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In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in Xenopus oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, strong coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems. Cells are microscopic biological factories that are constantly creating new proteins. To do so, a cell must first convert its master genetic blueprint, the DNA, into strands of messenger RNA or mRNA. These strands are subsequently translated to make proteins. Cells have two ways to adjust the number of proteins they generate so they do not produce too many or too few: by changing how many mRNA molecules are available for translation, and by regulating how efficiently they translate these mRNA molecules into proteins. In animals, both unfertilized eggs and early-stage embryos lack the ability to create or destroy mRNAs, and consequently cannot adjust the number of mRNA molecules available for translation. These cells can therefore only regulate how efficiently each mRNA is translated. They do this by changing the length of the so-called poly(A) tail at the end of each mRNA molecule, which is made up of a long stretch of repeating adenosine nucleotides. The mRNAs with longer poly(A) tails are translated more efficiently than those with shorter poly(A) tails. However, this difference disappears in older embryos, when both long and short poly(A) tails are translated with equal efficiency, and it is largely unknown why. To find out more, Xiang and Bartel studied frog eggs, and discovered that artificially raising levels of a protein that binds poly(A) tails, also known as PABPC, improved the translation of short-tailed mRNAs to create a situation in which both short- and long-tailed mRNAs were translated with near-equal efficiency. This suggested that short- and long-tailed mRNAs compete for limited amounts of the translation-enhancing PABPC, and that long-tailed mRNAs are better at it than short-tailed mRNAs. Further investigation revealed that eggs also had to establish the right conditions for PABPC to enhance translation and had to protect mRNAs not associated with PABPC from being destroyed before they could be translated. Overall, Xiang and Bartel found that in eggs and early embryos, PABPC and poly(A) tails enhanced the translation of mRNAs but did not influence their stability, whereas later in development, they enhanced mRNA stability but not translation. This research provides new insights into how protein production is controlled at different stages of animal development, from unfertilized eggs to older embryos. Understanding how this process is regulated during normal development is crucial for gaining insights into how it can become dysfunctional and cause disease. These findings may therefore have important implications for research into areas such as infertility, reproductive medicine and rare genetic diseases.

Alternative polyadenylation generates numerous 3′ mRNA isoforms that can differ in their stability, structure, and function. These isoforms can be used to map mRNA stabilizing and destabilizing elements within 3′ untranslated regions (3′UTRs). Here, we examine how environmental conditions affect 3′ mRNA isoform turnover and structure in yeast cells on a transcriptome scale. Isoform stability broadly increases when cells grow more slowly, with relative half-lives of most isoforms being well correlated across multiple conditions. Surprisingly, dimethyl sulfate probing reveals that individual 3′ isoforms have similar structures across different conditions, in contrast to the extensive structural differences that can exist between closely related isoforms in an individual condition. Unexpectedly, most mRNA stabilizing and destabilizing elements function only in a single growth condition. The genes associated with some classes of condition-specific stability elements are enriched for different functional categories, suggesting that regulated mRNA stability might contribute to adaptation to different growth environments. Condition-specific stability elements do not result in corresponding condition-specific changes in steady-state mRNA isoform levels. This observation is consistent with a compensatory mechanism between polyadenylation and stability, and it suggests that condition-specific mRNA stability elements might largely reflect condition-specific regulation of mRNA 3′ end formation.

The mechanistic/mammalian target of rapamycin (mTOR), a protein discovered in 1991, integrates a complex pathway with a key role in maintaining cellular homeostasis. By comprising two functionally distinct complexes, mTOR complex 1 (mTORC1) and mTORC2, it is a central cellular hub that integrates intra- and extracellular signals of energy, nutrient, and hormone availability, modulating the molecular responses to acquire a homeostatic state through the regulation of anabolic and catabolic processes. Accordingly, dysregulation of mTOR pathway has been implicated in a variety of human diseases. While major advances have been made regarding the regulators and effectors of mTOR signaling pathway, insights into the regulation of mTOR gene expression are beginning to emerge. Here, we present the current available data regarding the mTOR expression regulation at the level of transcription, translation and mRNA stability and systematize the current knowledge about the fluctuations of mTOR expression observed in several diseases, both cancerous and non-cancerous. In addition, we discuss whether mTOR expression changes can be used as a biomarker for diagnosis, disease progression, prognosis and/or response to therapeutics. We believe that our study will contribute for the implementation of new disease biomarkers based on mTOR as it gives an exhaustive perspective about the regulation of mTOR gene expression in both normal and pathological conditions.

Regulated changes in mRNA stability are critical drivers of gene expression adaptations to immunological cues. mRNA stability is controlled mainly by RNA-binding proteins (RBPs) which can directly cleave mRNA but more often act as adaptors for the recruitment of the RNA-degradation machinery. One of the most prominent RBPs with regulatory roles in the immune system is tristetraprolin (TTP). TTP targets mainly inflammation-associated mRNAs for degradation and is indispensable for the resolution of inflammation as well as the maintenance of immune homeostasis. Recent advances in the transcriptome-wide knowledge of mRNA expression and decay rates together with TTP binding sites in the target mRNAs revealed important limitations in our understanding of molecular mechanisms of TTP action. Such orthogonal analyses lead to the discovery that TTP binding destabilizes some bound mRNAs but not others in the same cell. Moreover, comparisons of various immune cells indicated that an mRNA can be destabilized by TTP in one cell type while it remains stable in a different cell linage despite the presence of TTP. The action of TTP extends from mRNA destabilization to inhibition of translation in a subset of targets. This article will discuss these unexpected context-dependent functions and their implications for the regulation of immune responses. Attention will be also payed to new insights into the role of TTP in physiology and tissue homeostasis.

The poly(A) tail on viral mRNAs plays an important role in gene expression, given the role of the 3′ mRNA tail in mRNA stability and translation. Viruses have developed several strategies to maintain the integrity of their poly(A) tails. These include attracting stabilizing proteins through elements in the 3′ untranslated regions of their mRNA, remodeling their poly(A) tails using terminal nucleotidyl transferases, and blocking deadenylase access to the terminal 3′ end of their poly(A) tails using protein–protein interactions or through triple helical RNA structures. Collectively, the presence of these multiple strategies illustrates the vital overall need for viruses to maintain and preserve their poly(A) tails, highlighting a potential avenue for broad-spectrum antiviral development. In addition, poly(A) tail preservation strategies used by viruses may also be applied to RNA vaccines and therapeutics.

HR + /HER2-low breast cancer is a significant subgroup of conventional HR + /HER2-negative breast cancer, and combination of CDK4/6 inhibitor and endocrine therapy is the standard first-line and second-line treatments for advanced HR + /HER2-low breast cancer. Nevertheless, it remains uncertain whether HER2 signaling affects the effectiveness of CDK4/6 inhibitor administered in combination with endocrine therapy for HR + /HER2-low breast cancer and suitable intervention measures. This study revealed poor efficacy for CDK4/6 inhibitor combined with endocrine therapy for HR + /HER2-low breast cancer in vitro and in vivo models. Secondly, suppression of HER2 gene expression in HR + /HER2-low breast cancer cells resulted in significantly improved efficacy for CDK4/6 inhibitor combined with endocrine therapy. Furthermore, the anti-HER inhibitor neratinib was administered to enhance the effectiveness of CDK4/6 inhibitor combined with endocrine therapy in HR + /HER2-low breast cancer by inhibiting the HER2 pathway and lowering HER2 mRNA expression. Strikingly, neratinib reversed the efficacy of CDK4/6 inhibitor and endocrine therapy by reducing HER2 mRNA stability in HR + /HER2-low breast cancer through the interaction of HER2 3’-UTR region with hsa-miR-23a-5p. Even after reducing neratinib dosage to the standard 1/2 dose (20 mg/kg), it remained highly effective and well-tolerated. This study provides a viable and well-tolerated triple combination therapy for clinical HR + /HER2-low breast cancer.

Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific. We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE) and AU-rich (ARE) elements are over-represented in the 3'UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES) cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip) to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3'UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3'UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1, consistent with the role of CUGBP1 as a destabilizing factor. Taken together, our results systematically establish cis-acting determinants of mRNA decay rates in C2C12 myoblast cells and demonstrate that CUGBP1 associates with GREs to regulate decay of a wide range of mRNAs including several that are critical for muscle development.

The primary function of terminators is to terminate transcription in gene expression. Although some studies have suggested that terminators also contribute positively to upstream gene expression, the extent and underlying mechanism of this effect remain largely unexplored. Here, the correlation between terminating strength and upstream mRNA stability was investigated by constructing a terminator mutation library through randomizing 5 nucleotides, assisted by FlowSeq technology, terminator variants were categorized based on the downstream fluorescence intensity, followed by high-throughput sequencing. To examine the impact of terminators on mRNA stability, the abundance of downstream gene transcripts for each terminator variant was quantified through cDNA sequencing. The results revealed that the transcript abundance controlled by strong terminators was, on average 2.2 times greater than those controlled by weak terminators on average. Moreover, several distinct features could be ascribed to high relative abundance of upstream gene transcript, including a high GC content at the base region of hairpin, and a high AT content in downstream of the U-tract. Additionally, these terminators showed a free energy between −28 and −22 kcal/mol, and a stem length of 14 nt. Finally, these features ascribed the upstream beneficial terminator were validated across various expression systems. By incorporating the optimal terminator downstream of RSF, GSH and HIS in three different strains, the fermentation productions-NMN SAM and VD13 exhibited a remarkable enhancement of 30 %–70 %. The findings presented here uncovered the terminator characteristics contributed to the upstream mRNA stability, providing guiding principles for gene circuit design. [Display omitted]

Abstract The rampant variability in codon bias existing between bacterial genomes is expected to interfere with horizontal gene transfer (HGT), a phenomenon that drives bacterial adaptation. However, delineating the constraints imposed by codon bias on functional integration of the transferred genes is complicated by multiple genomic and functional barriers controlling HGT, and by the dependence of the evolutionary outcomes of HGT on the host's environment. Here, we designed an experimental system in which codon composition of the transferred genes is the only variable triggering fitness change of the host. We replaced Escherichia coli's chromosomal folA gene encoding dihydrofolate reductase, an essential enzyme that constitutes a target for trimethoprim, with combinatorial libraries of synonymous codons of folA genes from trimethoprim-sensitive Listeria grayi and trimethoprim-resistant Neisseria sicca. The resulting populations underwent selection at a range of trimethoprim concentrations, and the ensuing changes in variant frequencies were used to infer the fitness effects of the individual combinations of codons. We found that when HGT causes overstabilization of the 5′-end mRNA, the fitness contribution of mRNA folding stability dominates over that of codon optimality. The 5′-end overstabilization can also lead to mRNA accumulation outside of the polysome, thus preventing the decay of the foreign transcripts despite the codon composition-driven reduction in translation efficiency. Importantly, the fitness effects of mRNA stability or codon optimality become apparent only at sub-lethal levels of trimethoprim individually tailored for each library, emphasizing the central role of the host's environment in shaping the codon bias compatibility of horizontally transferred genes.