Generally, mRNAs that prematurely terminate translation are abnormally low in abundance. In the case of mammalian cells, nonsense codons most often mediate a reduction in the abundance of newly synthesized, nucleus-associated mRNA by a mechanism that is not well understood. With the aim of defining cis-acting sequences that are important to the reduction process, the effects of particular beta-globin gene rearrangements on the metabolism of beta-globin mRNAs harboring one of a series of nonsense codons have been assessed. Results indicate that nonsense codons located 54 bp or more upstream of the 3'-most intron, intron 2, reduce the abundance of nucleus-associated mRNA to 10-15% of normal without altering the level of either of the two introns within pre-mRNA. The level of cytoplasmic mRNA is also reduced to 10-15% of normal, indicating that decay does not take place once the mRNA is released from an association with nuclei into the cytoplasm. A nonsense codon within exon 2 that does not reduce mRNA abundance can be converted to the type that does by (1) inserting a sufficiently large in-frame sequence immediately upstream of intron 2 or (2) deleting and reinserting intron 2 a sufficient distance downstream of its usual position. These findings indicate that only those nonsense codons located more than 54 bp upstream of the 3'-most intron reduce beta-globin mRNA abundance, which is remarkably consistent with which nonsense codons within the triosephosphate isomerase (TPI) gene reduce TPI mRNA abundance. We propose that the 3'-most exon-exon junction of beta-globin mRNA and, possibly, most mRNAs is marked by the removal of the 3'-most intron during pre-mRNA splicing and that the "mark" accompanies mRNA during transport to the cytoplasm. When cytoplasmic ribosomes terminate translation more than 54 nt upstream of the mark during or immediately after transport, the mRNA is subjected to nonsense-mediated decay. The finding that deletion of beta-globin intron 2 does not appreciably alter the effect of any nonsense codon on beta-globin mRNA abundance suggests that another cis-acting sequence functions in nonsense-mediated decay comparably to intron 2, at least in the absence of intron 2, possibly as a fail-safe mechanism. The analysis of deletions and insertions indicates that this sequence resides within the coding region and can be functionally substituted by intron 2.

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Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3-most intron from pre-mRNA "marks" the 3-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the "mark" mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5 untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.For all organisms that have been studied, the abundance of mRNAs harboring a nonsense codon generated by either a frameshift or a nonsense mutation is generally no more than 20 to 25% of normal (for reviews, see references 22, 23, 30, and 36). In the case of mammalian cells, exceptions to this generalization arise when nonsense codon recognition is prevented by inhibitors of translation such as (i) a suppressor tRNA (3, 21), (ii) ribosome-binding drugs, including anisomysin, cycloheximide, emetine, puromycin, or pactamycin (8, 26, 34); (iii) a secondary structure within the 5Ј untranslated region that blocks translation initiation (3); or (iv) polio virus infection, which inactivates cap-dependent translation (8). Exceptions also arise for nonsense codons followed by an in-frame translation reinitiation site (47) or residing within the distal end of the translational reading frame (reviewed in references 22 and 23).For mRNA encoding human triosephosphate isomerase (TPI), the boundary between distal nonsense codons that do and do not reduce mRNA abundance resides bet...

All eukaryotic cells analyzed have developed mechanisms to eliminate the production of mRNAs that prematurely terminate translation. The mechanisms are thought to exist to protect cells from the deleterious effects of in-frame nonsense codons that are generated by routine inefficiencies and inaccuracies in RNA metabolism such as pre-mRNA splicing. Depending on the particular mRNA and how it is produced, nonsense codons can mediate a reduction in mRNA abundance either (i) before its release from an association with nuclei into the cytoplasm, presumably but not certainly while the mRNA is being exported to the cytoplasm and translated by cytoplasmic ribosomes, or (ii) in the cytoplasm. Here, we provide evidence for a factor that functions to eliminate the production of nonsensecontaining RNAs in mammalian cells. The factor, variously referred to as Rent1 (regulator of nonsense transcripts) or HUPF1 (human Upf1 protein), was identified by isolating cDNA for a human homologue to Saccharomyces cerevisiae Upf1p, which is a group I RNA helicase that functions in the nonsensemediated decay of mRNA in yeast. Using monkey COS cells and human HeLa cells, we demonstrate that expression of human Upf1 protein harboring an arginine-to-cysteine mutation at residue 844 within the RNA helicase domain acts in a dominantnegative fashion to abrogate the decay of nonsense-containing mRNA that takes place (i) in association with nuclei or (ii) in the cytoplasm. These findings provide evidence that nonsensemediated mRNA decay is related mechanistically in yeast and in mammalian cells, regardless of the cellular site of decay.All eukaryotic cells are thought to be characterized by pathways that eliminate the production of mRNAs that encode truncated proteins (reviewed in refs. 1-4). Truncated proteins can arise as the consequence of inefficiencies or inaccuracies in RNA metabolism. In fact, errors in transcription initiation and splicing can result in the production of either nonfunctional protein or protein that acts in a dominant-negative or gain-of-function fashion and may have provided the selective pressure under which the nonsense-mediated decay pathway evolved (reviewed in ref.2). The nonsense-mediated decay pathway also eliminates nonsensecontaining mRNAs for Igs and T cell receptors that arise as a consequence of nonproductive gene rearrangements and hypermutations (reviewed in ref. 5), certain selenoprotein mRNAs when a UGA codon is recognized as a premature termination codon rather than a selenocysteine codon (6), and nonsensecontaining mRNAs that arise as the consequence of random germ-line or somatic defects (reviewed in ref.2).Trans-acting factors required for nonsense-mediated mRNA decay in S. cerevisiae have been defined by genetic analyses to include Upf1p͞Isf2p͞Sal2p͞Nam7p, Upf2p͞Nmd2p͞Sua1p͞Ifs1p, Upf3p͞Sua6p, Xrn1p, which is a 5Ј 3 3Ј exonuclease, and Dcp1p, which is a decapping enzyme (7-19). Evidence that the three Upf factors function in a common pathway that leads to the accelerated decay of nonsense-cont...

Most of the biomedical materials printed using 3D bioprinting are static and are unable to alter/transform with dynamic changes in the internal environment of the body. The emergence of four-dimensional (4D) printing addresses this problem. By preprogramming dynamic polymer materials and their nanocomposites, 4D printing is able to produce the desired shapes or transform functions under specific conditions or stimuli to better adapt to the surrounding environment. In this review, the current and potential applications of 4D-printed materials are introduced in different aspects of the biomedical field, e.g., tissue engineering, drug delivery, and sensors. In addition, the existing limitations and possible solutions are discussed. Finally, the current limitations of 4D-printed materials along with their future perspective are presented to provide a basis for future research.

Apoptosis in metazoans is often accompanied by the destruction of DNA replication initiation proteins, inactivation of checkpoints and activation of cyclin-dependent kinases, which are inhibited by checkpoints that directly or indirectly require initiation proteins. Here we show that, in the budding yeast Saccharomyces cerevisiae, mutations in initiation proteins that attenuate both the initiation of DNA replication and checkpoints also induce features of apoptosis similar to those observed in metazoans. The apoptosis-like phenotype of initiation mutants includes the production of reactive oxygen species (ROS) and activation of the budding-yeast metacaspase Yca1p. In contrast to a recent report that activation of Yca1p only occurs in lysed cells and does not contribute to cell death, we found that, in at least one initiation mutant, Yca1p activation occurs at an early stage of cell death (before cell lysis) and contributes to the lethal effects of the mutation harbored by this strain. Apoptosis in initiation mutants is probably caused by DNA damage associated with the combined effects of insufficient DNA replication forks to completely replicate the genome and defective checkpoints that depend on initiation proteins and/or replication forks to restrain subsequent cell-cycle events until DNA replication is complete. A similar mechanism might underlie the proapoptotic effects associated with the destruction of initiation and checkpoint proteins during apoptosis in mammals, as well as genome instability in initiation mutants of budding yeast.

Regulatory T (Treg) cells are a distinct T-cell lineage characterized by sustained Foxp3 expression and potent suppressor function, but the upstream dominant factors that preserve Treg lineage-specific features are mostly unknown. Here, we show that Lkb1 maintains Treg cell lineage identity by stabilizing Foxp3 expression and enforcing suppressor function. Upon T-cell receptor (TCR) stimulation Lkb1 protein expression is upregulated in Treg cells but not in conventional T cells. Mice with Treg cell-specific deletion of Lkb1 develop a fatal early-onset autoimmune disease, with no Foxp3 expression in most Treg cells. Lkb1 stabilizes Foxp3 expression by preventing STAT4-mediated methylation of the conserved noncoding sequence 2 (CNS2) in the Foxp3 locus. Independent of maintaining Foxp3 expression, Lkb1 programs the expression of a wide spectrum of immunosuppressive genes, through mechanisms involving the augmentation of TGF-β signalling. These findings identify a critical function of Lkb1 in maintaining Treg cell lineage identity.

The highly conserved Cdc6 protein is required for initiation of eukaryotic DNA replication and, in yeast and Xenopus, for the coupling of DNA replication to mitosis. Herein, we show that human Cdc6 is rapidly destroyed by a p53-independent, proteasome-, and ubiquitin-dependent pathway during early stages of programmed cell death induced by the DNA-damaging drug adozelesin, or by a separate caspase-dependent pathway in cells undergoing apoptosis through an extrinsic pathway induced by tumor necrosis factor-alpha and cycloheximide. The proteasome-dependent pathway induced by adozelesin is conserved in the budding yeast Saccharomyces cerevisiae. The destruction of Cdc6 may be a primordial programmed death response that uncouples DNA replication from the cell division cycle, which is reinforced in metazoans by the evolution of caspases and p53.