In vitro translation of mRNAs that are in their native ribonucleoprotein complexes

2015 ◽  
Vol 472 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Baptiste Panthu ◽  
Fabrice Mure ◽  
Henri Gruffat ◽  
Theophile Ohlmann
Keyword(s):  
Binding Proteins ◽  
In Vitro Translation ◽  
Ribosome Binding ◽  
Ribonucleoprotein Complexes ◽  
The Impact

The present study shows the impact of ribosome binding proteins on in vitro translation.

scholarly journals Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

2015 ◽  
Vol 308 (1) ◽  
pp. L58-L75 ◽  
Author(s):  
Nikolaos Tsotakos ◽  
Patricia Silveyra ◽  
Zhenwu Lin ◽  
Neal Thomas ◽  
Mudit Vaid ◽  
...  
Keyword(s):  
In Silico ◽  
Splice Variants ◽  
Surfactant Protein ◽  
In Vitro Translation ◽  
Start Codon ◽  
Translation Efficiency ◽  
Human Lung Tissue ◽  
In Vitro Techniques ◽  
The Impact

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.

scholarly journals Cell-free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs

2021 ◽  
Author(s):  
Hoa Quynh Do ◽  
Carla M Bassil ◽  
Elizabeth I Andersen ◽  
Michaela Jansen
Keyword(s):  
Tumor Cells ◽  
Plasma Membranes ◽  
In Vitro Translation ◽  
Translation System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Membrane Scaffold ◽  
The Impact

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA

1994 ◽  
Vol 14 (4) ◽  
pp. 2629-2639
Author(s):  
L K Read ◽  
H U Göringer ◽  
K Stuart
Keyword(s):  
Complex Formation ◽  
Rna Editing ◽  
Binding Proteins ◽  
Macromolecular Complex ◽  
Differential Distribution ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Recognition Elements

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.

scholarly journals 2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 106 ◽  
Author(s):  
Piero Monaco ◽  
Virginie Marcel ◽  
Jean-Jacques Diaz ◽  
Frédéric Catez
Keyword(s):  
Ribosomal Rna ◽  
Translational Control ◽  
Ribosome Biogenesis ◽  
In Vitro Translation ◽  
Ribosome Structure ◽  
Quantitative Mapping ◽  
And Function ◽  
The Impact ◽  
First Time

Ribosomal RNA (rRNA) undergoes post-transcriptional modification of over 200 nucleotides, predominantly 2′-O-methylation (2′-O-Me). 2′-O-Methylation protects RNA from hydrolysis and modifies RNA strand flexibility but does not contribute to Watson-Crick base pairing. The contribution of 2′-O-Me to the translational capacity of ribosomes has been established. Yet, how 2′-O-Me participates in ribosome biogenesis and ribosome functioning remains unclear. The development of 2′-O-Me quantitative mapping methods has contributed to the demonstration that these modifications are not constitutive but rather provide heterogeneity to the ribosomal population. Moreover, recent advances in ribosome structure analysis and in vitro translation assays have proven, for the first time, that 2′-O-Me contributes to regulating protein synthesis. This review highlights the recent data exploring the impact of 2′-O-Me on ribosome structure and function, and the emerging idea that the rRNA epitranscriptome is involved in translational control.

scholarly journals Human SP-A1 (SFTPA1) variant-specific 3′ UTRs and poly(A) tail differentially affect the in vitro translation of a reporter gene

2010 ◽  
Vol 299 (4) ◽  
pp. L523-L534 ◽  
Author(s):  
Patricia Silveyra ◽  
Guirong Wang ◽  
Joanna Floros
Keyword(s):  
Reporter Gene ◽  
Splice Variants ◽  
In Vitro Translation ◽  
Luciferase Reporter ◽  
Structure Stability ◽  
Sequence Variant ◽  
Translation Efficiency ◽  
Significant Difference ◽  
The Impact

Human surfactant protein A (SP-A) is encoded by two functional genes (SFTPA1, SFTPA 2) with a high degree of sequence identity. Sequence differences among these genes and their genetic variants have been observed at the 5′ and 3′ untranslated regions (UTRs). In this work, we studied the impact on translation of the SFTPA1 (hSP-A1) and SFTPA2 (hSP-A2) gene 5′ UTR splice variants and 3′ UTR sequence variants, in the presence or absence of poly(A) tail. We generated constructs containing the luciferase reporter gene flanked upstream by one of the hSP-A 5′ UTR splice variants and/or downstream by one hSP-A 3′ UTR sequence variant. mRNA transcripts were prepared by in vitro transcription and used for either in vitro translation with a rabbit reticulocyte lysate or transient transfection of the lung adenocarcinoma cell line NCI-H441. The luciferase activity results indicate that hSP-A 5′ UTR and 3′ UTR together have an additive effect on translation. In this context, the hSP-A1 6A3 and 6A4 3′ UTR variants exhibited higher translation efficiency than the 6A2 variant ( P <0.05), whereas no significant difference was observed between the two hSP-A2 3′ UTRs studied (1A0, 1A3). Further sequence analysis revealed that a deletion of an 11-nucleotide (nt) element in both the 6A3 and 6A4 3′ UTR variants changes the predicted secondary structure stability and the number of putative miRNA binding sites. Removal of this 11-nt element in the 6A2 3′ UTR resulted in increased translation, and the opposite effect was observed when the 11-nt element was cloned in a guest 3′ UTR (6A3, 6A4). These results indicate that sequence differences among hSP-A gene variants may account for differential regulation at the translational level.

scholarly journals Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.

1994 ◽  
Vol 14 (4) ◽  
pp. 2629-2639 ◽  
Author(s):  
L K Read ◽  
H U Göringer ◽  
K Stuart
Keyword(s):  
Complex Formation ◽  
Rna Editing ◽  
Binding Proteins ◽  
Macromolecular Complex ◽  
Differential Distribution ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Recognition Elements

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.

scholarly journals A novel role for poly(C) binding proteins in programmed ribosomal frameshifting

2016 ◽  
Vol 44 (12) ◽  
pp. 5491-5503 ◽  
Author(s):  
Sawsan Napthine ◽  
Emmely E. Treffers ◽  
Susanne Bell ◽  
Ian Goodfellow ◽  
Ying Fang ◽  
...  
Keyword(s):  
Translational Control ◽  
Binding Proteins ◽  
Cellular Protein ◽  
In Vitro Translation ◽  
Respiratory Syndrome Virus ◽  
Mobility Shift ◽  
Ribosomal Frameshifting ◽  
Protein Factor ◽  
Slippery Sequence

Abstract Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide ‘slippery’ sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both −2 and −1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus–host interactions.

scholarly journals Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0253184
Author(s):  
Hoa Quynh Do ◽  
Carla M. Bassil ◽  
Elizabeth I. Andersen ◽  
Michaela Jansen
Keyword(s):  
Tumor Cells ◽  
Plasma Membranes ◽  
In Vitro Translation ◽  
Translation System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Dimyristoyl Phosphatidylcholine ◽  
Membrane Scaffold ◽  
The Impact

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

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