scholarly journals Design of RNA hairpin modules that predictably tune translation in yeast

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Tim Weenink ◽  
Jelle van der Hilst ◽  
Robert M McKiernan ◽  
Tom Ellis

Abstract Modular parts for tuning translation are prevalent in prokaryotic synthetic biology but lacking for eukaryotic synthetic biology. Working in Saccharomyces cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5′ untranslated region (5′UTR) of mRNAs can be used to tune expression levels by 100-fold by inhibiting translation. We determine the relationship between the calculated free energy of folding in the 5′UTR and in vivo protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this new tool, computational RNA sequence design can be used to predictably fine-tune protein production for genes expressed in yeast.

2017 ◽  
Author(s):  
Tim Weenink ◽  
Robert M. McKiernan ◽  
Tom Ellis

AbstractPredictable tuning of gene expression is essential for engineering genetic circuits and for optimising enzyme levels in metabolic engineering projects. In bacteria, gene expression can be tuned at the stage of transcription, by exchanging the promoter, or at stage of translation by altering the ribosome binding site sequence. In eukaryotes, however, only promoter exchange is regularly used, as the tools to modulate translation are lacking. Working in S. cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5’ untranslated region (5’UTR) of mRNAs can be used to tune expression levels by altering the efficiency of translation initiation. We demonstrate a direct link between the calculated free energy of folding in the 5’UTR and protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and it outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this tool, computational RNA sequence design can be used to predictably fine-tune protein production, providing a new way to modulate gene expression in eukaryotes.


Membranes ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 299
Author(s):  
Monica De Caroli ◽  
Fabrizio Barozzi ◽  
Luciana Renna ◽  
Gabriella Piro ◽  
Gian-Pietro Di Sansebastiano

Plants rely on both actin and microtubule cytoskeletons to fine-tune sorting and spatial targeting of membranes during cell growth and stress adaptation. Considerable advances have been made in recent years in the comprehension of the relationship between the trans-Golgi network/early endosome (TGN/EE) and cytoskeletons, but studies have mainly focused on the transport to and from the plasma membrane. We address here the relationship of the cytoskeleton with different endoplasmic reticulum (ER) export mechanisms toward vacuoles. These emergent features of the plant endomembrane traffic are explored with an in vivo approach, providing clues on the traffic regulation at different levels beyond known proteins’ functions and interactions. We show how traffic of vacuolar markers, characterized by different vacuolar sorting determinants, diverges at the export from the ER, clearly involving different components of the cytoskeleton.


2020 ◽  
Author(s):  
Xiao Liu ◽  
Jean-Denis Beaudoin ◽  
Carrie Ann Davison ◽  
Sara G. Kosmaczewski ◽  
Benjamin I. Meyer ◽  
...  

AbstractThe xbp-1 mRNA encodes the XBP-1 transcription factor, a critical part of the unfolded protein response. Here we report that an RNA fragment produced from xbp-1 mRNA cleavage is a biologically active non-coding RNA (ncRNA) in Caenorhabditis elegans neurons, providing the first example of ncRNA derived from mRNA cleavage. We show that the xbp-1 ncRNA is crucial for axon regeneration in vivo, and that it acts independently of the protein-coding function of the xbp-1 transcript. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; and this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding but not coding function of the endogenous xbp-1 transcript. Thus, cleavage of the xbp-1 mRNA bifurcates it into a coding and a non-coding pathway; modulation of the two pathways may allow neurons to fine-tune their response to injury and other stresses.Graphic abstract


2021 ◽  
Author(s):  
Antje Kruger ◽  
Andrew M Watkins ◽  
Roger Wellington-Oguri ◽  
Jonathan Romano ◽  
Camila Kofman ◽  
...  

Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.


2019 ◽  
Vol 8 (9) ◽  
pp. 1415 ◽  
Author(s):  
Farhad Md. Hossain ◽  
Yunkyung Hong ◽  
Yunho Jin ◽  
Jeonghyun Choi ◽  
Yonggeun Hong

Osteoarthritis (OA), the most common form of arthritis, may be triggered by improper secretion of circadian clock-regulated hormones, such as melatonin, thyroid-stimulating hormone (TSH), or cortisol. The imbalance of these hormones alters the expression of pro-inflammatory cytokines and cartilage degenerative enzymes in articular cartilage, resulting in cartilage erosion, synovial inflammation, and osteophyte formation, the major hallmarks of OA. In this review, we summarize the effects of circadian melatonin, TSH, and cortisol on OA, focusing on how different levels of these hormones affect OA pathogenesis and recovery with respect to the circadian clock. We also highlight the effects of melatonin, TSH, and cortisol at different concentrations both in vivo and in vitro, which may help to elucidate the relationship between circadian hormones and OA.


Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.


1988 ◽  
Vol 59 (02) ◽  
pp. 273-276 ◽  
Author(s):  
J Dawes ◽  
D A Pratt ◽  
M S Dewar ◽  
F E Preston

SummaryThrombospondin, a trimeric glycoprotein contained in the platelet α-granules, has been proposed as a marker of in vivo platelet activation. However, it is also synthesised by a range of other cells. The extraplatelet contribution to plasma levels of thrombospondin was therefore estimated by investigating the relationship between plasma thrombospondin levels and platelet count in samples from profoundly thrombocytopenic patients with marrow hypoplasia, using the platelet-specific α-granule protein β-thromboglobulin as control. Serum concentrations of both proteins were highly correlated with platelet count, but while plasma β-thromboglobulin levels and platelet count also correlated, there was no relationship between the number of platelets and thrombospondin concentrations in plasma. Serial sampling of patients recovering from bone marrow depression indicated that the plasma thrombospondin contributed by platelets is superimposed on a background concentration of at least 50 ng/ml probably derived from a non-platelet source, and plasma thrombospondin levels do not simply reflect platelet release.


1979 ◽  
Vol 42 (03) ◽  
pp. 825-831 ◽  
Author(s):  
Jean-Pierre Allain

SummaryIn order to determine the correlation between different doses of F. VIII and their clinical effect,. 70 children with severe hemophilia A were studied after treatment with single doses of cryoprecipitate. The relationship between plasma F. VIII levels or doses calculated in u/ kg of body weight and clinical results followed an exponential curve. Plasma F. VIII levels of 0.35 and 0.53 u/ml corresponded to 95 and 99% satisfactory treatment, respectively. Similar clinical results were obtained with 20 and 31 u/kg. When the in vivo recovery of F. VIII after lyophilized cryoprecipitate was 0.015 u/ml for each u/kg injected, plasma F. VIII levels of 0.30 and 0.47 u/ml respectively were achieved. Since home treatment is largely based on single infusions of F. VIII, it is suggested that moderate and severe hemorrhages be treated with a dose which will provide a plasma F. VIII level of 0.5 u/ml.


2016 ◽  
pp. 15
Author(s):  
Mariana Giaretto ◽  
Victoria Naffa

ResumenEn este trabajo analizamos las relaciones entre tomas de tierras y Estado, en elAlto Valle de Río Negro en Argentina. En un contexto general de especulacióninmobiliaria, por la que se encarecen los precios de alquileres y terrenos, y depolíticas de vivienda selectivas y acotadas, los sectores populares acceden aun espacio en la ciudad mediante tomas de tierras. Frente a estos conflictos,el Estado, en sus diferentes niveles y poderes, despliega una forma deintervención basada en la criminalización de las luchas por tierra y vivienda.Al mismo tiempo, el poder ejecutivo nacional crea la Secretaría de Acceso alHábitat, para posibilitar la intervención política orientada a la regularizaciónde los asentamientos. Sin embargo, esta intervención tiende a reinscribir laproblemática habitacional en el campo político, para reducirla a un conflicto“entre partes”, eludiendo la responsabilidad estatal.Desde un enfoque metodológico cualitativo, seleccionamos como referenteempírico el caso del asentamiento de Villa Obrera en Fiske, Menuco (Gral.Roca), y las técnicas de recolección de datos se basan en fuentes secundarias,como expedientes judiciales y normativa, y en fuentes primarias como son losrelatos de los protagonistas mediante entrevistas individuales y colectivas.Palabras clave: tomas de tierras, Estado, criminalización de los conflictos.Conflicts caused by land occupation and modesof State intervention: analysis of an experience ofAlto Valle in Río Negro (Argentina)AbstractThis paper analyzes the relationship between State and land occupation,at Alto Valle in Río Negro, Argentina. In a general context of real estatespeculation, where rents and land prices are expensive, and a policy ofselective and limited housing, popular sectors can have access to a spacein the city through land occupation. In front of these conflicts, the Statein its different levels and powers, displays a form of intervention basedon the criminalization of struggles for land and housing. At the sametime, the national executive creates the Secretariat of Access to Habitat,oriented to allow political intervention for the regularization of settlements.However, this intervention tends to re-register the housing problems in thepolitical arena, reducing it to a conflict “between parties” and eluding theresponsibility of the State.From a qualitative methodological approach, we selected as empirical referencethe case of the settlement of Villa Obrera in Fiske, Menuco (GeneralRoca). The techniques of data collection are based on secondary sources, suchas policy and legal records, and primary sources as the stories of protagoniststhrough individual and collective interviews.Keywords: land occupation, State, criminalization of conflicts.Conflitos sobre ocupações de terras e modos deintervenção do estado: análise de uma experiênciado Alto Valle do Rio Preto (Argentina)ResumoEste trabalho analisa as relações entre a posse de terra e o Estado, no AltoValle do Rio Preto, na Argentina. Num contexto geral da especulaçãoimobiliária, por qual se encarecem os preços do aluguel e das terras, e depolíticas de habitação seletivas e limitadas, os setores populares acedem aum espaço na cidade através de ocupações de terras. Frente a estes conflitos,o Estado, em seus diferentes níveis e poderes, desenvolve uma forma deintervenção com base na criminalização das lutas pela terra e vivenda. Aomesmo tempo, o poder executivo nacional cria a Secretaria de Acesso àHabitat para possibilitar à intervenção política orientada a regularização dosassentamentos. No entanto, esta intervenção tende a registrar os problemasde habitação no campo político, para reduzi-la a um conflito “entre aspartes”, iludindo a responsabilidade do Estado.A partir de uma abordagem metodológica qualitativa, foi selecionado comoreferência empírica o caso do Assentamento de Villa Obrera em Fiske,Menuco (Gral. Roca), e as técnicas de recolecção de dados são baseados emfontes secundárias, como expedientes judiciais e normativos, e em fontes primárias como são as histórias dos protagonistas através de entrevistasindividuais e coletivas.Palavras-chave: tomada de terras, Estado, criminalização dos conflitos.


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