The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).     

SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human–yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein–protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56–74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.     

Insulin-like growth factor-binding protein-1: an evolutionarily conserved fine tuner of insulin-like growth factor action under catabolic and stressful conditions

The insulin-like growth factor-binding proteins (IGFBPs) are evolutionarily conserved components of the insulin-like growth factor (IGF) system. The six forms of IGFBPs (IGFBP-1–6) bind the IGF ligands (IGF-1 and -2) with high affinity and regulate the IGFs available to their receptors, therefore providing additional flexibilities in regulating IGF signalling. IGFBP-1, the first identified member of the IGFBP family is highly inducible under a variety of catabolic conditions, such as food deprivation, malnutrition, stress, injury and hypoxia. Recent in vivo studies have indicated that the induced IGFBP-1 serves as a molecular switch by restricting IGF signalling and diverts the limited energy resources away from growth and development towards those metabolic processes essential for survival. This article reviews the recent understandings of the molecular basis of IGFBP-1 regulation and its biological functions, as revealed through research in mammalian and fish models.     

An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2.

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and colocalizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1-G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.     

Identification of an altered elongation factor in temperature-sensitive mutant ts 7'-14 of Saccharomyces cerevisiae

Postpolysomal extracts from wild-type (wt A364A) and temperature-sensitive (ts 7'-14) yeast cells were preincubated for short periods of time at the nonpermissive temperature (37-41 degrees C) prior to incubations for protein synthesis at 20 degrees C. Whereas wt A364A extracts were relatively unaffected by preincubation at the elevated temperature, mutant extracts lost their ability to translate exogenous natural mRNA and poly(U). Phe-tRNA synthetase and ribosomes from ts 7'-14 cells were not inactivated by preincubation at 37-41 degrees C, but a cytosolic component required for chain elongation, as measured by poly(U) translation, was extensively inactivated. The three elongation factors (EF-1, EF-2, and EF-3) required for chain elongation in yeast were resolved chromatographically. Only one factor, EF-3, was able to restore the poly(U)-translational activity of mutant extracts inactivated at the elevated temperature. Heat-inactivated yeast cytosols, which did not support protein synthesis with yeast ribosomes, were perfectly able to translate poly(U) with rat liver ribosomes, which require only EF-1 and EF-2. These and other experiments indicated that the genetically altered component in 7'-14 mutant cells is EF-3.     

Human Cdt1 lacking the evolutionarily conserved region that interacts with MCM2-7 is capable of inducing re-replication

Replication initiation must be a carefully regulated process to avoid genomic instability caused by aberrant replication. In eukaryotic cells, distinct steps of protein loading (origin licensing) and replication activation are choreographed such that a cell can replicate only once per cell cycle. The first proteins recruited to the origins form the pre-replication complex. Of these proteins, Cdt1 is of interest, as it is the focus of several pathways to control replication initiation. It is degraded by two different pathways, mediated by the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) or with cyclin-Cdk2 and inhibited by geminin once cells are in S-phase, presumably to prevent reloading of pre-replication complexes once S-phase has begun. Although the requirement of Cdt1 in loading MCM2-7 is known, the mechanism by which overexpressed Cdt1 stimulates re-replication is unclear. In this study we have designed various mutations in Cdt1 to determine which portion of Cdt1 is important for re-replication, providing insight into possible mechanisms. Surprisingly, we found that mutants of Cdt1 that do not interact with MCM2-7 are able to induce re-replication when overexpressed. The re-replication is not due to titration of geminin from endogenous Cdt1 and is not accompanied by stabilization of endogenous Cdt1. Additionally, the N-terminal one-third of Cdt1 is sufficient to induce re-replication. The N terminus contains the PCNA- and cyclin-interacting motifs, and deletion of both motifs simultaneously in the overexpressed Cdt1 prevents re-replication. These findings suggest that exogenous Cdt1 induces re-replication by de-repressing endogenous Cdt1 through the titration of PCNA and cyclin.     

Evidence of an evolutionarily conserved LMBR1 domain-containing protein that associates with endocytic cups and plays a role in cell migration in dictyostelium discoideum

The ampA gene plays a role in Dictyostelium discoideum cell migration. Loss of ampA function results in reduced ability of growing cells to migrate to folic acid and causes small plaques on bacterial lawns, while overexpression of AmpA results in a rapid-migration phenotype and correspondingly larger plaques than seen with wild-type cells. To help understand how the ampA gene functions, second-site suppressors were created by restriction enzyme-mediated integration (REMI) mutagenesis. These mutants were selected for their ability to reduce the large plaque size of the AmpA overexpresser strain. The lmbd2B gene was identified as a suppressor of an AmpA-overexpressing strain. The lmbd2B gene product belongs to the evolutionarily conserved LMBR1 protein family, some of whose known members are endocytic receptors associated with human diseases, such as anemia. In order to understand lmbd2B function, mRFP fusion proteins were created and lmbd2B knockout cell lines were established. Our findings indicate that the LMBD2B protein is found associated with endocytic cups. It colocalizes with proteins that play key roles in endocytic events and is localized to ruffles on the dorsal surfaces of growing cells. Vegetative lmbd2B-null cells display defects in cell migration. These cells have difficulty sensing the chemoattractant folic acid, as indicated by a decrease in their chemotactic index. lmbd2B-null cells also appear to have difficulty establishing a front/back orientation to facilitate migration. A role for lmbd2B in development is also suggested. Our research gives insight into the function of a previously uncharacterized branch of the LMBR1 family of proteins. We provide evidence of an LMBR1 family plasma membrane protein that associates with endocytic cups and plays a role in chemotaxis.     

Eucaryotic elongation factors Ts is an integral component of rabbit reticulocyte elongation factor 1.

Elongation factor 1 (EF-1) was purified from rabbit reticulocytes and found to contain at least two distinct polypeptides: one of Mr 53 000 and one of Mr 30 000. The 30 000-Mr polypeptide was purified from EF-1 by treatment of the factor with 5.4 M guanidine . HCl and subsequent chromatography on DEAE-BioGel A in the presence of 5 M urea. By a number of functional criteria, the 30 000-Mr polypeptide was found to be the eucaryotic elongation factor Ts (eEF-Ts). These criteria include the ability of the polypeptide to stimulate Artemia salina eEF-Tu-dependent binding of aminoacyl-tRNA to 80-S ribosomes as well as eEF-Tu + EF-2-dependent polyphenylalanine synthesis. The reticulocyte factor also markedly increased the rate of exchange of eEF-Tu . gdp complexes with free GTP. Furthermore, rabbit antibodies to EF-1 from A. salina which was previously shown to contain eEF-Ts [Slobin, L. I. and M?ller, W. (1978) Eur. J. Biochem. 84, 69--77] were found to cross-react with reticulocyte eEF-Ts, suggesting extensive structural homology between brine shrimp and rabbit eEF-Ts. The demonstration that eEF-Ts is and integral component of EF-1 from such diverse sources as brine shrimp and rabbit reticulocytes supports the conclusion that the factor is universally present in eucaryotic EF-1.     

Sperm protein "DE" mediates gamete fusion through an evolutionarily conserved site of the CRISP family

The first member of the cysteine-rich secretory protein (CRISP) family was described by our laboratory in the rat epididymis, and it is known as DE or CRISP-1. Since then, numerous CRISPs exhibiting a high amino acid sequence similarity have been identified in animals, plants and fungi, although their functions remain largely unknown. CRISP-1 proteins are candidates to mediate gamete fusion in the rat, mouse and human through their binding to complementary sites on the egg surface. To elucidate the molecular mechanisms underlying CRISP-1 function, in the present work, deletion mutants of protein DE were generated and examined for their ability to bind to the rat egg and interfere with gamete fusion. Results revealed that the egg-binding ability of DE resides within a 45-amino acid N-terminal region containing the two motifs of the CRISP family named Signature 1 and Signature 2. Subsequent assays using synthetic peptides and other CRISPs support that the egg-binding site of DE falls in the 12-amino-acid region corresponding to Signature 2. The interesting finding that the binding site of DE resides in an evolutionarily conserved region of the molecule provides novel information on the molecular mechanisms underlying CRISP-1 function in gamete fusion with important implications on the structure-function relationship of other members of the widely distributed CRISP family.     

MHO1, an evolutionarily conserved gene, is synthetic lethal with PLC1; Mho1p has a role in invasive growth

The novel protein Memo (Mediator of ErbB2 driven cell motility) was identified in a screen for ErbB2 interacting proteins and found to have an essential function in cell motility. Memo is evolutionarily conserved with homologs found in all branches of life; the human and yeast proteins have a similarity of >50%. In the present study we used the model organism S. cerevisiae to characterize the Memo-homologue Mho1 (Yjr008wp) and to investigate its function in yeast. In a synthetic lethal screen we found MHO1 as a novel synthetic lethal partner of PLC1, which encodes the single phospholipase C in yeast. Double-deleted cells lacking MHO1 and PLC1, proliferate for up to ten generations. Introduction of human Memo into the memoΔplc1Δ strain rescued the synthetic lethal phenotype suggesting that yeast and human proteins have similar functions. Mho1 is present in the cytoplasm and the nucleus of yeast cells; the same distribution of Memo was found in mammalian cells. None of the Memo homologues have a characteristic nuclear localization sequence, however, a conserved nuclear export sequence is found in all. In mammalian cells, blocking nuclear export with Leptomycin B led to nuclear Memo accumulation, suggesting that it is actively exported from the nucleus. In yeast MHO1 expression is induced by stress conditions. Since invasive growth in S. cerevisiea is also stress-induced, we tested Mho1's role in this response. MHO1 deletion had no effect on invasion induced by nutrient deprivation, however, Mho1 overexpression blocked the invasive ability of yeast cells, suggesting that Mho1 might be acting in a dominant negative manner. Taken together, our results show that MHO1 is a novel synthetic lethal interactor with PLC1, and that both gene products are required for proliferation. Moreover, a role for Memo in cell motility/invasion appears to be conserved across species.     

The ability to form intrastrand tetraplexes is an evolutionarily conserved feature of the 3' end of L1 retrotransposons

Mammalian genomes contain many thousands of members of a family of retrotransponsons known as L1 (or LINE-1) elements. These elements lack long terminal repeats (LTRs), and are thought to use a retroposition mechanism than differs from that of retroviruses and other LTR- containing retroelements. In order to define those regions of the L1 element that may be important for L1 retroposition, we examined the 3' untranslated regions (UTRs) of L1 elements from a diverse group of mammals. We show that while the 3' UTRs of L1 elements from different species share little if any sequence homology, they all contain a G- rich polypurine tract of variable length and sequence which can form one or more intrastrand tetraplexes. This conservation over the 100 Myr since the mammalian radiation suggests that either the G-rich motif itself or a conserved structure such as the tetraplex that can be formed by this motif is a significant structural feature of L1 elements and may play a role in their propagation.      

Properties of elongation factor G: its interaction with the ribosomal peptidyl-site

EF-G bound to poly(U)·ribosomes prevents enzymatic or nonenzymatic binding of charged tRNA not only to the A-site but also to the P-site. In turn, charged tRNA bound either to the P- or A-site prevents formation of EF-G·GMPPCP·ribosome complex. Ribosomes carrying newly synthetized peptidyl-tRNA in pretranslocative state are also unable to form stable complexes with EF-G. The functional implications of these observations are discussed and it is suggested that tRNA plays a regulatory role in the interaction of EF-G with ribosomes during the cyclic process of elongation.