Two independent mechanisms promote expression of an N-terminal truncated USP18 isoform with higher DeISGylation activity in the nucleus

Expression of the ISG15 specific protease USP18 is highly induced by type I interferons. The two main functions of USP18, i.e. its enzymatic activity and down-regulation of type I interferon signaling, are well characterized. However, to date all functional studies focused on full-length USP18. Here, we report that translation of human USP18 is initiated by a rare start codon (CUG). Usage of this non-canonical initiation site with its weak translation initiation efficiency promotes expression of an N-terminal truncated isoform (USP18-sf). In addition, an internal ribosome entry site (IRES) located in the 5'-coding region of USP18 also contributes to translation of USP18-sf. Functionally, both isoforms exhibit enzymatic activity and interfere with type I interferon signaling. However, USP18-sf shows different subcellular distribution compared with the full-length protein and enhanced deISGylation activity in the nucleus. Taken together, we report the existence of an N-terminal truncated isoform of USP18, whose expression is controlled on translational level by two independent mechanisms providing translational flexibility as well as cell type-specific resistance to inhibition of cap-dependent translation.     

Escherichia coli rpoS gene has an internal secondary translation initiation region

Sigma S (sigma(s)) encoded by rpoS in Escherichia coli is a stationary phase specific sigma subunit of the RNA polymerase holoenzyme. Widespread among the E. coli K12 strains is an amber mutation that prematurely terminates sigma(s). These rpoSAm mutants would be expected to show no sigma(s) activity. However, suppressor free rpoSAm mutants retain an intermediate catalase activity, a sigma S controlled function. By analyzing the sequence of the rpoS gene we hypothesize that a 277 amino acids long delta1-53 sigma(s) of about 30 kDa can be translated from an internal secondary translation initiation region (STIR, AGGGAGN11GUG) that is located downstream of the amber codon. By cloning this rpoSAm gene, following the expression, function, and N-terminal sequence of this mutant protein, we report the presence of a functional internal STIR in E. coli rpoS, from where a truncated but nevertheless functional form of sigma(s) can be synthesized.     

Alteration of protein subcellular location and domain formation by alternative translational initiation

Alternative translation is an important cellular mechanism contributing to the generation of proteins and the diversity of protein functions. Instead of studying individual cases, we systematically analyzed the alteration of protein subcellular location and domain formation by alternative translational initiation in eukaryotes. The results revealed that 85.7% of alternative translation events generated biological diversity, attributed to different subcellular localizations and distinct domain contents in alternative isoforms. Analysis of isoelectric point values revealed that most N-terminal truncated isoforms significantly lowered their isoelectric point values targeted at different subcellular localizations, whereas they had conserved domain contents the same as the full-length isoforms. Furthermore, Fisher's exact test indicated that the two ways-targeting at different cellular compartments and changing domain contents-were negatively associated. The N-term truncated isoforms should have only one way to diversify their functions distinct from the full-length ones. The peculiar consequence of subcellular relocation as well as change of domain contents reflected the very high level of biological complexity as alternative usage of initiation codons.     

Binding of N- and C-terminal anti-prion protein antibodies generates distinct phenotypes of cellular prion proteins (PrPC) obtained from human, sheep, cattle and mouse

Prion diseases are neurodegenerative disorders which cause Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. The infectious agent is a protease resistant isoform (PrP(Sc)) of a host encoded prion protein (PrP(C)). PrP(Sc) proteins are characterized according to size and glycoform pattern. We analyzed the glycoform patterns of PrP(C) obtained from humans, sheep, cattle and mice to find interspecies variability for distinct differentiation among species. To obtain reliable results, the imaging technique was used for measurement of the staining band intensities and reproducible profiles were achieved by many repeated immunoblot analysis. With a set of antibodies, we discovered two distinct patterns which were not species-dependent. One pattern is characterized by high signal intensity for the di-glycosylated isoform using antibodies that bind to the N-terminal region, whereas the other exhibits high intensity for protein bands at the size of the nonglycosylated isoform using antibodies recognizing the C-terminal region. This pattern is the result of an overlap of the nonglycosylated full-length and the glycosylated N-terminal truncated PrP(C) isoforms. Our data demonstrate the importance of antibody selection in characterization of PrP(C).     

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads to a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.     

Molecular cloning of a splicing variant of human RECQL helicase

A cDNA was isolated from the human heart library. This cDNA variant was produced by the deletion of 176 bases at the 5(') end of human RecQL gene, presumably by an alternative mRNA splicing. The cDNA contains two open reading frames and so may encode two isoforms of human RECQL. The first isoform is a 105 amino acid protein with the first 53 N-terminal amino acids identical to the known sequence of RECQL protein and followed by 52 amino acids introduced by in-frame premature stop codon. The second isoform is a 537 amino acid protein that has the same sequence as the published human RECQL helicase, except the first 112 amino acids at the N-terminal end were absent. The possible roles of both of these proteins are discussed.     

Proteolytic processing of the receptor-type protein tyrosine phosphatase PTPBR7

The single-copy mouse gene Ptprr gives rise to different protein tyrosine phosphatase (PTP) isoforms in neuronal cells through the use of distinct promoters, alternative splicing, and multiple translation initiation sites. Here, we examined the array of post-translational modifications imposed on the PTPRR protein isoforms PTPBR7, PTP-SL, PTPPBSgamma42 and PTPPBSgamma37, which have distinct N-terminal segments and localize to different parts of the cell. All isoforms were found to be short-lived, constitutively phosphorylated proteins. In addition, the transmembrane isoform, PTPBR7, was subject to N-terminal proteolytic processing, in between amino acid position 136 and 137, resulting in an additional, 65-kDa transmembrane PTPRR isoform. Unlike for some other receptor-type PTPs, the proteolytically produced N-terminal ectodomain does not remain associated with this PTPRR-65. Shedding of PTPBR7-derived polypeptides at the cell surface further adds to the molecular complexity of PTPRR biology.     

Two rat surfactant protein A isoforms arise by a novel mechanism that includes alternative translation initiation

A single gene for rat surfactant protein A (SP-A) encodes two isoforms that are distinguished by an isoleucine-lysine-cysteine (IKC) N-terminal extension (SP-A and IKC-SP-A). Available evidence suggests that the variants are generated by alternative signal peptidase cleavage of the nascent polypeptide at a primary site (Cys(-)(1)-Asn(1)) and a secondary site (Gly(-)(4)-Ile(-)(3)). In this study, we used site-directed mutagenesis and heterologous expression in vitro and in insect cells to the examine mechanisms that may lead to alternative signal peptidase cleavage including alternative translation initiation at two in-frame AUGs (Met(-)(30) and Met(-)(20)), a suboptimal context for hydrolysis at the primary cleavage site, or cotranslational protein modifications that expose an otherwise cryptic secondary cleavage site. In vitro translation of a rat cDNA for SP-A resulted in both 28 and 29 kDa primary translation products on SDS-PAGE analysis, while translation of cDNAs encoding Met-30Ala and Met-20Ala mutations resulted in only the single 28 and 29 kDa molecular mass species, respectively. These data are consistent with translation initiation at both Met(-)(30) and Met(-)(20) during in vitro synthesis of SP-A. The Met-30Ala mutation reduced expression of the longer isoform in insect cells, indicating that the Met(-)(30) site also contributes to eucaryotic protein expression. Forcing translation initiation at Met(-)(30) by optimizing the Kozak consensus sequence surrounding that codon or by mutating the Met(-)(20) codon resulted in preferential expression of the longer SP-A isoform but reduced overall expression of the protein almost 10-fold. Both isoforms were generated to some degree whether translation was initiated at the codon for Met(-)(30) or Met(-)(20), indicating that the site of translation initiation is not the sole determinant of isoform generation and suggesting that either the context of the primary cleavage site is suboptimal or that cotranslational modifications affect cleavage. Preventing N-terminal glycosylation at Asn(1) did not affect the site of signal peptidase cleavage. Disruption of interchain disulfide formation at Cys(-)(1) by substitution with serine markedly enhanced cleavage at the Gly(-)(4)-Ile(-)(3) bond, but substitution with alanine enhanced cleavage at the Cys(-)(1)-Asn(1) bond. We conclude that rat SP-A isoforms arise by a novel mechanism that includes both alternative translation initiation at two in-frame AUGs and a suboptimal context for signal peptidase hydrolysis at the primary cleavage site.     

Polypyrimidine tract binding protein regulates IRES-mediated translation of p53 isoforms

The p53 tumor suppressor protein plays a key role in maintaining genomic integrity. Enhanced expression of p53 during genotoxic stress is due to both increased protein stability and translational up regulation. Previous reports have shown that p53 mRNA is translated from an alternative initiation codon to produce N-terminal truncated isoform (ΔN-p53) besides full-length p53. We have demonstrated that two internal ribosome entry sites (IRESs) regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Here, we report that polypyrimidine tract-binding protein (PTB) is a p53 IRES interacting trans-acting factor. PTB protein binds specifically to both the p53 IRESs but with differential affinity. siRNA-mediated knockdown of PTB protein results in reduction of activity of both IRESs and also the levels of both the isoforms. It is well known that DNA-damaging agents such as doxorubicin enhance the expression of p53. Our results indicate that during doxorubicin treatment, PTB protein translocates from nucleus to the cytoplasm, probably to facilitate IRES mediated p53 translation. These observations suggest that the relative cytoplasmic abundance of PTB protein, under DNA-damaging conditions, might contribute to regulating the coordinated expression of the p53 isoforms, owing to the differential affinity of PTB binding to the two p53 IRESs.     

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein

The HIV-2 genomic RNA serves both as a messenger for protein synthesis and as a genome for viral assembly and particle production. Our previous work has shown that the HIV-2 genomic RNA encodes two additional Gag proteins that are N-terminal truncated isoforms of the p57 Gag polyprotein. In this study, by the use of mono- and bicistronic RNAs we show that translation at the three AUGs is driven by three distinct and independent internal ribosome entry segments both in vitro and ex vivo. Furthermore we used the recombinant Gag and HIV-2 protease to show that, in vitro, translation is tightly regulated by these two viral proteins. This regulation is exerted both at the level of protein production and also on the selection of the AUG initiation site which changes the ratio at which the three different Gag isoforms are produced.