The molecular basis of translation initiation and its regulation in eukaryotes.

The regulation of gene expression is fundamental for life. Whereas the role of transcriptional regulation of gene expression has been studied for several decades, it has been clear over the past two decades that post-transcriptional regulation of gene expression, of which translation regulation is a major part, can be equally important. Translation can be divided into four main stages: initiation, elongation, termination and ribosome recycling. Translation is controlled mainly during its initiation, a process which culminates in a ribosome positioned with an initiator tRNA over the start codon and, thus, ready to begin elongation of the protein chain. mRNA translation has emerged as a powerful tool for the development of innovative therapies, yet the detailed mechanisms underlying the complex process of initiation remain unclear. Recent studies in yeast and mammals have started to shed light on some previously unclear aspects of this process. In this Review, we discuss the current state of knowledge on eukaryotic translation initiation and its regulation in health and disease. Specifically, we focus on recent advances in understanding the processes involved in assembling the 43S pre-initiation complex and its recruitment by the cap-binding complex eukaryotic translation initiation factor 4F (eIF4F) at the 5' end of mRNA. In addition, we discuss recent insights into ribosome scanning along the 5' untranslated region of mRNA and selection of the start codon, which culminates in joining of the 60S large subunit and formation of the 80S initiation complex.

A novel allelic donkey ß-lactoglobulin I protein isoform generated by a non-AUG translation initiation codon is associated with a nonsynonymous SNP.

ß-lactoglobulin I (ß-LG I) is one of the most important whey proteins in donkey milk. However, to our knowledge, there has been no study focusing on the full nucleotide sequences of this gene (BLG I). Current investigation of donkey BLG I gene is very limited with only 2 variants (A and B) characterized so far at the protein level. Recently, a new ß-LG I variant, with a significantly higher mass (+1,915 Da) than known variants has been detected. In this study, we report the whole nucleotide sequence of the BLG I gene from 2 donkeys, whose milk samples are characterized by the ß-LG I SDS-PAGE band with a normal electrophoretic mobility (18,514.25 Da, ß-LG I B1 form) the first, and by the presence of a unique ß-LG I band with a higher electrophoretic mobility (20,428.5 Da, ß-LG I D form) the latter. A high genetic variability was found all over the 2 sequenced BLG I alleles. In particular, 16 polymorphic sites were found in introns, one in the 5' flanking region, 3 SNPs in the 5' untranslated region and one SNP in the coding region (g.1871G > A) located at the 40th nucleotide of exon 2 and responsible for the AA substitutions p.Asp28 > Asn in the mature protein. Two SNPs (g.920-922CAC > TGT and g.1871G/A) were genotyped in 93 donkeys of 2 Italian breeds (60 Ragusana and 33 Amiatina, respectively) and the overall frequencies of g.920-922CAC and g.1871A were 0.3065 and 0.043, respectively. Only the rare allele g.1871A was observed to be associated with the slower migrating ß-LG I. Considering this genetic diversity and those found in the database, it was possible to deduce at least 5 different alleles (BLG I A, B, B1, C, D) responsible for 4 potential ß-LG I translations. Among these alleles, B1 and D are those characterized in the present research, with the D allele of real novel identification. Haplotype data analysis suggests an evolutionary pathway of donkey BLG I gene and a possible phylogenetic map is proposed. Analyses of mRNA secondary structure showed relevant changes in the structures, as consequence of the g.1871G > A polymorphism, that might be responsible for the recognition of an alternative initiation site providing an additional signal peptide. The extension of 19 AA sequence to the mature protein, corresponding to the canonical signal peptide with an additional alanine residue, is sufficient to provide the observed molecular weight of the slower migrating ß-LG I encoded by the BLG I D allele.

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit.

The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5' untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine-Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2'-OMe oligonucleotides.

Patterns of codon usage in two ciliates that reassign the genetic code: Tetrahymena thermophila and Paramecium tetraurelia.

We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms; we have analyzed codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along the length of the genes and in a subset of highly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate some specific observations. In both genomes we found a strong bias toward codons with low GC content; however, in highly expressed genes this bias is smaller and codons ending in GC tend to be more frequent. We also found that codon bias increases along gene segments and in highly expressed genes and that the context surrounding initiation and termination codons are always AT rich. Our results also suggest differences in the efficiency of translation of the reassigned stop codons between the two species and between the reassigned codons. Finally, we discuss some of the possible causes for such translational efficiency differences.

Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124.

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galbeta1-->3GlcNAc, LNB) and galacto-N-biose (Galbeta1-->3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBPBl). In the presence of alpha-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a kcat/Km value for GNB that was approximately 50 times higher than that for LNB, whereas LNBPBl showed similar kcat/Km values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-alpha-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.

The complete mitochondrial genome of the black coral Chrysopathes formosa (Cnidaria:Anthozoa:Antipatharia) supports classification of antipatharians within the subclass Hexacorallia.

Black corals comprise a globally distributed shallow- and deep-water taxon whose phylogenetic position within the Anthozoa has been debated. We sequenced the complete mitochondrial genome of the antipatharian Chrysopathes formosa to further evaluate its phylogenetic relationships. The circular mitochondrial genome (18,398 bp) consists of 13 energy pathway protein-coding genes and two ribosomal RNAs, but only two transfer RNA genes (trnM and trnW), as well as a group I intron within the nad5 gene that contains the only copies of nad1 and nad3. No novel genes were found in the antipatharian mitochondrial genome. Gene order and genome content are most similar to those of the sea anemone Metridium senile (subclass Hexacorallia), with differences being the relative location of three contiguous genes (cox2-nad4-nad6) and absence (from the antipatharian) of a group I intron within the cox1 gene. Phylogenetic analyses of multiple protein-coding genes support classifying the Antipatharia within the subclass Hexacorallia and not the subclass Ceriantipatharia; however, the sister-taxon relationships of black corals within Hexacorallia remain inconclusive.

Impact of nonsense-mediated mRNA decay on the global expression profile of budding yeast.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd(+)) and mutant (Nmd(-)) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% +/- 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd(-) strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available.

A CCAAT/enhancer-binding protein site at -87 is required for the activation of a novel murine melanocortin 2-receptor promoter at late stages during adipogenesis.

The peptide hormone ACTH stimulates lipolysis and suppresses leptin production in adipocytes via the G protein-coupled receptor, melanocortin 2 receptor (MC2-R). We have shown previously that peroxisome proliferator-activated receptor-gamma2 is the primary factor responsible for transactivation of the already identified murine MC2-R promoter in the differentiating 3T3-L1 adipocyte cell line. In this study we show that despite the activity of this promoter being transient during differentiation, MC2-R message remains elevated at later time points during adipogenesis. Analysis of the late transcripts reveals that they initiate from a transcriptional start site in the first intron of the murine MC2-R. The genomic sequence upstream of this start site acts as an adipocyte-specific promoter whose activation is delayed in differentiation, compared with the upstream promoter. A CCAAT/enhancer-binding protein binding site, 87 bp upstream of the transcriptional initiation site, is necessary for the activity of this promoter, and protein binding analyses reveal that this site is bound by CCAAT/enhancer-binding protein factors. Real-time PCR analysis of mRNA initiating from the two start sites shows that there is a switch in promoter usage from the 5' to the 3' promoter around d 5, indicating the complex regulation of the murine MC2-R during adipogenesis.

Characterization of sams genes of Amoeba proteus and the endosymbiotic X-bacteria.

As a result of harboring obligatory bacterial endosymbionts, the xD strain of Amoeba proteus no longer produces its own S-adenosylmethionine synthetase (SAMS). When symbiont-free D amoebae are infected with symbionts (X-bacteria), the amount of amoeba SAMS decreases to a negligible level within four weeks, but about 47% of the SAMS activity, which apparently comes from another source, is still detected. Complete nucleotide sequences of sams genes of D and xD amoebae are presented and show that there are no differences between the two. Long-established xD amoebae contain an intact sams gene and thus the loss of xD amoeba's SAMS is not due to the loss of the gene itself. The open reading frame of the amoeba's sams gene has 1,281 nucleotides, encoding SAMS of 426 amino acids with a mass of 48 kDa and pI of 6.5. The amino acid sequence of amoeba SAMS is longer than the SAMS of other organisms by having an extra internal stretch of 28 amino acids. The 5'-flanking region of amoeba sams contains consensus-binding sites for several transcription factors that are related to the regulation of sams genes in E. coli and yeast. The complete nucleotide sequence of the symbiont's sams gene is also presented. The open reading frame of X-bacteria sams is 1,146 nucleotides long, encoding SAMS of 381 amino acids with a mass of 41 kDa and pI of 6.0. The X-bacteria SAMS has 45% sequence identity with that of A. proteus.

The product of the UL12.5 gene of herpes simplex virus type 1 is not essential for lytic viral growth and is not specifically associated with capsids.

The herpes simplex virus type 1 UL12 gene encodes a pH-dependent deoxyribonuclease termed alkaline nuclease. An N-terminally truncated version of the UL12 gene, called UL12.5, was shown to be translated independently from a subgenic mRNA which shares its 3' terminus with the full-length UL12 mRNA. We showed previously that the UL12.5 gene product cannot compensate for the absence of the full-length UL12 gene product (R. Martinez, L. Shao, J. C. Bronstein, P. C. Weber, and S. K. Weller, 1996, Virology 215, 152-164); however, it was not known whether UL12.5 itself performs an essential function during lytic viral growth. In this article the initiation codon for the UL12.5 gene product was mapped and altered to create a gene no longer capable of producing UL12.5. This mutation was introduced into the viral genome to create a virus which was capable of producing full-length UL12 but not UL12.5. The growth properties of this virus indicate that UL12.5 is not essential for viral growth in culture. UL12.5 was previously reported to represent a capsid-associated form of alkaline nuclease (J. C. Bronstein, S. K. Weller, and P. C. Weber, 1997, J. Virol. 71, 3039-3047). Sucrose sedimentation analysis of capsids from cells infected with wild-type or mutant viruses indicates that both UL12 and UL12.5 are found in fractions from across the sucrose gradient which do not always correlate with the presence of viral capsids. Furthermore, UL12.5 is found in fractions across the gradient even in cells infected under conditions in which no capsids are formed. These results indicate that UL12.5 does not specifically associate with viral capsids. Taken together, these data indicate that UL12.5 is not likely to play an important role in lytic viral infection.

A novel bacterial gene-finding system with improved accuracy in locating start codons.

Although a number of bacterial gene-finding programs have been developed, there is still room for improvement especially in the area of correctly detecting translation start sites. We developed a novel bacterial gene-finding program named GeneHacker Plus. Like many others, it is based on a hidden Markov model (HMM) with duration. However, it is a 'local' model in the sense that the model starts from the translation control region and ends at the stop codon of a coding region. Multiple coding regions are identified as partial paths, like local alignments in the Smith-Waterman algorithm, regardless of how they overlap. Moreover, our semiautomatic procedure for constructing the model of the translation control region allows the inclusion of an additional conserved element as well as the ribosome-binding site. We confirmed that GeneHacker Plus is one of the most accurate programs in terms of both finding potential coding regions and precisely locating translation start sites. GeneHacker Plus is also equipped with an option where the results from database homology searches are directly embedded in the HMM. Although this option does not raise the overall predictability, labeled similarity information can be of practical use. GeneHacker Plus can be accessed freely at http://elmo.ims.u-tokyo.ac.jp/GH/.

Machine learning approaches for the prediction of signal peptides and other protein sorting signals.

Prediction of protein sorting signals from the sequence of amino acids has great importance in the field of proteomics today. Recently, the growth of protein databases, combined with machine learning approaches, such as neural networks and hidden Markov models, have made it possible to achieve a level of reliability where practical use in, for example automatic database annotation is feasible. In this review, we concentrate on the present status and future perspectives of SignalP, our neural network-based method for prediction of the most well-known sorting signal: the secretory signal peptide. We discuss the problems associated with the use of SignalP on genomic sequences, showing that signal peptide prediction will improve further if integrated with predictions of start codons and transmembrane helices. As a step towards this goal, a hidden Markov model version of SignalP has been developed, making it possible to discriminate between cleaved signal peptides and uncleaved signal anchors. Furthermore, we show how SignalP can be used to characterize putative signal peptides from an archaeon, Methanococcus jannaschii. Finally, we briefly review a few methods for predicting other protein sorting signals and discuss the future of protein sorting prediction in general.

Characterization of gene expression in mouse blastocyst using single-pass sequencing of 3995 clones.

To study the gene expression profile in the mouse blastocyst and to identify embryonic stage-specific genes, we randomly selected cDNAs derived from mouse blastocysts and sequenced a total of 3995 clones from one or both ends. Excluding the uninformative clones, 3395 clones were grouped as 937 different kinds of genes. Among these, 465 and 406 species showed similarity to known genes and expressed sequence tags (ESTs), respectively, whereas 66 species showed no significant similarity to any genes in known databases. Analysis of these cDNAs revealed that this library contained a variety of functional genes as well as genes that have not been detected in the human EST database; it should provide us with a useful resource for molecular analysis of developmental mechanisms. Although the human EST project is considered to represent roughly half of all genes, our findings indicate that many early stage developmental genes remain to be identified.