Nonsense-mediated mRNA reduction and pre-mRNA processing in drosophila

From bacteria to mammalian cells, the presence of a nonsense mutation causes a reduction in the level of the mRNA of the corresponding gene. The reduction is not, contrary to initial expectations, due to a passive mechanism by which non translated mRNAs are degraded; rather it is a active process in which active translation, cis-acting sequences and specific trans-acting factors are required. It is generally accepted that this phenomenon is the consequence of an evolutionary conserved mechanism that evolved to protect cells from the potentially deleterious effect of truncated proteins - this is often referred to as the mRNA surveillance system or nonsense mediated mRNA decay (NMD). This phenomenon has been extensively studied in budding yeast and in mammalian systems and to a lesser extent in C. elegans. In yeast the recognition of the nonsense codon appears to occur during cytoplasmic translation and premature translation termination is thought to activate a specific protein complex - called the surveillance complex - which in tum triggers an accelerated decay of the aberrant mRNA. However, contrary to the expectation that the recognition of the nonsense codon should occur during cytoplasmic translation, several studies in mammalian cells indicate that NMD may take place in the nucleus by a mechanism that is independent of cytoplasmic translation. For example, several reports indicate that this reduction occurs while the mRNA is still associated with the nucleus, and that the stability of the cytoplasmic mRNA is unchanged relative to a wild-type allele. The common view in the field is that these apparently discordant results between NMD in yeast and in mammalian cells will eventually be accommodated in a single model in which translation in the cytoplasm plays a prominent role. For example, a commonly given explanation is that the recognition of the nonsense codon takes place during nuclear export, and it has been implied that the apparent effects on nuclear RNA are in fact triggered by the premature abortion of translation at the cytoplasmic side of the nuclear envelope. However not all the data from mammalian systems can be so easily explained by the above model. For example, several reports indicate that nonsense mutations affect the splicing of the corresponding pre-mRNA, which makes it difficult to imagine how premature translation in the cytoplasm could effect such an early event in mRNA biogenesis

Splicing-dependent NMD does not require the EJC in Schizosaccharomyces pombe

Nonsense-mediated mRNA decay (NMD) is a translation-linked process that destroys mRNAs with premature translation termination codons (PTCs). In mammalian cells, NMD is also linked to pre-mRNA splicing, usually PTCs trigger strong NMD only when positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems. Here, we report that in Schizosaccharomyces pombe splicing also enhances NMD, but against the EJC model prediction, an intron stimulated NMD regardless of whether it is positioned upstream or downstream of the PTC and EJC components are not required. Still the effect of splicing seems to be direct—we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing. On the basis of these results, we propose a new model to explain how splicing could affect NMD

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.Published versio

The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from gene loci

UPF1 is an RNA helicase that is required for efficient nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3'UTRs of mRNAs that are directed for NMD in the cytoplasm. Here we offer evidence, obtained from Drosophila, that UPF1 constantly moves between the nucleus and cytoplasm by a mechanism that requires its RNA helicase activity. UPF1 is associated, genome-wide, with nascent RNAs at most of the active Pol II transcription sites and at some Pol III-transcribed genes, as demonstrated microscopically on the polytene chromosomes of salivary glands and by ChIP-seq analysis in S2 cells. Intron recognition seems to interfere with association and translocation of UPF1 on nascent pre-mRNAs, and cells depleted of UPF1 show defects in the release of mRNAs from transcription sites and mRNA export from the nucleus

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive, correlates with Pol II transcription and mRNA expression levels. Changes in Pol II occupancy were detected in a Upf1 deficient (upf1∆) strain, prevalently at genes showing a high Upf1 relative to Pol II association in wild-type. Additionally, an increased Ser2 Pol II signal was detected at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1cells are hypersensitive to the transcription elongation inhibitor 6-azauracil. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1. These data envisage that by operating on the nascent transcript, Upf1 might influence Pol II phosphorylation and transcription

Pre-mRNA processing: Insights from nonsense

AbstractIn eukaryotic cells, translation is thought to be confined to cytoplasm, but two recent studies have challenged this notion, one showing that an mRNA's open reading frame influences nuclear events as early as release from the site of transcription, and the other by providing evidence for protein synthesis within the nucleus