Cotranscriptional effect of a premature termination codon revealed by live-cell imaging

Albert Einstein College of Medicine, Bronx, New York 10461, USA.
RNA (Impact Factor: 4.94). 12/2011; 17(12):2094-107. DOI: 10.1261/rna.02918111
Source: PubMed


Aberrant mRNAs with premature translation termination codons (PTCs) are recognized and eliminated by the nonsense-mediated mRNA decay (NMD) pathway in eukaryotes. We employed a novel live-cell imaging approach to investigate the kinetics of mRNA synthesis and release at the transcription site of PTC-containing (PTC+) and PTC-free (PTC-) immunoglobulin-μ reporter genes. Fluorescence recovery after photobleaching (FRAP) and photoconversion analyses revealed that PTC+ transcripts are specifically retained at the transcription site. Remarkably, the retained PTC+ transcripts are mainly unspliced, and this RNA retention is dependent upon two important NMD factors, UPF1 and SMG6, since their depletion led to the release of the PTC+ transcripts. Finally, ChIP analysis showed a physical association of UPF1 and SMG6 with both the PTC+ and the PTC- reporter genes in vivo. Collectively, our data support a mechanism for regulation of PTC+ transcripts at the transcription site.

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    • "ZB . Four hours after transfection , the transfection medium was replaced by 1 mL of fresh , 37°C prewarmed medium containing 1ug / mL Dox , and cells were incubated overnight to reach steady - state NORM LacZA and TER LacZB expression as described above . Anti - RENT1 UPF1 antibody ( Bethyl Laboratories , A300 - 036A ) was used for Western Blot ( de Turris et al . 2011 ) . b - Actin , detected by rabbit anti - actin antibody ( Sigma , A2103 ) was used as a loading control ."
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    ABSTRACT: Nonsense-mediated mRNA decay (NMD) is a quality control mechanism responsible for "surveying" mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. To address these issues, we used single-RNA fluorescent in situ hybridization (FISH) and measured the NMD of PTC-containing β-globin mRNA in intact single cells after the induction of β-globin gene transcription. This approach preserves temporal and spatial information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing β-globin mRNA occurs soon after its export into the cytoplasm, with a half-life of <1 min; the remainder is degraded with a half-life of >12 h, similar to the half-life of normal PTC-free β-globin mRNA, indicating that it had evaded NMD. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear degradation of PTC-containing transcripts. We provide a spatial and temporal model for the biphasic decay of NMD targets.
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    • "In situ hybridization to detect a PTC-containing Ig-μ premRNA in the nucleus showed the level of PTC-containing pre-mRNA was higher than the level of PTC-free pre-mRNA near the transcription site, although transcription rates were indistinguishable (69). These results were reproducible by photobleaching (FRAP) and photoconversion analyses, showing that unspliced PTC-containing transcripts were more condensed at the transcription site than unspliced PTC-free transcripts (70). Notably, downregulation of Upf1 or SMG6 released nuclear PTC-containing transcripts to cytoplasm, suggesting that Upf1, which is a NAS factor, and SMG6 play a key role in holding PTC-containing pre-mRNA in the nucleus. "
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    ABSTRACT: In mammalian cells, aberrant transcripts harboring a premature termination codon (PTC) can be generated by abnormal or inefficient biogenesis of mRNAs or by somatic mutation. Truncated polypeptides synthesized from these aberrant transcripts could be toxic to normal cellular functions. However, mammalian cells have evolved sophisticated mechanisms for monitoring the quality of mRNAs. The faulty transcripts harboring PTC are subject to nonsense-mediated mRNA decay (NMD), nonsense-mediated translational repression (NMTR), nonsense-associated alternative splicing (NAS), or nonsense-mediated transcriptional gene silencing (NMTGS). In this review, we briefly outline the molecular characteristics of each pathway and suggest mRNA quality control mechanisms as a means to regulate normal gene expression. [BMB Reports 2013; 46(1): 009-016].
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    • "In mammalian cells, unspliced variants of PTC-containing mRNAs (and not their PTC-free counterparts) are retained at their site of transcription and this retention depends on UPF1 and SMG6 [138] [139]. Interestingly, UPF1 and SMG6 do not discriminate targets and using ChIP they immunoprecipitate with both PTC-containing and PTC-free genes, yet in an RNA-independent manner [138]. In some cases, a PTC can also induce a reversible transcriptional silencing of its cognate gene [140]. "
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