Repurposing of the enhancer-promoter communication underlies the compensation of Mesp2 by Mesp1

Organisms are inherently equipped with buffering systems against genetic perturbations. Genetic compensation, the compensatory response by upregulating another gene or genes, is one such buffering mechanism. Recently, a well-conserved compensatory mechanism was proposed: transcriptional adaptation of homologs under the nonsense-mediated mRNA decay pathways. However, this model cannot explain the onset of all compensatory events. We report a novel genetic compensation mechanism operating over the Mesp gene locus. Mesp1 and Mesp2 are paralogs located adjacently in the genome. Mesp2 loss is partially rescued by Mesp1 upregulation in the presomitic mesoderm (PSM). Using a cultured PSM induction system, we reproduced the compensatory response in vitro and found that the Mesp2-enhancer is required to promote Mesp1. We revealed that the Mesp2-enhancer directly interacts with the Mesp1 promoter, thereby upregulating Mesp1 expression upon the loss of Mesp2. Of note, this interaction is established by genomic arrangement upon PSM development independently of Mesp2 disruption. We propose that the repurposing of this established enhancer-promoter communication is the mechanism underlying this compensatory response for the upregulation of the adjacent gene.

Nonsense-Mediated mRNA Decay, a Finely Regulated Mechanism

Nonsense-mediated mRNA decay (NMD) is both a mechanism for rapidly eliminating mRNAs carrying a premature termination codon and a pathway that regulates many genes. This implies that NMD must be subject to regulation in order to allow, under certain physiological conditions, the expression of genes that are normally repressed by NMD. Therapeutically, it might be interesting to express certain NMD-repressed genes or to allow the synthesis of functional truncated proteins. Developing such approaches will require a good understanding of NMD regulation. This review describes the different levels of this regulation in human cells.

Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5ʹ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5ʹ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.

Towards Synthesis of Simplified Analogues of Pateamine A

<p>Pateamine A (22) is a natural product that was isolated from a marine sponge inhabiting the coast of New Zealand. It exhibits potent inhibition of protein synthesis and nonsense-mediated mRNA decay through binding with eIF4A isoforms. Due to the scarcity of pateamine A (22) in the natural source and the low yield of total synthesis of pateamine A, it is necessary to prepare structurally simplified analogues which would allow further research on structure-activity relationships (SAR) of pateamine A (22). Based on the structure-activity relationship studies reported by Romo and co-workers, a simplified triazole analogue 182 lacking methyl groups was synthesized by Hemi Cumming, a previous Ph.D. student who studied at Victoria University of Wellington. The antiproliferative activity of this analogue was found to be significantly lower than that of pateamine A, suggesting that the thiazole embedded within the molecule or the excised methyl groups are crucial for its potency. Therefore, to further explore the necessary features for its selective activity for eIF4A isoforms, new thiazole analogues 183 – 186 and triazole analogues (10S)-and (10R)-analogue 187 were targeted in this project. The preparation of the thiazole-containing macrocyclic core of analogues 183 and 184 was achieved. It features: (1) gold-catalysed thiazole formation through coupling between an alkyne fragment and a thioamide fragment; (2) preparation of the Z,E-dienoate moiety by base-induced ring-opening of a δ-substituted-α, β-unsaturated lactone; and (3) a modified Mukaiyama macrolactonisation. The synthesis of the triazole-containing macrocyclic core of (10S)-analogue 187 was completed. It features: (1) a copper-catalysed triazole formation through 1,3-dipolar cycloaddition between an alkyne fragment and an azide fragment; (2) preparation of the Z,E-dienoate moiety by base-induced ring-opening of δ-substituted-α, β-unsaturated lactone; and (3) a modified Mukaiyama macrolactonisation. Studies on the preparation of a side-chain fragment with suitable functionalities to allow coupling with the various macrocycles through olefination reactions were also conducted. The attachment of the side-chain fragment onto the macrocyclic cores for the synthesis of the targeted analogues 183 and 184 and (10S)-analogue 187 will be investigated in future work. These experimental results will inform the synthesis of new generation analogues to further study the key structures required for effective binding to the protein target eIF4A and selectivity between isoforms.</p>

Towards Synthesis of Simplified Analogues of Pateamine A

<p>Pateamine A (22) is a natural product that was isolated from a marine sponge inhabiting the coast of New Zealand. It exhibits potent inhibition of protein synthesis and nonsense-mediated mRNA decay through binding with eIF4A isoforms. Due to the scarcity of pateamine A (22) in the natural source and the low yield of total synthesis of pateamine A, it is necessary to prepare structurally simplified analogues which would allow further research on structure-activity relationships (SAR) of pateamine A (22). Based on the structure-activity relationship studies reported by Romo and co-workers, a simplified triazole analogue 182 lacking methyl groups was synthesized by Hemi Cumming, a previous Ph.D. student who studied at Victoria University of Wellington. The antiproliferative activity of this analogue was found to be significantly lower than that of pateamine A, suggesting that the thiazole embedded within the molecule or the excised methyl groups are crucial for its potency. Therefore, to further explore the necessary features for its selective activity for eIF4A isoforms, new thiazole analogues 183 – 186 and triazole analogues (10S)-and (10R)-analogue 187 were targeted in this project. The preparation of the thiazole-containing macrocyclic core of analogues 183 and 184 was achieved. It features: (1) gold-catalysed thiazole formation through coupling between an alkyne fragment and a thioamide fragment; (2) preparation of the Z,E-dienoate moiety by base-induced ring-opening of a δ-substituted-α, β-unsaturated lactone; and (3) a modified Mukaiyama macrolactonisation. The synthesis of the triazole-containing macrocyclic core of (10S)-analogue 187 was completed. It features: (1) a copper-catalysed triazole formation through 1,3-dipolar cycloaddition between an alkyne fragment and an azide fragment; (2) preparation of the Z,E-dienoate moiety by base-induced ring-opening of δ-substituted-α, β-unsaturated lactone; and (3) a modified Mukaiyama macrolactonisation. Studies on the preparation of a side-chain fragment with suitable functionalities to allow coupling with the various macrocycles through olefination reactions were also conducted. The attachment of the side-chain fragment onto the macrocyclic cores for the synthesis of the targeted analogues 183 and 184 and (10S)-analogue 187 will be investigated in future work. These experimental results will inform the synthesis of new generation analogues to further study the key structures required for effective binding to the protein target eIF4A and selectivity between isoforms.</p>

Nonsense-mediated mRNA decay uses complementary mechanisms to suppress mRNA and protein accumulation

Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons. Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to eightfold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.

Progressive polysomal association of Upf1, Upf2, and Upf3 as ribosomes traverse mRNA coding regions

SUMMARYUpf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.

Cellular variability of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two β-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.

Deciphering the nonsense-mediated mRNA decay pathway to identify cancer cell vulnerabilities for effective cancer therapy

Nonsense-mediated mRNA decay (NMD) is a highly conserved cellular surveillance mechanism, commonly studied for its role in mRNA quality control because of its capacity of degrading mutated mRNAs that would produce truncated proteins. However, recent studies have proven that NMD hides more complex tasks involved in a plethora of cellular activities. Indeed, it can control the stability of mutated as well as non-mutated transcripts, tuning transcriptome regulation. NMD not only displays a pivotal role in cell physiology but also in a number of genetic diseases. In cancer, the activity of this pathway is extremely complex and it is endowed with both pro-tumor and tumor suppressor functions, likely depending on the genetic context and tumor microenvironment. NMD inhibition has been tested in pre-clinical studies showing favored production of neoantigens by cancer cells, which can stimulate the triggering of an anti-tumor immune response. At the same time, NMD inhibition could result in a pro-tumor effect, increasing cancer cell adaptation to stress. Since several NMD inhibitors are already available in the clinic to treat genetic diseases, these compounds could be redirected to treat cancer patients, pending the comprehension of these variegated NMD regulation mechanisms. Ideally, an effective strategy should exploit the anti-tumor advantages of NMD inhibition and simultaneously preserve its intrinsic tumor suppressor functions. The targeting of NMD could provide a new therapeutic opportunity, increasing the immunogenicity of tumors and potentially boosting the efficacy of the immunotherapy agents now available for cancer treatment.