Not only do such mutations result in the production of a truncated and usually inactive protein product, but premature stop codon mutations that occur upstream of the last exon-exon junction are also ...known to activate nonsense mediated decay (NMD) which results in the specific degradation of the affected mRNA. 1, 2 However, several mechanisms may allow either fully or partially functional protein to be produced from alleles containing a premature stop codon mutation and this phenomenon may lead to considerable amelioration of the resulting phenotype. 3 Second, an internally truncated form of the protein may be expressed due to altered processing of the mRNA in which the stop codon containing exon (and sometimes adjacent exons) are removed from the mature mRNA. 6 A correlation between exon skipping and suppression of disease symptoms has been documented in several cases of Becker muscular dystrophy (MIM 300376), a milder form of Duchenne muscular dystrophy. 7, 8 Third, protein expression in some genes can be rescued by translational initiation at internal start codons downstream from a premature stop codon or frameshift mutation.
Previous studies with mice overproducing ornithine decarboxylase have demonstrated the importance of polyamine homeostasis for normal mammalian spermatogenesis. The present study introduces a likely ...key player in the maintenance of proper polyamine homeostasis during spermatogenesis. Antizyme 3 is a paralog of mammalian ornithine decarboxylase antizymes. Like its previously described counterparts, antizymes 1 and 2, it inhibits ornithine decarboxylase, which catalyzes the synthesis of putrescine. Earlier work has shown that the coding sequences for antizymes 1 and 2 are in two different, partially overlapping reading frames. Ribosomes translate the first reading frame, and just before the stop codon for that frame, they shift to the second reading frame to synthesize a trans-frame product. The efficiency of this frameshifting depends on polyamine concentration, creating an autoregulatory circuit. Antizyme 3 cDNA has the same arrangement of reading frames and a potential shift site with definite, although limited, homology to its evolutionarily distant antizyme 1 and 2 counterparts. In contrast to antizymes 1 and 2, which are widely expressed throughout the body, antizyme 3 transcription is restricted to testis germ cells. Expression starts early in spermiogenesis and finishes in the late spermatid phase. The potential significance of antizyme 3 expression during spermatogenesis is discussed in this paper.
Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast ...Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.
It is generally believed that significant ribosomal frameshifting during translation does not occur without a functional purpose. The distribution of two frameshift‐prone sequences, A_AAA_AAG and ...CCC_TGA, in coding regions of Escherichia coli has been analyzed. Although a moderate level of selection against the first sequence is evident, 68 genes contain A_AAA_AAG and 19 contain CCC_TGA. The majority of those tested in their genomic context showed >1% frameshifting. Comparative sequence analysis was employed to assess a potential biological role for frameshifting in decoding these genes. Two new candidates, in pheL and ydaY, for utilized frameshifting have been identified in addition to those previously known in dnaX and nine insertion sequence elements. For the majority of the shift‐prone sequences no functional role can be attributed to them, and the frameshifting is likely erroneous. However, none of frameshift sequences is in the 306 most highly expressed genes. The unexpected conclusion is that moderate frameshifting during expression of at least some other genes is not sufficiently harmful for cells to trigger strong negative evolutionary pressure.
Optimization of probe design for array‐based experiments requires improved predictability of oligonucleotide hybridization behavior. Currently, designing oligonucleotides capable of interacting ...efficiently and specifically with the relevant target is not a routine procedure. Multiple examples demonstrate that oligonucleotides targeting different regions of the same RNA differ in their hybridization ability. The present work shows how thermodynamic evaluations of oligo‐target duplex or oligo self‐structure stabilities can facilitate probe design. Statistical analysis of large sets of hybridization data reveals that thermodynamic evaluation of oligonucleotide properties can be used to avoid poor RNA binders. Thermodynamic criteria for the selection of 20 and 21mers, which, with high probability, interact efficiently and specifically with their targets, are suggested. The design of longer oligonucleotides can also be facilitated by the same calculations of ΔG°T values for oligo‐target duplex or oligo self‐structure stabilities and similar selection schemes.
The efficiency of programmed ribosomal frameshifting in decoding antizyme mRNA is the sensor for an autoregulatory circuit that controls cellular polyamine levels in organisms ranging from the yeast ...Schizosaccharomyces pombe to Drosophila to mammals. Comparison of the frameshift sites and flanking stimulatory signals in many organisms now permits a reconstruction of the likely evolutionary path of the remarkably conserved mRNA sequences involved in the frameshifting.
Individuals with body dysmorphic disorder (BDD) and clinically concerning body-image concern (BIC) appear to possess abnormalities in the way they perceive visual information in the form of a bias ...towards local visual processing. As inversion interrupts normal global processing, forcing individuals to process locally, an upright-inverted stimulus discrimination task was used to investigate this phenomenon. We examined whether individuals with nonclinical, yet high levels of BIC would show signs of this bias, in the form of reduced inversion effects (i.e., increased local processing). Furthermore, we assessed whether this bias appeared for general visual stimuli or specifically for appearance-related stimuli, such as faces and bodies. Participants with high-BIC (n = 25) and low-BIC (n = 30) performed a stimulus discrimination task with upright and inverted faces, scenes, objects, and bodies. Unexpectedly, the high-BIC group showed an increased inversion effect compared to the low-BIC group, indicating perceptual abnormalities may not be present as local processing biases, as originally thought. There was no significant difference in performance across stimulus types, signifying that any visual processing abnormalities may be general rather than appearance-based. This has important implications for whether visual processing abnormalities are predisposing factors for BDD or develop throughout the disorder.
A key regulator of cellular polyamine levels from yeasts to mammals is the protein antizyme. The antizyme gene consists of two overlapping reading frames with ORF2 in the +1 frame relative to ORF1. A ...programmed +1 ribosomal frameshift occurs at the last codon of ORF1 and results in the production of full-length antizyme protein. The efficiency of frameshifting is proportional to the concentration of polyamines, thus creating an autoregulatory circuit for controlling polyamine levels. The mRNA recoding signals for frameshifting include an element 5′ and a pseudoknot 3′ of the shift site. The present work illustrates that the ORF1 stop codon and the 5′ element are critical for polyamine sensing, whereas the 3′ pseudoknot acts to stimulate frameshifting in a polyamine independent manner. We also demonstrate that polyamines are required to stimulate stop codon readthrough at the MuLV redefinition site required for normal expression of the GagPol precursor protein.
Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1-239) that contains one selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain ...(residues 240-361) that contains nine selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up by renal proximal convoluted tubule (PCT) cells via megalin-mediated endocytosis. We purified Sepp1 forms from the urine of megalin(-/-) mice using a monoclonal antibody to the N-terminal domain. Mass spectrometry revealed that the purified urinary Sepp1 consisted of N-terminal fragments terminating at 11 sites between residues 183 and 208. They were therefore designated Sepp1(UF). Because the N-terminal domain of Sepp1 has a thioredoxin fold, Sepp1(UF) were compared with full-length Sepp1, Sepp1(Δ240-361), and Sepp1(U40S) as a substrate of thioredoxin reductase-1 (TrxR1). All forms of Sepp1 except Sepp1(U40S), which contains serine in place of the selenocysteine, were TrxR1 substrates, catalyzing NADPH oxidation when coupled with H2O2 or tert-butylhydroperoxide as the terminal electron acceptor. These results are compatible with proteolytic cleavage freeing Sepp1(UF) from full-length Sepp1, the form that has the role of selenium transport, allowing Sepp1(UF) to function by itself as a peroxidase. Ultimately, plasma Sepp1(UF) and small selenium-containing proteins are filtered by the glomerulus and taken up by PCT cells via megalin-mediated endocytosis, preventing loss of selenium in the urine and providing selenium for the synthesis of glutathione peroxidase-3.
Bacterial genome annotations contain a number of coding sequences (CDSs) that, in spite of reading frame disruptions, encode a single continuous polypeptide. Such disruptions have different origins: ...sequencing errors, frameshift, or stop codon mutations, as well as instances of utilization of nontriplet decoding. We have extracted over 1,000 CDSs with annotated disruptions and found that about 75% of them can be clustered into 64 groups based on sequence similarity. Analysis of the clusters revealed deep phylogenetic conservation of open reading frame organization as well as the presence of conserved sequence patterns that indicate likely utilization of the nonstandard decoding mechanisms: programmed ribosomal frameshifting (PRF) and programmed transcriptional realignment (PTR). Further enrichment of these clusters with additional homologous nucleotide sequences revealed over 6,000 candidate genes utilizing PRF or PTR. Analysis of the patterns of conservation apparently associated with nontriplet decoding revealed the presence of both previously characterized frameshift-prone sequences and a few novel ones. Since the starting point of our analysis was a set of genes with already annotated disruptions, it is highly plausible that in this study, we have identified only a fraction of all bacterial genes that utilize PRF or PTR. In addition to the identification of a large number of recoded genes, a surprising observation is that nearly half of them are expressed via PTR-a mechanism that, in contrast to PRF, has not yet received substantial attention.
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