Posttranscriptional Regulation by the Upstream Open Reading Frame of the Phosphoethanolamine N-Methyltransferase Gene

Phosphoethanolamine N-methyltransferase (PEAMT) is involved in choline biosynthesis in plants. The 5′ untranslated region (UTR) of several PEAMT genes was found to contain an upstream open reading frame (uORF). We generated transgenic Arabidopsis calli that expressed a chimeric gene constructed by fusing the 5′ UTR of the Arabidopsis PEAMT gene (AtNMT1) upstream of the β-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream β-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.     

Sucrose Induction of Anthocyanin Biosynthesis Mediated by DELLA

Dear Editor, While they affect all aspects of plant life, the exact molecu- lar mechanisms involved in sugar sensing and signaling are still mostly unknown. However, using the induction of antho- cyanin biosynthesis as a convenient readout and tool, the DELLA proteins can be identified as key positive regulators in sucrose-specific signaling and a point of integration of diverse metabolic and hormonal signals.     

Reinitiation after Translation of Two Upstream Open Reading Frames (ORF) Governs Expression of the ORF35-37 Kaposi's Sarcoma-Associated Herpesvirus Polycistronic mRNA

The Kaposi''s sarcoma-associated herpesvirus (KSHV) ORF36 protein kinase is translated as a downstream gene from the ORF35-37 polycistronic mRNA via a unique mechanism involving short upstream open reading frames (uORFs) located in the 5′ untranslated region. Here, we confirm that ORF35-37 is functionally dicistronic during infection and demonstrate that mutation of the dominant uORF restricts KSHV replication. Leaky scanning past the uORFs facilitates ORF35 expression, while a reinitiation mechanism after translation of the uORFs enables ORF36 expression.     

膜穿透肽介导的核酸转运

以能通透生物膜的膜穿透肽为载体,使其携带DNA或寡核苷酸进入细胞,这种新型的核酸转运方法不仅体内外均有效,而且毒性很低。预计该方法在未来的生物工程和基因治疗中将发挥重要作用。     

原核生物基因组肽编码sORFs分布及功能特征

近期,从非编码RNA中发现具有肽编码能力的小开放阅读框(sORFs),激发了人们对这种长期被忽略的基因组元件的研究兴趣,sORFs迅速成为当前重点研究领域.由于表达水平及丰度低、序列短等因素,对肽编码sORFs的有效研究方法及数据资源还很缺乏,现有研究仅集中在少数真核模式生物,对自然界中广泛存在的原核生物研究非常少,肽编码sORFs的发现为目前精准背景下的基因组注释提出严峻挑战.在此背景下,本文首先系统研究了80余种不同类型原核生物中长度小于100个氨基酸的肽编码sORFs分布及功能特征,并对不同长度区间sORFs的序列组成、分布及进化特征进行了对比分析.结果表明,肽编码sORFs在原核生物基因组普遍存在,随着序列长度的降低,其序列复杂度降低,行使的生物功能也相对集中.在此基础上,进一步结合当前肽编码sORFs研究现状,深入总结了肽编码sORFs研究存在的问题及挑战,为今后肽编码sORFs研究奠定了坚实理论基础.     

Surrogate Antigen Processing Mediated by TAP-dependent Antigenic Peptide Secretion

MHC class I proteins assemble with peptides in the ER. The peptides are predominantly generated from cytoplasmic proteins, probably by the action of the proteasome, a multicatalytic proteinase complex. Peptides are translocated into the ER by the transporters associated with antigen processing (TAP), and bind to the MHC class I molecules before transport to the cell surface. Here, we use a new functional assay to demonstrate that peptides derived from vesicular stomatitis virus nucleoprotein (VSV-N) antigen are actively secreted from cells. This secretion pathway is dependent on the expression of TAP transporters, but is independent of the MHC genotype of the donor cells. Furthermore, the expression and transport of MHC class I molecules is not required. This novel pathway is sensitive to the protein secretion inhibitors brefeldin A (BFA) and a temperature block at 21°C, and is also inhibited by the metabolic poison, azide, and the protein synthesis inhibitor, emetine. These data support the existence of a novel form of peptide secretion that uses the TAP transporters, as opposed to the ER translocon, to gain access to the secretion pathway. Finally, we suggest that this release of peptides in the vicinity of uninfected cells, which we term surrogate antigen processing, could contribute to various immune and secretory phenomena.Protein secretion has traditionally been thought to involve the action of the translocon located in the membrane of the ER of eukaryotic cells. Proteins are recognized cotranslationally when a signal sequence or a signal–anchor sequence emerges from the ribosome (Walter and Johnson, 1994). These sequences are recognized and bound by the signal recognition particle, and the resulting ribosomal complex then interacts with the signal recognition particle receptor on the ER membrane at the translocon (Andrews and Johnson, 1996). This results in the inclusion of proteins within the secretory pathway. This pathway is by far the best described route of protein secretion in eukaryotic cells. Recently, it has been proposed that some proteins are recognized by a component of the translocon, sec 61, exit the ER, and are transported into the cytoplasm where they are degraded (Wiertz et al., 1996).The translocation into the ER of antigenic peptides for presentation by major histocompatibility complex (MHC)¹ class I molecules is largely independent of the translocon. This form of translocation involves the action of two gene products that are members of the ATP binding cassette family. These genes encode transporters associated with antigen processing 1 and 2 (TAP-1 and -2), and have been implicated in the translocation of peptides from the cytoplasm to the lumen of the ER (Deverson et al., 1990; Bahram et al., 1991; Spies and DeMars, 1991; Spies et al., 1992; Gabathuler et al., 1994). After translocation into the ER, antigenic peptides bind to MHC class I molecules composed of a heavy chain (46-kD) and a light chain (12-kD) called β₂m (Nuchtern et al., 1989; Yewdell and Bennink, 1989; van Bleek and Nathenson, 1990; Matsumura et al., 1992), before transport to the cell surface. The assembly and transport of MHC class I molecules appears to be regulated by a series of chaperones that includes calnexin (Degen and Williams, 1991), calreticulin, and tapasin (Sadasivan et al., 1996).High performance liquid chromatography analysis of peptides eluted from acid-treated whole cells or MHC class I molecules has allowed the identification and characterization of the peptides associated with MHC class I molecules (Falk et al., 1990; Rötzschke et al., 1990; van Bleek and Nathenson, 1990). It is proposed that MHC class I molecules determine the final identity of MHC- restricted peptides and have an instructive role, in addition to a selective role, in peptide selection (Wallny et al., 1992). MHC binding to larger peptides followed by protected proteolytic trimming is a possible mechanism that could account for the observed MHC dependency of cellular peptides (Falk et al., 1990). Peptides unable to bind MHC class I because they are in excess or lack the correct MHC binding motif for the MHC haplotype are thus far undetectable by the techniques commonly used in the field, and are presumed to be short lived and degraded (Falk et al., 1990; Rötzschke et al., 1990). Recent results, however, suggest that peptides not able to bind to a MHC class I molecule intracellularly may be found bound to heat shock proteins (HSPs) such as gp96 (grp94; Arnold et al., 1995). These authors show that cellular antigens are represented by peptides associated with gp96 molecules independently of the MHC class I expressed, confirming earlier results (Udono and Srivastava, 1993, 1994). Gp96 extracted from a specific cell is able to induce cross-priming (Udono and Srivastava, 1993, 1994). Finally, two studies have demonstrated that peptides transported into the lumen of the ER, and do not bind to MHC class I molecules, can be transported out of the ER into the cytoplasm again by a process called “efflux” (Momburg et al., 1994; Schumacher et al., 1994), which may involve the action of the TAP molecules or the sec 61 protein associated with the translocon (Wiertz et al., 1996).We have developed a new bioassay to test the hypothesis that peptides translocated into the ER by the action of the TAP molecules become secreted. Using this assay, we present evidence of an alternative secretion pathway that exists in various mammalian cell types. These observations revise the model of peptide catabolism, and may provide an explanation for various immune and secretion phenomena.     

Prion Protein Peptide Neurotoxicity Can Be Mediated by Astrocytes

A peptide based on amino acids 106-126 of the sequence of human prion protein (PrP106-126) is neurotoxic in culture. A role for astrocytes mediating PrP106-126 toxicity was investigated. The toxicity of PrP106-126 to cerebellar cell cultures was reduced by aminoadipate, a gliotoxin. Normally, PrP106-126 is not toxic to cultures containing neurones deficient in the cellular isoform of prion protein (PrPc). However, PrP106-126 was toxic to cerebellar cells derived from Prnp(0/0) mice (deficient in PrPc expression) when those cerebellar cells were cocultured with astrocytes. This toxicity was found to occur only in the presence of PrPc-positive astrocytes and to be mediated by glutamate. Furthermore, PrPc-positive astrocytes were shown to protect Prnp(0/0) cerebellar cells from glutamate toxicity. This effect could be inhibited by PrP106-126. PrP106-126 did not enhance the toxicity of glutamate to neurones directly. When cerebellar cells were cocultured with astrocytes, the neurones became dependent on astrocytes for protection from glutamate toxicity and expressed an increased sensitivity to glutamate. In such a system, the protective effects of astrocytes against glutamate toxicity to neurones were inhibited by PrP106-126, resulting in a greater reduction in neuronal survival than would have been caused by PrP106-126 when astrocytes were not present. This new model provides a possible mechanism by which the gliosis in prion disease may accelerate the neurodegeneration seen in the later stages of the disease.     

An Upstream Open Reading Frame in Phosphatase and Tensin Homolog Encodes a Circuit Breaker of Lactate Metabolism

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Kinetically Controlled Peptide Synthesis Mediated by Papain Using the Carbamoylmethyl Ester as an Acyl Donor

A series of dipeptides were synthesized generally in good yields with carbamoylmethyl (Cam) esters as acyl donors in the presence of a cysteine protease, papain, immobilized on Celite. Several segment condensations were also achieved generally in high yields without danger of racemization and formation of the secondary-hydrolysis product. Moreover, partial sequences of some bioactive peptides were prepared through segment condensations, and aimed-at peptides were obtained generally in high yields without the racemization of C-terminal residues of the carboxyl components. Thus, the superiority of the Cam ester in the kinetically controlled peptide synthesis was once again ascertained in couplings mediated by the cysteine protease as in those catalyzed by the serine proteases reported earlier.     

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.