Timing molecular motion and production with a synthetic transcriptional clock

The realization of artificial biochemical reaction networks with unique functionality is one of the main challenges for the development of synthetic biology. Due to the reduced number of components, biochemical circuits constructed in vitro promise to be more amenable to systematic design and quantitative assessment than circuits embedded within living organisms. To make good on that promise, effective methods for composing subsystems into larger systems are needed. Here we used an artificial biochemical oscillator based on in vitro transcription and RNA degradation reactions to drive a variety of “load” processes such as the operation of a DNA-based nanomechanical device (“DNA tweezers”) or the production of a functional RNA molecule (an aptamer for malachite green). We implemented several mechanisms for coupling the load processes to the oscillator circuit and compared them based on how much the load affected the frequency and amplitude of the core oscillator, and how much of the load was effectively driven. Based on heuristic insights and computational modeling, an “insulator circuit” was developed, which strongly reduced the detrimental influence of the load on the oscillator circuit. Understanding how to design effective insulation between biochemical subsystems will be critical for the synthesis of larger and more complex systems

Interventions for supporting the initiation and continuation of breastfeeding among women who are overweight or obese

This is the protocol for a review and there is no abstract. The objectives are as follows: The main objective of this review is to evaluate the effectiveness of interventions to improve breastfeeding rates in women who are overweight or obese. We will also examine the effectiveness of different types of interventions based on the intervention delivery format (individual or group and face-to-face or mobile technology); style (proactive or reactive); intensity; provider (peer or professional workers); setting (community or hospital, Baby Friendly Initiative accredited; background breastfeeding initiation rate); timing (antenatal, postnatal or both); and co-morbidities (without complications or with gestational diabetes mellitus or pre-existing diabetes, caesarean section, preterm birth)

G-quadruplexes: the beginning and end of UTRs

Molecular mechanisms that regulate gene expression can occur either before or after transcription. The information for post-transcriptional regulation can lie within the sequence or structure of the RNA transcript and it has been proposed that G-quadruplex nucleic acid sequence motifs may regulate translation as well as transcription. Here, we have explored the incidence of G-quadruplex motifs in and around the untranslated regions (UTRs) of mRNA. We observed a significant strand asymmetry, consistent with a general depletion of G-quadruplex-forming RNA. We also observed a positional bias in two distinct regions, each suggestive of a specific function. We observed an excess of G-quadruplex motifs towards the 5′-ends of 5′-UTRs, supportive of a hypothesis linking 5′-UTR RNA G-quadruplexes to translational control. We then analysed the vicinity of 3′-UTRs and observed an over-representation of G-quadruplex motifs immediately after the 3′-end of genes, especially in those cases where another gene is in close proximity, suggesting that G-quadruplexes may be involved in the termination of gene transcription

Controlling the rate of organic reactions: rational design of allosteric Diels-Alderase ribozymes

Allosteric mechanisms are widely used in nature to control the rates of enzymatic reactions, but little is known about RNA catalysts controlled by these principles. The only natural allosteric ribozyme reported to date catalyzes an RNA cleavage reaction, and so do almost all artificial systems. RNA has, however, been shown to accelerate a much wider range of chemical reactions. Here we report that RNA catalysts for organic reactions can be put under the stringent control of effector molecules by straight-forward rational design. This approach uses known RNA sequences with catalytic and ligand-binding properties, and exploits weakly conserved sequence elements and available structural information to induce the formation of alternative, catalytically inactive structures. The potential and general applicability is demonstrated by the design of three different systems in which the rate of a catalytic carbon–carbon bond forming reaction is positively regulated up to 2100-fold by theophylline, tobramycin and a specific mRNA sequence, respectively. Although smaller in size than a tRNA, all three ribozymes show typical features of allosteric metabolic enzymes, namely high rate acceleration and tight allosteric regulation. Not only do these findings demonstrate RNA's power as a catalyst, but also highlight on RNA's capabilities as signaling components in regulatory networks

Light-up properties of complexes between thiazole orange-small molecule conjugates and aptamers

The full understanding of dynamics of cellular processes hinges on the development of efficient and non-invasive labels for intracellular RNA species. Light-up aptamers binding fluorogenic ligands show promise as specific labels for RNA species containing those aptamers. Herein, we took advantage of existing, non-light-up aptamers against small molecules and demonstrated a new class of light-up probes in vitro. We synthesized two conjugates of thiazole orange dye to small molecules (GMP and AMP) and characterized in vitro their interactions with corresponding RNA aptamers. The conjugates preserved specific binding to aptamers while showing several 100-fold increase in fluorescence of the dye (the ‘light-up’ property). In the presence of free small molecules, conjugates can be displaced from aptamers serving also as fluorescent sensors. Our in vitro results provide the proof-of-concept that the small-molecule conjugates with light-up properties can serve as a general approach to label RNA sequences containing aptamers

The malignant phenotype in breast cancer is driven by eIF4A1-mediated changes in the translational landscape

Human mRNA DeXD/H-box helicases are ubiquitous molecular motors that are required for the majority of cellular processes that involve RNA metabolism. One of the most abundant is eIF4A, which is required during the initiation phase of protein synthesis to unwind regions of highly structured mRNA that would otherwise impede the scanning ribosome. Dysregulation of protein synthesis is associated with tumorigenesis, but little is known about the detailed relationships between RNA helicase function and the malignant phenotype in solid malignancies. Therefore, immunohistochemical analysis was performed on over 3000 breast tumors to investigate the relationship among expression of eIF4A1, the helicase-modulating proteins eIF4B, eIF4E and PDCD4, and clinical outcome. We found eIF4A1, eIF4B and eIF4E to be independent predictors of poor outcome in ER-negative disease, while in contrast, the eIF4A1 inhibitor PDCD4 was related to improved outcome in ER-positive breast cancer. Consistent with these data, modulation of eIF4A1, eIF4B and PCDC4 expression in cultured MCF7 cells all restricted breast cancer cell growth and cycling. The eIF4A1-dependent translatome of MCF7 cells was defined by polysome profiling, and was shown to be highly enriched for several classes of oncogenic genes, including G-protein constituents, cyclins and protein kinases, and for mRNAs with G/C-rich 5′UTRs with potential to form G-quadruplexes and with 3′UTRs containing microRNA target sites. Overall, our data show that dysregulation of mRNA unwinding contributes to the malignant phenotype in breast cancer via preferential translation of a class of genes involved in pro-oncogenic signaling at numerous levels. Furthermore, immunohistochemical tests are promising biomarkers for tumors sensitive to anti-helicase therapies

Human SHBG mRNA Translation Is Modulated by Alternative 5′-Non-Coding Exons 1A and 1B

BACKGROUND: The human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1, -1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines, as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed translated in the human prostate and whether 5' UTR exons 1A and 1B differently regulate SHBG translation. RESULTS: Cis-regulatory elements that could potentially regulate translation were identified within the 5'UTRs of SHBG TU-1A and TU-1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG constructs containing exons 2 to 8 but lacking the 5'UTR sequence did show SHBG translation, whereas inclusion of the 5'UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG did not vary between cells transfected with constructs with or without the 5'UTR sequence, thus confirming that the first in-frame ATG of exon 2 is the translation start site of TU-1A and TU-1B. CONCLUSIONS: The use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence

Altered translation of GATA1 in Diamond-Blackfan anemia

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA)[superscript 1, 2], congenital asplenia[superscript 3] and T cell leukemia[superscript 4]. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type– and tissue-specific defects remains unknown[superscript 5]. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.National Institutes of Health (U.S.) (Grant P01 HL32262)National Institutes of Health (U.S.) (Grant U54 HG003067-09

Alternatively spliced isoforms of the human elk-1 mRNA within the 5′ UTR: implications for ELK-1 expression

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5′ UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5′ UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5′ UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions

Ribosomal scanning on the 5′-untranslated region of the human immunodeficiency virus RNA genome

Translation initiation on most eukaryotic mRNAs occurs via a cap-dependent scanning mechanism and its efficiency is modulated by their 5′-untranslated regions (5′-UTR). The human immunodeficiency virus type 1 (HIV-1) 5′-UTR contains a stable TAR hairpin directly at its 5′-end, which possibly masks the cap structure. In addition, the 5′-UTR is relatively long and contains several stable RNA structures that are essential for viral replication. These characteristics may interfere with ribosomal scanning and suggest that translation is initiated via internal entry of ribosomes. Literature on the HIV-1 5′-UTR-driven translation initiation mechanism is controversial. Both scanning and internal initiation have been shown to occur in various experimental systems. To gain further insight in the translation initiation process, we determined which part of the 5′-UTR is scanned. To do so, we introduced upstream AUGs at various positions across the 5′-UTR and determined the effect on expression of a downstream reporter gene that was placed under control of the gag start codon. This strategy allowed us to determine the window of ribosomal scanning on the HIV-1 5′-UTR