Elements regulating Potato leafroll virus sgRNA1 translation are located within the coding sequences of the coat protein and read-through domain.

Translation of viral proteins from subgenomic RNAs (sgRNAs) is a common strategy among positive-stranded RNA viruses. Unlike host mRNA, sgRNA of Potato leafroll virus (PLRV) does not possess a cap at its 5' end nor a poly(A) tail at the 3' terminus, both of which are known to be crucial for translation of RNA in eukaryotic cells. Here, we demonstrate, that in wheat germ extract (WGE) truncation of the sgRNA1 5' UTR increases translation efficiency, as it has previously been observed in rabbit reticulocyte lysate (RRL), whereas removal of the 3' UTR does not affect translation. We also describe two regulatory elements located within the coding sequence of the coat protein (CP) gene and its read-through domain (RTD) and are responsible for regulation of in vitro translation of the PLRV sgRNA1. The frst element is composed of the purine sequence AAAGGAAA located between the AUG codons of the CP and 17K genes. Deletion of this domain or its substitution by pyrimidines reduced by half the translation of both genes, whereas deletion of the RTD resulted in a 3.6-fold reduction in translation efficiency. This is the first report of translation regulatory elements of plant viruses located within a coding region.

Download Full-text

Nucleotide sequence of the potato leafroll virus coat protein gene

Nucleic Acids Research ◽

10.1093/nar/17.4.1768 ◽

1989 ◽

Vol 17 (4) ◽

pp. 1768-1768 ◽

Cited By ~ 2

Author(s):

B. Prill ◽

E. Maiss ◽

U. Timpe ◽

R. Casper

Keyword(s):

Nucleotide Sequence ◽

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Potato Leafroll Virus ◽

Virus Coat Protein ◽

Virus Coat ◽

Leafroll Virus

Download Full-text

Translational regulation of human beta interferon mRNA: association of the 3' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3487-3493.1993 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3487-3493

Author(s):

G Grafi ◽

I Sela ◽

G Galili

Keyword(s):

Translational Regulation ◽

Binding Activity ◽

Rnase H ◽

Translational Efficiency ◽

Translation Efficiency ◽

Protection Assay ◽

Rich Region ◽

Reticulocyte Lysate ◽

Rna Probe

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.

Download Full-text

Nucleotide sequence and in vitro translation of the coat protein gene of cymbidium mosaic virus

Virus Genes ◽

10.1007/bf01702396 ◽

1993 ◽

Vol 7 (2) ◽

pp. 157-170 ◽

Cited By ~ 2

Author(s):

Koh-Koh Neo ◽

Sek-Man Wong ◽

Mian Wu

Keyword(s):

Nucleotide Sequence ◽

Coat Protein ◽

Mosaic Virus ◽

Coat Protein Gene ◽

Protein Gene ◽

In Vitro Translation ◽

Cymbidium Mosaic Virus

Download Full-text

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1848-1855.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1848-1855

Author(s):

G M Small ◽

T Imanaka ◽

H Shio ◽

P B Lazarow

Keyword(s):

Candida Tropicalis ◽

Proteolytic Processing ◽

In Vitro Translation ◽

Moderate Number ◽

Brij 35 ◽

Reticulocyte Lysate ◽

Molecular Information ◽

Acyl Coenzyme A

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

Download Full-text

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00058.2014 ◽

2015 ◽

Vol 308 (1) ◽

pp. L58-L75 ◽

Cited By ~ 3

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.

Download Full-text

Potyvirus Genome-linked Protein, VPg, Directly Affects Wheat Germ in Vitro Translation

Journal of Biological Chemistry ◽

10.1074/jbc.m703356200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1340-1349 ◽

Cited By ~ 67

Author(s):

Mateen A. Khan ◽

Hiroshi Miyoshi ◽

Daniel R. Gallie ◽

Dixie J. Goss

Keyword(s):

Translation Initiation ◽

Wheat Germ ◽

Mrna Translation ◽

Kinetic Studies ◽

Dissociation Rate ◽

In Vitro Translation ◽

Translation Efficiency ◽

Viral Mrna ◽

Initiation Factors

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show ∼2.6-fold faster association for eIFiso4F·VPg as compared with eIF4F·VPg. The dissociation rate was ∼2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F·VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.

Download Full-text

Ribozymes that cleave potato leafroll virus RNA within the coat protein and polymerase genes

Journal of General Virology ◽

10.1099/0022-1317-71-10-2257 ◽

1990 ◽

Vol 71 (10) ◽

pp. 2257-2264 ◽

Cited By ~ 16

Author(s):

J. W. Lamb ◽

R. T. Hay

Keyword(s):

Coat Protein ◽

Potato Leafroll Virus ◽

Virus Rna ◽

Leafroll Virus

Download Full-text

Studies on the translation mechanism of subgenomic RNA of potato leafroll virus.

Acta Biochimica Polonica ◽

10.18388/abp.1997_4441 ◽

1997 ◽

Vol 44 (1) ◽

pp. 69-77 ◽

Cited By ~ 1

Author(s):

M Juszczuk ◽

W Zagórski-Ostoja ◽

D M Hulanicka

Keyword(s):

Stop Codon ◽

In Vitro Transcription ◽

Open Reading Frames ◽

Translation System ◽

Translation Efficiency ◽

Subgenomic Rna ◽

Expression Of Genes ◽

Initiation Of Translation ◽

Leafroll Virus

The expression of open reading frames located on the subgenomic RNA (sgRNA) has been studied in an in vitro transcription and translation system. The obtained results indicate: a) translation of sgRNA occurs according to the scanning model since the insertion of palindrome (delta G0 = -61 kcal/mol) prevents the initiation of translation; b) ORF6 is translated by suppression of the stop codon separating ORF4 from ORF6 and the presence of suppressor tRNA is necessary for the readthrough; c) the presence of leader sequence of sgRNA (212 nucleotides) decreases the translation efficiency of ORFs located downstream and it affects the ratio of products of ORF4 and ORF5; d) 3'UTR does not influence on an expression of genes located on the sgRNA.

Download Full-text

Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

10.1101/092445 ◽

2016 ◽

Cited By ~ 2

Author(s):

Molly Gasperini ◽

Gregory M. Findlay ◽

Aaron McKenna ◽

Jennifer H. Milbank ◽

Choli Lee ◽

...

Keyword(s):

Large Scale ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Mendelian Disease ◽

Regulatory Sequences ◽

Coding Region ◽

Endogenous Gene ◽

Distal Regulatory Elements

AbstractThe extent to which distal non-coding mutations contribute to Mendelian disease remains a major unknown in human genetics. Given that a gene’s in vivo function can be appropriately modeled in vitro, CRISPR/Cas9 genome editing enables the large-scale perturbation of distal non-coding regions to identify functional elements in their native context. However, early attempts at such screens have relied on one individual guide RNA (gRNA) per cell, resulting in sparse mutagenesis with minimal redundancy across regions of interest. To address this, we developed a system that uses pairs of gRNAs to program thousands of kilobase-scale deletions that scan across a targeted region in a tiling fashion (“ScanDel”). As a proof-of-concept, we applied ScanDel to program 4,342 overlapping 1- and 2- kilobase (Kb) deletions that tile a 206 Kb region centered on HPRT1, the gene underlying Lesch-Nyhan syndrome, with median 27-fold redundancy per base. Programmed deletions were functionally assayed by selecting for loss of HPRT1 function with 6-thioguanine. HPRT1 exons served as positive controls, and all were successfully identified as functionally critical by the screen. Remarkably, HPRT1 function appeared robust to deletion of any intergenic or deeply intronic non-coding region across the 206 Kb locus, indicating that proximal regulatory sequences are sufficient for its expression. A sparser mutagenesis screen of the same 206 Kb with individual gRNAs also failed to identify critical distal regulatory elements. Although our screen did find programmed deletions and individual gRNAs with putative functional consequences that targeted exon-proximal non-coding sequences (e.g. the promoter), long-read sequencing revealed that this signal was driven almost entirely by rare, unexpected deletions that extended into exonic sequence. These targeted validation experiments defined a small region surrounding the transcriptional start site as the only non-coding sequence essential to HPRT1 function. Overall, our results suggest that distal regulatory elements are not critical for HPRT1 expression, and underscore the necessity of comprehensive edited-locus genotyping for validating the results of CRISPR screens. The application of ScanDel to additional loci will enable more insight into the extent to which the disruption of distal non-coding elements contributes to Mendelian diseases. In addition, dense, redundant, large-scale deletion scanning with gRNA pairs will facilitate a deeper understanding of endogenous gene regulation in the human genome.

Download Full-text