examined using plants grown in hydroponic medium. Salt stress inhibited microsporogenesis and stamen filament elongation. Because plants grown in hydroponic media can be rapidly and transiently stressed, the minimum inductive treatment to cause ovule abortion could be determined. Nearly 90% of the ovules aborted when roots were incubated for 12 h in a hydroponic medium supplemented with 200 mm NaCl. The anatomical effects of salt stress on maternal organs were distinct from those in the gametophyte. A fraction of cells in the chalaza and integuments underwent DNA fragmentation and programmed cell death. While three-fourths of the gametophytes aborted prior to fertilization, DNA fragmentation was not detected in these cells. Those gametophytes that survived were fertilized and formed embryos. However, very few of these developing embryos formed seeds; most senesced during seed development. Thus, during seed formation, there were multiple points where stress could prematurely terminate plant reproduction. These decreases in fecundity are discussed with respect to the hypothesis of serial adjustment of maternal investment.     

Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to ribosomal stress

Ribosomal proteins play a critical role in tightly coordinating p53 signaling with ribosomal biogenesis. Several ribosomal proteins have been shown to induce and activate p53 via inhibition of MDM2. Here, we report that S27a, a small subunit ribosomal protein synthesized as an 80-amino acid ubiquitin C-terminal extension protein (CEP80), functions as a novel regulator of the MDM2-p53 loop. S27a interacts with MDM2 at the central acidic domain of MDM2 and suppresses MDM2-mediated p53 ubiquitination, leading to p53 activation and cell cycle arrest. Knockdown of S27a significantly attenuates the p53 activation in cells in response to treatment with ribosomal stress-inducing agent actinomycin D or 5-fluorouracil. Interestingly, MDM2 in turn ubiquitinates S27a and promotes proteasomal degradation of S27a in response to actinomycin D treatment, thus forming a mutual-regulatory loop. Altogether, our results reveal that S27a plays a non-redundant role in mediating p53 activation in response to ribosomal stress via interplaying with MDM2.     

The gene for mitochondrial ribosomal protein S14 has been transferred to the nucleus in Arabidopsis thaliana

The transfer of genetic information from the mitochondrion to the nucleus is thought to be still underway in higher plants. The mitochondrial genome of Arabidopsis thaliana contains only one rps14 pseudogene. In this paper we show that the functional gene encoding mitochondrial ribosomal protein S14 has been translocated to the nucleus. This gene transfer is a recent evolutionary event, which occurred within Cruciferae, probably after the divergence of Arabidopsis and Brassica napus. A 5′ extension of the rps14 reading frame encodes a presequence which, in?vitro, targets the polypeptide to isolated mitochondria and is cleaved off during or after import. No intron was found at the junction of the targeting presequence with the mitochondrially derived sequence, which are directly connected. By contrast, a 90-bp intron, which is removed by splicing to give a mature poly(A)⁺mRNA of 0.9 kb, is located in the 3′ non-coding region. To our knowledge, this is the first report of an intron in such a position in a functional transferred gene in higher plants, and suggests that exon shuffling may have been involved in the acquisition of elements necessary for expression in the nucleus. Putative roles of this intron in polyadenylation and enhancement of gene expression are discussed.     

Two evolutionarily divergent genes encode a cytoplasmic ribosomal protein of Arabidopsis thaliana

Two clones of Arabidopsis thaliana possessing high sequence identity to the yeast gene encoding ribosomal (r) protein L3 were isolated by heterologous DNA hybridization. The coding regions of these two clones have approx. 63% amino acid (aa) sequence identity to the yeast L3 r-protein and 85% aa sequence identity to each other. Both genes are expressed in shoots. The presence of two divergent genes in A. thaliana raises the possibility that the gene products participate in the formation of functionally distinct ribosomes.     

Involvement of ribosomal protein S1 in the assembly of the initiation complex.

Antibodies against ribosomal protein S1 (anti-S1) have been used to determine the function of S1 in the partial reactions involved in the translation of MS2 RNA in vitro. Vacant ribosomes are fully sensitive to the antibodies, whereas elongating ribosomes are resistant. We have determined at which stage of translation the resistance to anti-S1 is acquired. We find that insensitivity to anti-S1 already arises upon mixing 30-S subunits with MS2 RNA. Apparently the two particles form a complex in which S1 is functionally protected against its antibody. Complex formation depends on elevated temperature, a suitable ionic environment and it is stimulated by the initiation factor IF-3. It does not depend on IF-1, IF-2 or fMet-tRNA. Thus ribosomes have the potential to recognize the messenger in the absence of fMet-tRNA. Protein S1 appears directly involved in this primary recognition reaction.