Showing papers on "Ribosomal protein published in 2005"

Structures of the bacterial ribosome at 3.5 Å resolution

Abstract: Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 A. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.

Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis

Abstract: The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, we have established viable and fertile knock-in mice, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6P-/-). Here we show that contrary to the widely accepted model, this mutation does not affect the translational control of TOP mRNAs. rpS6P-/- mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6P+/+ MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6P-/- MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, as it also selectively characterizes pancreatic β-cells in adult rpS6P-/- mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance.

Inactivation of S6 ribosomal protein gene in T lymphocytes activates a p53-dependent checkpoint response

Abstract: Ribosome biogenesis has been associated with regulation of cell growth and cell division, but the molecular mechanisms that integrate the effect of ribosome biogenesis on these processes in mammalian cells remain unknown. To study the effect of impaired ribosome functions in vivo, we conditionally deleted one or two alleles of the 40S ribosomal protein S6 gene in T cells in the mouse. While complete deletion of S6 abrogated T-cell development, hemizygous expression did not have any effect on T-cell maturation in the thymus, but inhibited the accumulation of T cells in the spleen and lymph nodes, as a result of their decreased survival in the peripheral lymphoid organs. Additionally, TCR-mediated stimulation of S6-heterozygous T cells induced a normal increase in their size, but cell cycle progression was impaired. Genetic inactivation of p53 tumor suppressor rescued development of S6-homozygous null thymocytes and proliferative defect of S6-heterozygous T cells. These results demonstrate the existence of a p53-dependent checkpoint mechanism that senses changes in the fidelity of the translational machinery to prevent aberrant cell division or eliminate defective T cells in vivo. Failure to activate this checkpoint response could potentially lead to a development of pathological processes such as tumors and autoimmune diseases.

Arabidopsis TCP20 links regulation of growth and cell division control pathways.

Abstract: During postembryonic plant development, cell division is coupled to cell growth. There is a stringent requirement to couple these processes in shoot and root meristems. As cells pass through meristems, they transit through zones with high rates of cell growth and proliferation during organogenesis. This transition implies a need for coordinate regulation of genes underpinning these two fundamental cell functions. Here, we report a mechanism for coregulation of cell division control genes and cell growth effectors. We identified a GCCCR motif necessary and sufficient for high-level cyclin CYCB1;1 expression at G2/M. This motif is overrepresented in many ribosomal protein gene promoters and is required for high-level expression of the S27 and L24 ribosomal subunit genes we examined. p33TCP20, encoded by the Arabidopsis TCP20 gene, binds to the GCCCR element in the promoters of cyclin CYCB1;1 and ribosomal protein genes in vitro and in vivo. We propose a model in which organ growth rates, and possibly shape in aerial organs, are regulated by the balance of positively and negatively acting teosinte-branched, cycloidea, PCNA factor (TCP) genes in the distal meristem boundary zone where cells become mitotically quiescent before expansion and differentiation.

An assembly landscape for the 30S ribosomal subunit.

Abstract: Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, mRNA processing, translation, DNA replication, and cellular transport The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes Our understanding of 30S assembly is incomplete, due to the challenges of monitoring the association of many components simultaneously We have developed a new method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S rRNA during formation of the functional particle These data represent the first detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex By measuring the protein binding rates at a range of temperatures, we have found that local transformations throughout the assembling subunit have similar but distinct activation energies This observation shows that the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to a view in which the assembly of the complex traverses a landscape dotted with a variety of local conformational transitions

Structure of the E. coli protein-conducting channel bound to a translating ribosome

Abstract: Secreted and membrane proteins are translocated across or into cell membranes through a protein-conducting channel (PCC). Here we present a cryo-electron microscopy reconstruction of the Escherichia coli PCC, SecYEG, complexed with the ribosome and a nascent chain containing a signal anchor. This reconstruction shows a messenger RNA, three transfer RNAs, the nascent chain, and detailed features of both a translocating PCC and a second, non-translocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYEbeta structure into the PCC electron microscopy densities favours a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. On the basis of our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.

ANTIBIOTICS TARGETING RIBOSOMES: Resistance, Selectivity, Synergism, and Cellular Regulation

Abstract: Antibiotics target ribosomes at distinct locations within functionally relevant sites. They exert their inhibitory action by diverse modes, including competing with substrate binding, interfering with ribosomal dynamics, minimizing ribosomal mobility, facilitating miscoding, hampering the progression of the mRNA chain, and blocking the nascent protein exit tunnel. Although the ribosomes are highly conserved organelles, they possess subtle sequence and/or conformational variations. These enable drug selectivity, thus facilitating clinical usage. The structural implications of these differences were deciphered by comparisons of high-resolution structures of complexes of antibiotics with ribosomal particles from eubacteria resembling pathogens and from an archaeon that shares properties with eukaryotes. The various antibiotic-binding modes detected in these structures demonstrate that members of antibiotic families possessing common chemical elements with minute differences might bind to ribosomal pockets in significantly different modes, governed by their chemical properties. Similarly, the nature of seemingly identical mechanisms of drug resistance is dominated, directly or via cellular effects, by the antibiotics' chemical properties. The observed variability in antibiotic binding and inhibitory modes justifies expectations for structurally based improved properties of existing compounds as well as for the discovery of novel drug classes.

Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

Abstract: The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37°C and 32°C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32°C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

Immunopurification of Polyribosomal Complexes of Arabidopsis for Global Analysis of Gene Expression

Abstract: Immunoaffinity purification of polyribosomes (polysomes) from crude leaf extracts of Arabidopsis (Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein (RP) with a His(6)-FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic RPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% +/- 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters.

Ribosomal Proteins in the Spotlight

Abstract: The assignment of specific ribosomal functions to individual ribosomal proteins is difficult due to the enormous cooperativity of the ribosome; however, important roles for distinct ribosomal proteins are becoming evident. Although rRNA has a major role in certain aspects of ribosomal function, such as decoding and peptidyl-transferase activity, ribosomal proteins are nevertheless essential for the assembly and optimal functioning of the ribosome. This is particularly true in the context of interactions at the entrance pore for mRNA, for the translation-factor binding site and at the tunnel exit, where both chaperones and complexes associated with protein transport through membranes bind.

Ribosomal protein L30 is a component of the UGA- selenocysteine recoding machinery in eukaryotes

Abstract: The translational recoding of UGA as selenocysteine (Sec) is directed by a SECIS element in the 3' untranslated region (UTR) of eukaryotic selenoprotein mRNAs. The selenocysteine insertion sequence (SECIS) contains two essential tandem sheared G.A pairs that bind SECIS-binding protein 2 (SBP2), which recruits a selenocysteine-specific elongation factor and Sec-tRNA(Sec) to the ribosome. Here we show that ribosomal protein L30 is a component of the eukaryotic selenocysteine recoding machinery. L30 binds SECIS elements in vitro and in vivo, stimulates UGA recoding in transfected cells and competes with SBP2 for SECIS binding. Magnesium, known to induce a kink-turn in RNAs that contain two tandem G.A pairs, decreases the SBP2-SECIS complex in favor of the L30-SECIS interaction. We propose a model in which SBP2 and L30 carry out different functions in the UGA recoding mechanism, with the SECIS acting as a molecular switch upon protein binding.

Central role of Ifh1p–Fhl1p interaction in the synthesis of yeast ribosomal proteins

Abstract: The 138 genes encoding the 79 ribosomal proteins (RPs) of Saccharomyces cerevisiae form the tightest cluster of coordinately regulated genes in nearly all transcriptome experiments. The basis for this observation remains unknown. We now provide evidence that two factors, Fhl1p and Ifh1p, are key players in the transcription of RP genes. Both are found at transcribing RP genes in vivo. Ifh1p, but not Fhl1p, leaves the RP genes when transcription is repressed. The occupancy of the RP genes by Ifh1p depends on its interaction with the phospho-peptide recognizing forkhead-associated domain of Fhl1p. Disruption of this interaction is severely deleterious to ribosome synthesis and cell growth. Loss of functional Fhl1p leads to cells that have only 20% the normal amount of RNA and that synthesize ribosomes at only 5–10% the normal rate. Homeostatic mechanisms within the cell respond by reducing the transcription of rRNA to match the output of RPs, and by reducing the global transcription of mRNA to match the capacity of the translational apparatus.

Large-scale analysis of the human ubiquitin-related proteome.

Abstract: Protein ubiquitylation contributes to the regulation of many cellular processes including protein degradation, receptor internalization, and repair of DNA damage. We now present a comprehensive characterization of ubiquitin-conjugated and ubiquitin-associated proteins in human cells. The proteins were purified by immunoaffinity chromatography under denaturing or native conditions. They were then digested with trypsin, and the resulting peptides were analyzed by 2-D LC and MS/MS. A total of 670 distinct proteins were identified; 345 proteins (51%) were classified as Urp-D (ubiquitin-related proteome under the denaturing condition) and comprised ubiquitin-conjugated molecules, whereas 325 proteins (49%) were classified as Urp-N (ubiquitin-related proteome only under the native condition) and included molecules that associated with ubiquitylated proteins. The proportions of proteins in various functional categories differed substantially between Urp-D and Urp-N. Many ribosomal subunits were detected in the Urp-D group of proteins and several of these subunits were directly shown to be ubiquitylated by mass spectrometric analysis, suggesting that ubiquitylation might play an important role in the regulation and/or quality control of ribosomal proteins. Our results demonstrate the potential of proteomics analysis of protein ubiquitylation to provide important insight into the regulation of protein stability and other ubiquitin-related cellular functions.

Nuclear export and cytoplasmic processing of precursors to the 40S ribosomal subunits in mammalian cells

Abstract: It is generally assumed that, in mammalian cells, preribosomal RNAs are entirely processed before nuclear exit. Here, we show that pre-40S particles exported to the cytoplasm in HeLa cells contain 18S rRNA extended at the 3′ end with 20–30 nucleotides of the internal transcribed spacer 1. Maturation of this pre-18S rRNA (which we named 18S-E) involves a cytoplasmic protein, the human homolog of the yeast kinase Rio2p, and appears to be required for the translation competence of the 40S subunit. By tracking the nuclear exit of this precursor, we have identified the ribosomal protein Rps15 as a determinant of preribosomal nuclear export in human cells. Interestingly, inhibition of exportin Crm1/Xpo1 with leptomycin B strongly alters processing of the 5′-external transcribed spacer, upstream of nuclear export, and reveals a new cleavage site in this transcribed spacer. Completion of the maturation of the 18S rRNA in the cytoplasm, a feature thought to be unique to yeast, may prevent pre-40S particles from initiating translation with pre-mRNAs in eukaryotic cells. It also allows new strategies for the study of preribosomal transport in mammalian cells.

Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p

Abstract: In eukaryotes, nuclear export of the large (60S) ribosomal subunit requires the adapter protein Nmd3p to provide the nuclear export signal. Here, we show that in yeast release of Nmd3p from 60S subunits in the cytoplasm requires the ribosomal protein Rpl10p and the G-protein, Lsg1p. Mutations in LSG1 or RPL10 blocked Nmd3-GFP shuttling into the nucleus and export of pre-60S subunits from the nucleus. Overexpression of NMD3 alleviated the export defect, indicating that the block in 60S export in lsg1 and rpl10 mutants results indirectly from failing to recycle Nmd3p. The defect in Nmd3p recycling and the block in 60S export in both lsg1 and rpl10 mutants was also suppressed by mutant Nmd3 proteins that showed reduced binding to 60S subunits in vitro. We propose that the correct loading of Rpl10p into 60S subunits is required for the release of Nmd3p from subunits by Lsg1p. These results suggest a coupling between recycling the 60S export adapter and activation of 60S subunits for translation.

Specific Binding of Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA Is Essential for Replication

Abstract: Plus-strand RNA viruses replicate their genomes in infected cells by using a replicase complex comprised of viral and host proteins that assembles in association with cellular membranes (1, 4, 15). In infected cells, viral replicases are able to specifically recognize and selectively replicate their cognate viral RNAs from a heterogeneous pool of cellular RNA molecules. In contrast, in vitro studies have shown that many purified viral replicase complexes are able to utilize heterologous promoter or initiation elements quite efficiently (11, 28, 39). These conflicting findings between in vivo and in vitro data have led to models that attribute selective recognition of viral templates to host proteins (4, 13). Phage Qbeta utilizes this type of mechanism whereby the S1 ribosomal protein and elongation factor Tu in the four-subunit replicase complex mediate viral template recognition (3, 13). There is also evidence that virally encoded proteins can facilitate selective template recruitment to the viral replicase complex. Examples in this category include the 1a protein of Brome mosaic virus and the 126-kDa protein of Tomato mosaic virus (5, 17, 33). However, in both of these cases it is not known whether the respective viral RNAs are recognized directly by these replicase proteins or require assistance from host proteins (6). In Poliovirus, specific binding of 3CD protein to the 5′-terminal cloverleaf-like structure has been reported, and this interaction likely contributes to template selection into replication (9, 38). However, the host poly(C) binding protein 2 also interacts specifically with the same 5′-terminal RNA structure, and it, too, is proposed to be involved in mediating template selection (9, 38). In general, the contribution of viral and cellular proteins to RNA template recognition by cognate replicases is largely unknown in most viral systems. Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the large family Tombusviridae. Its genome encodes two proteins involved in viral RNA replication, the prereadthrough product p33 and the readthrough product p92 (Fig. 1A and B). Both of these proteins are essential for RNA replication (18, 22), they are part of the viral replicase complex (23), and they accumulate in vivo at a ratio of 20:1, respectively (23, 31). The less plentiful p92 functions as the viral RNA-dependent RNA polymerase (RdRp), while the role of the more abundant prereadthrough p33 is undefined (37). In other tombusviruses, the orthologues of TBSV p33 have been shown to be targeted to mitochondrial or peroxisomal membranes, the presumed sites of tombusvirus RNA replication (29). In TBSV, both p33 and p92 are membrane associated (23, 31). RNA-binding domains have also been identified in these proteins (22, 26), and the ability of p33 to interact with itself and p92 has been demonstrated (27). The cumulative data support an essential role for both TBSV p33 and p92 in viral RNA replication, with p92 comprising the catalytic subunit responsible for RNA synthesis and p33 playing a critical but unknown auxiliary role. FIG. 1. Specific binding of the recombinant p33 replicase protein to RII(+)-SL in vitro. (A) Schematic representation of the p33 and p92 replicase proteins of TBSV. The sequence of p33 is identical to the N-terminal overlapping (prereadthrough) domain ... In this paper we tested the binding of a recombinant TBSV p33 to four conserved regions of the viral genome known to affect replication (37). We demonstrate that p33 binds selectively in vitro to a conserved RNA motif within the p92 coding region of the viral genome. The specific recognition of this RNA element is dependent on a C · C mismatch positioned within a helix, and this key determinant of p33 binding is also essential for TBSV replication in host cells. We propose that this interaction directs viral template recruitment into replication and suggest that this mechanism may also apply to other members of the large family Tombusviridae.

Detection and Localization of a Class of Proteins Immunologically Related to a 100‐kDa Nucleolar Protein

Abstract: A high-molecular-mass nucleolar protein (100-kDa protein) is associated with nascent pre-rRNA and pre-ribosomes in Chinese hamster ovary cells. We have prepared antiserum against the 100-kDa protein and we have used it to study the intracellular localization and the possible processing of this protein. Serologically related proteins were detected in the nucleolus and in ribosomes. Proteins of various subcellular fractions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, reacted with serum and the protein-immunoglobulin complexes were revealed by 125I-labeled protein A. In the nucleolus, four proteins with molecular masses of 100 kDa, 95 kDa, 76 kDa and 70 kDa were thus visualized. In the ribosomes, two proteins (of 100 kDa and 76 kDa) gave a strong reaction while six others (of 70 kDa, 60 kDa, 50 kDa, 30 kDa, 21 kDa, 18 kDa) reacted slightly. By immunological precipitation of total cell extracts, we have shown that the 100-kDa protein antiserum cross-reacts with five proteins with molecular masses of 120 kDa, 100 kDa, 95 kDa, 70 kDa and 60 kDa. Specific degradation of the 100-kDa protein into similar peptides with molecular masses of 95 kDa, 76 kDa, 70 kDa, 60 kDa and 50 kDa can be achieved by incubation of isolated nucleoli or of purified 100-kDa protein in vitro. Cleavage of the protein is due to a thiol endoprotease which is tightly bound to the 100-kDa protein. Possible relations between the maturation of this preribosomal protein into ribosomal proteins and the processing of preribosomal RNA into mature ribosomal RNA are discussed.

Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes.

Abstract: Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one- and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.

Ribosomal protein S2 is a substrate for mammalian PRMT3 (protein arginine methyltransferase 3).

Abstract: PRMT3 (protein arginine methyltransferase 3) is one of four type I arginine methyltransferases that catalyse the formation of asymmetric dimethylarginine. PRMT3 is unique in that its N-terminus harbours a C2H2 zinc-finger domain that is proposed to confer substrate specificity. In addition, PRMT3 is the only type I enzyme that is restricted to the cytoplasm. Known in vitro substrates for PRMT3 include GST-GAR (a glutathione S-transferase fusion protein containing the glycine- and arginine-rich N-terminal region of fibrillarin), Sam68 (Src-associated substrate during mitosis 68 kDa) and PABP-N1 [poly(A)-binding protein-N1; PABP2]. Here we report the identification of an in vivo substrate for mammalian PRMT3. We found that FLAG-tagged PRMT3 can 'pull down' a protein with a molecular mass of 30 kDa from HeLa cell extracts. MS identified this PRMT3-interacting protein as rpS2 (ribosomal protein S2). In vitro studies showed that the zinc-finger domain of PRMT3 is necessary and sufficient for binding to rpS2. In addition, rpS2 is methylated by PRMT3 in vitro and is also methylated in cell lines. Deletion analysis of the rpS2 amino acid sequence identified a N-terminal Arg-Gly repeat as the methylation site. Furthermore, both PRMT3 and rpS2 co-sediment with free ribosomal subunits. These studies implicate PRMT3 in ribosomal function and in the regulation of protein synthesis.

Scanning the human proteome for calmodulin-binding proteins.

Abstract: The calcium ion (Ca2+) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca2+ are mediated by intracellular Ca2+-binding proteins, also known as Ca2+ sensors. A key obstacle to studying many Ca2+-sensing proteins is the difficulty in identifying the numerous downstream target interactions that respond to Ca2+-induced conformational changes. Among a number of Ca2+ sensors in the eukaryotic cell, calmodulin (CaM) is the most widespread and the best studied. Employing the mRNA display technique, we have scanned the human proteome for CaM-binding proteins and have identified and characterized a large number of both known and previously uncharacterized proteins that interact with CaM in a Ca2+-dependent manner. The interactions of several identified proteins with Ca2+/CaM were confirmed by using pull-down assays and coimmunoprecipitation. Many of the CaM-binding proteins identified belong to protein families such as the DEAD/H box proteins, ribosomal proteins, proteasome 26S subunits, and deubiquitinating enzymes, suggesting the possible involvement of Ca2+/CaM in different signaling pathways. The selection method described herein could be used to identify the binding partners of other calcium sensors on the proteome-wide scale.

AU-rich sequences within 5' untranslated leaders enhance translation and stabilize mRNA in Escherichia coli.

Abstract: We have shown previously that when the Escherichia coli chromosomal lacZ gene is put under the control of an extended Shine-Dalgarno (SD) sequence (10 or 6 nucleotides in length), the translation efficiency can be highly variable, depending on the presence of AU-rich targets for ribosomal protein S1 in the mRNA leader. Here, the same strains have been used to examine the question of how strong ribosome binding to extended SD sequences affects the stability of lacZ mRNAs translated with different efficiencies. The steady-state concentration of the lacZ transcripts has been found to vary over a broad range, directly correlating with translation efficiency but not with the SD duplex stability. The observed strain-to-strain variations in lacZ mRNA level became far less marked in the presence of the rne-1 mutation, which partially inactivates RNase E. Together, the results show that (i) an SD sequence, even one that is very long, cannot stabilize the lacZ mRNA in E. coli if translation is inefficient; (ii) inefficiently translated lacZ transcripts are sensitive to RNase E; and (iii) AU-rich elements inserted upstream of a long SD sequence enhance translation and stabilize mRNA, despite the fact that they constitute potential RNase E sites. These data strongly support the idea that the lacZ mRNA in E. coli can be stabilized only by translating, and not by stalling, ribosomes.

Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua)

Abstract: The epidermal mucus of fish species has been found to contain antimicrobial proteins and peptides, which is of interest in regard to fish immunity. An acidic extract from the epidermal mucus of the Atlantic cod (Gadus morhua) was found to exhibit antimicrobial activity against Bacillus megaterium, Escherichia coli and Candida albicans. This activity varied significantly when salt was added to the antimicrobial assay, and was eliminated by pepsin digestion. No lysozyme activity was detected in the extract. By using weak cationic exchange chromatography together with reversed-phase chromatography, and monitoring the antimicrobial activity, we have isolated four cationic proteins from the mucus extract. Using N-terminal and C-terminal amino acid sequence analysis, together with MS, the antimicrobial proteins were identified as histone H2B (13 565 Da), ribosomal protein L40 (6397 Da), ribosomal protein L36A (12 340 Da) and ribosomal protein L35 (14 215 Da). The broad spectra of antimicrobial activities in the cod mucus and the characterization of four antimicrobial polypeptides suggest that mucus compounds contribute to the innate host defence of cod.

The RNA polymerase II subunit Rpb4p mediates decay of a specific class of mRNAs

Abstract: Gene expression is a complex process that involves several distinct stages, each of which is subject to tight controls. Previous works from many laboratories revealed that the regulation of the various stages is coordinated or “coupled”. Thus, each stage can be regarded as a subdivision of a continuous gene expression process. Coupling between mRNA synthesis and its export (Vinciguerra and Stutz 2004) and between translation and mRNA degradation (Jacobson and Peltz 1996; Schwartz and Parker 1999; Coller and Parker 2004; Wilusz and Wilusz 2004) have been demonstrated in several laboratories. However, relatively little is known about the possible coupling between transcription in the nucleus and mRNA degradation in the cytoplasm. mRNA turnover is an essential and well-controlled component of the gene expression process. The half-lives of yeast mRNAs vary substantially, from 3 min to >100 min (e.g., see Wang et al. 2002; Grigull et al. 2004). However, the degradation rates within a group of mRNAs are sometimes found to be alike. Examples of mRNA groups are mRNAs encoding proteins that constitute common complexes and mRNAs encoding proteins that have overlapping cellular roles (Wang et al. 2002). Moreover, mRNAs encoding functionally related proteins exhibit a similar degradation profile in response to environmental signals (Garcia-Martinez et al. 2004; Grigull et al. 2004). These observations highlight the significant influence of mRNA decay on regulation of gene expression and raised the concept of “decay regulons” (Garcia-Martinez et al. 2004). One prominent decay regulon in yeast consists of mRNAs encoding ribosomal proteins (RP-mRNAs) (Wang et al. 2002; Garcia-Martinez et al. 2004; Grigull et al. 2004). In yeast, a major pathway of mRNA degradation initiates with deadenylation of the mRNA poly(A) tail whose full length in yeast is usually 60–80 bases (Brown and Sachs 1998; Mangus et al. 2003). When the length of the poly(A) tail reaches 10–12 bases or less, the mRNA 5′ cap [m(7)GpppN] is removed (Decker and Parker 1993) followed by an exonucleolytic digestion of the unprotected RNA. Deadenylation is carried out by Pan2p/Pan3p and the Ccr4p/Caf1p poly(A) nuclease complexes (Tucker et al. 2001, 2002), while decapping by Dcp1p/Dcp2p (Parker and Song 2004). Decapping is probably a point of no return in the degradation process, as it exposes the 5′ end of the mRNA to the major 5′-to-3′ exonuclease, Xrn1p, and facilitates its activity. Several proteins regulate the decapping process, including Pat1p. Pat1p is considered to be the first decay factor recruited to mRNA, at a time when the mRNA is still associated with translation factors (Tharun and Parker 2001). Subsequently, Pat1p recruits the hepta-heterodimer Lsm1–7 complex (Coller and Parker 2004). Lsm1–7 complex and its associated factors are important for efficient decapping, probably due to their influence on RNP architecture and on accessibility of the cap structure (Coller and Parker 2004) and possibly by their direct effect on Dcp1/Dcp2 activity (Zhang et al. 1999). It is still unclear how the degradation factors are recruited to mRNA and what controls this recruitment. In particular, it is not clear how Pat1p is recruited to the mRNA. Recently, it was discovered that mRNA decay intermediates are found in specific yeast cytoplasmic foci, termed P bodies (Sheth and Parker 2003; Teixeira et al. 2005). These foci also accommodate proteins involved in mRNA decapping and 5′-to-3′ exonucleolytic degradation (Sheth and Parker 2003). Hence, P bodies likely represent sites wherein mRNAs are decapped and degraded. Analogous structures have been observed in mammalian cells, suggesting that P bodies are required for a conserved mechanism (Bashkirov et al. 1997; Ingelfinger et al. 2002; Lykke-Andersen 2002; van Dijk et al. 2002). The decay rates of mRNA are affected by a variety of stimuli, including hormones (Ross 1996), starvation (Jona et al. 2000; Yin et al. 2003; Garcia-Martinez et al. 2004), cell cycle progression (Morris et al. 1991), and cell differentiation (Jack and Wabl 1988). Nevertheless, we have begun to appreciate only recently that some degradation factors might have a selective role in the decay program in that they act only on a subset of mRNAs (Badis et al. 2004; Grigull et al. 2004). In particular, the degradation of mRNAs encoding ribosomal proteins (RP) and ribosomal biogenesis regulons (Ribi) seems to be mediated by specific factors. For example, the decay factor Ccr4p contributes to the degradation of this group of mRNAs more than it does to other mRNAs (Grigull et al. 2004). Interestingly, Ccr4p is also a transcription factor (Collart 2003). The RNA polymerase II (pol II) is composed of 12 subunits (termed Rpb1p–Rpb12p) (Young 1991). Crystal structures of the 12 subunits of the yeast pol II have been resolved recently (Armache et al. 2003; Bushnell and Kornberg 2003). The structures reveal that the 12-subunit enzyme consists of two distinct parts. The first is a 10-subunit core that constitutes the bulk of the pol II structure and includes the catalytic active site. The second part is a two-subunit heterodimeric complex composed of Rpb4p and Rpb7p (for a recent review, see Choder 2004). The 10-subunit core structure has been studied intensively, whereas Rpb4/7 has received less attention. Rpb7p is an essential protein (like most pol II subunits) (McKune et al. 1993; Mitsuzawa et al. 2003). Rpb4p is essential for transcription only under some adverse conditions, but is dispensable under optimal environmental conditions (Woychik and Young 1989; Choder and Young 1993; Khazak et al. 1995; Sheffer et al. 1999; Tan et al. 2000; Miyao et al. 2001; Farago et al. 2003). Rpb4p is present in excess over Rpb7p, suggesting that the former might carry out some of its functions independently of the latter (Choder 2004). Previously, we found that Rpb4p is required for mRNA export to the cytoplasm (Farago et al. 2003). Here we report two novel functions of Rpb4p in mRNA decay. The effect of Rpb4p on mRNA turnover is specific to mRNAs whose products are involved in protein synthesis. Our observations implicate Rpb4p as a factor that couples and possibly coordinates between the nuclear and cytoplasmic stages of gene expression, but that influences only a subset of mRNAs.

Defining the order in which Nmd3p and Rpl10p load onto nascent 60S ribosomal subunits.

Abstract: The large ribosomal subunit protein Rpl10p is required for subunit joining and 60S export in yeast. We have recently shown that Rpl10p as well as the cytoplasmic GTPase Lsg1p are required for releasing the 60S nuclear export adapter Nmd3p from subunits in the cytoplasm. Here, we more directly address the order of Nmd3p and Rpl10p recruitment to the subunit. We show that Nmd3p can bind subunits in the absence of Rpl10p. In addition, we examined the basis of the previously reported dominant negative growth phenotype caused by overexpression of C-terminally truncated Rpl10p and found that these Rpl10p fragments are not incorporated into subunits in the nucleus but instead sequester the WD-repeat protein Sqt1p. Sqt1p is an Rpl10p binding protein that is proposed to facilitate loading of Rpl10p into the 60S subunit. Although Sqt1p normally only transiently binds 60S subunits, the levels of Sqt1p that can be coimmunoprecipitated by the 60S-associated GTPase Lsg1p are significantly increased by a dominant mutation in the Walker A motif of Lsg1p. This mutant Lsg1 protein also leads to increased levels of Sqt1p in complexes that are coimmunoprecipitated with Nmd3p. Furthermore, the dominant LSG1 mutant also traps a mutant Rpl10 protein that does not normally bind stably to the subunit. These results support the idea that Sqt1p loads Rpl10p onto the Nmd3p-bound subunit after export to the cytoplasm and that Rpl10p loading involves the GTPase Lsg1p.

Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli

Abstract: During the stationary phase of Escherichia coli growth, ribosomal structure changes drastically. Proteins RMF, YhbH, YfiA and SRA are expressed and bind to ribosome particles. In a process named 'ribosomal hibernation,' RMF binding induces the dimerization and subsequent inactivation of 70S ribosomes. Here, we examined the functions of YhbH and YfiA in the formation of 70S dimers using deletion mutants of YhbH and YfiA. The yfiA deletion mutant expressed YhbH and RMF in the stationary phase and formed a greater number of 100S particles than the wild-type, showing that YhbH promotes and stabilizes 100S formation. In contrast, the yhbH deletion mutant expressed YfiA and RMF and produced no 70S dimers, suggesting that YfiA prevents 70S dimer formation. Thus, YhbH and YfiA have opposite functions in 70S dimer formation. YhbH and YfiA share 40% sequence homology, suggesting that their binding sites overlap and they compete for a region proximal to the P- and A-sites on 30S subunits. In the yhbH and yfiA double deletion mutant, which expresses only RMF, 70S dimers were observed as 90S particles. Since 100S particles were seen in the yfiA deletion mutant containing RMF and YhbH, YhbH probably converts immature 90S ribosomes into mature 100S particles.

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association.

Abstract: The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j−, respectively). Although unlike eIF3j+, eIF3j− does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2•GTP•Met-tRNAiMet ternary complex, eIF3j−/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j− nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.