Nonsense‐mediated mRNA decay (NMD) is a cellular surveillance pathway that recognizes and degrades mRNAs with premature termination codons (PTCs). The mechanisms underlying translation termination ...are key to the understanding of RNA surveillance mechanisms such as NMD and crucial for the development of therapeutic strategies for NMD‐related diseases. Here, we have used a fully reconstituted in vitro translation system to probe the NMD proteins for interaction with the termination apparatus. We discovered that UPF3B (i) interacts with the release factors, (ii) delays translation termination and (iii) dissociates post‐termination ribosomal complexes that are devoid of the nascent peptide. Furthermore, we identified UPF1 and ribosomes as new interaction partners of UPF3B. These previously unknown functions of UPF3B during the early and late phases of translation termination suggest that UPF3B is involved in the crosstalk between the NMD machinery and the PTC‐bound ribosome, a central mechanistic step of RNA surveillance.
Synopsis
Nonsense‐Mediated Decay (NMD)‐factor UPF3B plays a dual role in early and late translation termination, suggesting a role in coordinating the crosstalk between the NMD machinery and the PTC‐bound ribosome.
NMD‐factor UPF3B interferes with translation termination in vitro when release factors are limiting.
UPF3B dissolves post‐termination complexes after peptidyl‐tRNA hydrolysis by the release factors.
UPF3B interacts with eRF3a in vivo and directly binds to eRF3a in vitro.
UPF1 interaction with release factors is indirect and may be mediated by UPF3B.
In vitro reconstitution assays reveal that UPF3B plays a direct role in translation termination and sheds light on the mechanism of nonsense‐mediated mRNA decay in mammals.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
IS6110 restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of Mycobacterium tuberculosis complex. We have previously described ...mixed-linker PCR, an IS6110-based PCR method that favorably compared with other typing methods for M. tuberculosis complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131 M. tuberculosis complex and nontuberculous isolates showed the ability to differentiate 81 types among 90 M. tuberculosis complex isolates with 84 different IS6110 RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS6110 RFLP. It may as well serve as a secondary typing technique for other, non-IS6110-based methods.
The degradation of nonsense-mutated β-globin mRNA by nonsense-mediated mRNA decay (NMD) limits the synthesis of C-terminally truncated dominant negative β-globin chains and thus protects the majority ...of heterozygotes from symptomatic β-thalassemia. β-globin mRNAs with nonsense mutations in the first exon are known to bypass NMD, although current mechanistic models predict that such mutations should activate NMD. A systematic analysis of this enigma reveals that (1) β-globin exon 1 is bisected by a sharp border that separates NMD-activating from NMD-bypassing nonsense mutations and (2) the ability to bypass NMD depends on the ability to reinitiate translation at a downstream start codon. The data presented here thus reconcile the current mechanistic understanding of NMD with the observed failure of a class of nonsense mutations to activate this important mRNA quality-control pathway. Furthermore, our data uncover a reason why the position of a nonsense mutation alone does not suffice to predict the fate of the affected mRNA and its effect on protein expression.
Progress in the systemic control of osteosarcoma has been limited over the past decades thus indicating the urgent clinical need for the development of novel treatment strategies. Therefore, we have ...recently developed new preclinical models to study promising novel agents for the treatment of pediatric osteosarcoma. The checkpoint kinase (chk) inhibitor prexasertib (LY2606368) and its salt form (LSN2940930) have recently been shown to be active in adult and pediatric malignancies, including sarcoma. We have now tested the potency of prexasertib in clonogenic survival assays in two new lines of primary patient‐derived osteosarcoma cells and in two established osteosarcoma cell lines as a single agent and in combination with cisplatin and the poly ADP‐ribose polymerase (PARP) inhibitor talazoparib. Prexasertib alone results in strongly reduced clonogenic survival at low nanomolar concentrations and acts by affecting cell cycle progression, induction of apoptosis and induction of double‐stranded DNA breakage at concentrations that are well below clinically tolerable and safe plasma concentrations. In combination with cisplatin and talazoparib, prexasertib acts in a synergistic fashion. Chk1 inhibition by prexasertib and its combination with the DNA damaging agent cisplatin and the PARP‐inhibitor talazoparib thus emerges as a potential new treatment option for pediatric osteosarcoma which will now have to be tested in preclinical primary patient derived in vivo models and clinical studies.
What's new?
Treatment options and outcomes for osteosarcoma have changed very little in decades. New treatment strategies are thus urgently needed. In this study, the authors found that the checkpoint inhibitor ‘prexasertib’ increased apoptosis and DNA damage in patient‐derived osteosarcoma cells, at very low concentrations. In addition, when combined with cisplatin and talazoparib, prexasertib acted in a synergistic fashion. This drug thus represents a promising therapeutic candidate for further preclinical and clinical development in osteosarcoma.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract
Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation ...by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3′CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1’s accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
ABSTRACT
IS
6110
restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of
Mycobacterium tuberculosis
complex. We have previously ...described mixed-linker PCR, an IS
6110
-based PCR method that favorably compared with other typing methods for
M. tuberculosis
complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131
M. tuberculosis
complex and nontuberculous isolates showed the ability to differentiate 81 types among 90
M. tuberculosis
complex isolates with 84 different IS
6110
RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS
6110
RFLP. It may as well serve as a secondary typing technique for other, non-IS
6110
-based methods.
ABSTRACT
A PCR assay based on the simultaneous detection of IS
1245
and IS
1311
was developed and used to determine the host range of these insertion elements. Specific PCR products were observed in
...Mycobacterium malmoense
,
Mycobacterium scrofulaceum,
and
Mycobacterium nonchromogenicum
, indicating that IS
1245
and IS
1311
are not limited to the
Mycobacterium avium
complex.
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