Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting

1992 ◽  
Vol 12 (9) ◽  
pp. 4242-4248 ◽  
Author(s):  
H Ilves ◽  
O Kahre ◽  
M Speek
Keyword(s):  
Reverse Transcriptase ◽  
Rna Binding ◽  
Genomic Structure ◽  
Protein A ◽  
Open Reading Frames ◽  
Initiation Of Translation ◽  
Reticulocyte Lysate ◽  
Internal Initiation ◽  
Overlapping Open Reading Frames

The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.

scholarly journals Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting.

1992 ◽  
Vol 12 (9) ◽  
pp. 4242-4248 ◽  
Author(s):  
H Ilves ◽  
O Kahre ◽  
M Speek
Keyword(s):  
Reverse Transcriptase ◽  
Rna Binding ◽  
Genomic Structure ◽  
Protein A ◽  
Open Reading Frames ◽  
Initiation Of Translation ◽  
Reticulocyte Lysate ◽  
Internal Initiation ◽  
Overlapping Open Reading Frames

The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.

scholarly journals An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (7) ◽  
pp. 4485-4492 ◽  
Author(s):  
B A Dombroski ◽  
Q Feng ◽  
S L Mathias ◽  
D M Sassaman ◽  
A F Scott ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Consensus Sequence ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Long Terminal Repeats ◽  
Base Pairs ◽  
In Vivo Assay ◽  
Reading Frames

L1 elements constitute a highly repetitive human DNA family (50,000 to 100,000 copies) lacking long terminal repeats and ending in a poly(A) tail. Some L1 elements are capable of retrotransposition in the human genome (Kazazian, H. H., Jr., C. Wong, H. Youssoufian, A. F. Scott, D. G. Phillips, and S.E. Antonarakis, Nature (London) 332:164-166, 1988). Although most are 5' truncated, a consensus sequence of complete L1 elements is 6 kb long and contains two open reading frames (ORFs) (Scott, A. F., B. J. Schmeckpeper, M. Abdelrazik, C. T. Comey, B. O'Hara, J. P. Rossiter, T. Cooley, P. Health, K. D. Smith, and L. Margolet, Genomics 1:113-125, 1987). The protein encoded by ORF2 has reverse transcriptase (RT) activity in vitro (Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel, Science 254:1808-1810, 1991). Because L1 elements are so numerous, efficient methods for identifying active copies are required. We have developed a simple in vivo assay for the activity of L1 RT based on the system developed by Derr et al. (Derr, L. K., J. N. Strathern, and D. J. Garfinkel, Cell 67:355-364, 1991) for yeast HIS3 pseudogene formation. L1 ORF2 displays an in vivo RT activity similar to that of yeast Ty1 RT in this system and generates pseudogenes with unusual structures. Like the HIS3 pseudogenes whose formation depends on Ty1 RT, the HIS3 pseudogenes generated by L1 RT are joined to Ty1 sequences and often are part of complex arrays of Ty1 elements, multiple HIS3 pseudogenes, and hybrid Ty1/L1 elements. These pseudogenes differ from those previously described in that there are base pairs of unknown origin inserted at several of the junctions. In two of three HIS3 pseudogenes studied, the L1 RT appears to have jumped from the 5' end of a Ty1/L1 transcript to the poly(A) tract of the HIS3 RNA.

scholarly journals Human U4/U6 snRNP Recycling Factor p110: Mutational Analysis Reveals the Function of the Tetratricopeptide Repeat Domain in Recycling

2004 ◽  
Vol 24 (17) ◽  
pp. 7392-7401 ◽  
Author(s):  
Jan Medenbach ◽  
Silke Schreiner ◽  
Sunbin Liu ◽  
Reinhard Lührmann ◽  
Albrecht Bindereif
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Protein A ◽  
Tetratricopeptide Repeat ◽  
Terminal Sequence ◽  
Recognition Motifs ◽  
Tpr Domain

ABSTRACT After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

scholarly journals Elevated Levels of a U4/U6.U5 snRNP-Associated Protein, Spp381p, Rescue a Mutant Defective in Spliceosome Maturation

1999 ◽  
Vol 19 (1) ◽  
pp. 577-584 ◽  
Author(s):  
Suzanne Lybarger ◽  
Kristopher Beickman ◽  
Vicky Brown ◽  
Neetu Dembla-Rajpal ◽  
Kristin Morey ◽  
...  
Keyword(s):  
Rna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Rna Transcripts ◽  
Binding Domains ◽  
Elevated Expression ◽  
Immune Precipitation ◽  
Ts Mutant

ABSTRACT U4 snRNA release from the spliceosome occurs through an essential but ill-defined Prp38p-dependent step. Here we report the results of a dosage suppressor screen to identify genes that contribute toPRP38 function. Elevated expression of a previously uncharacterized gene, SPP381, efficiently suppresses the growth and splicing defects of a temperature-sensitive (Ts) mutantprp38-1. This suppression is specific in that enhancedSPP381 expression does not alter the abundance of intronless RNA transcripts or suppress the Ts phenotypes of otherprp mutants. Since SPP381 does not suppress aprp38::LEU2 null allele, it is clear that Spp381p assists Prp38p in splicing but does not substitute for it. YeastSPP381 disruptants are severely growth impaired and accumulate unspliced pre-mRNA. Immune precipitation studies show that, like Prp38p, Spp381p is present in the U4/U6.U5 tri-snRNP particle. Two-hybrid analyses support the view that the carboxyl half of Spp381p directly interacts with the Prp38p protein. A putative PEST proteolysis domain within Spp381p is dispensable for the Spp381p–Prp38p interaction and for prp38-1 suppression but contributes to Spp381p function in splicing. Curiously, in vitro, Spp381p may not be needed for the chemistry of pre-mRNA splicing. Based on the in vivo and in vitro results presented here, we propose that two small acidic proteins without obvious RNA binding domains, Spp381p and Prp38p, act in concert to promote U4/U5.U6 tri-snRNP function in the spliceosome cycle.

scholarly journals RNAs Extracted from Herpes Simplex Virus 1 Virions: Apparent Selectivity of Viral but Not Cellular RNAs Packaged in Virions

2001 ◽  
Vol 75 (17) ◽  
pp. 8105-8116 ◽  
Author(s):  
Maria-Teresa Sciortino ◽  
Mikiko Suzuki ◽  
Brunella Taddeo ◽  
Bernard Roizman
Keyword(s):  
Reverse Transcription ◽  
Rna Binding ◽  
Cdna Array ◽  
Open Reading Frames ◽  
Herpes Simplex Virus 1 ◽  
Cdna Arrays ◽  
Series Of Experiments ◽  
Packaging Rna ◽  
Simplex Virus

ABSTRACT Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, we investigated the presence and identity of RNAs from purified virions of herpes simple virus 1. To facilitate these studies, we designed primers for all known open reading frames (ORFs) and also constructed cDNA arrays containing probes designed to detect all known transcripts. In the first series of experiments, labeled DNA made by reverse transcription of poly(A)+ RNA extracted from infected HEp-2 or rabbit skin cells hybridized to all but two of the probes in the cDNA array. A similar analysis of the RNA extracted from purified extracellular virions derived from infected HEp-2 cells hybridized to probes representing 24 of the ORFs. In the second series of analyses, we reverse transcribed and amplified by PCR RNAs from purified intracellular or extracellular virions derived from infected HEp-2 or Vero cell lines. The positive RNAs were retested by PCR with and without prior reverse transcription to ensure that the samples tested were free of contaminating DNA. The results were as follows. (i) Only a fraction of viral ORF transcripts were represented in virion RNA, and only nine RNAs (UL10, UL34/UL35, UL36, UL42, UL48, UL51, US1/US1.5, US8.5, and US10/US11) were positive in all RT PCR assays. Of these, seven were positive by hybridization to cDNA arrays. (ii) RNA extracted from cells infected with a mutant virus lacking the US8 to US12 genes yielded results similar to those described above, indicating that US11, a known RNA binding protein, does not play a role in packaging RNA in virions. (iii) Cellular RNAs detected in virions were representative of the abundant cellular RNAs. Last, RNA extracted from virions was translated in vitro and the translation products were reacted with antibody to αTIF (VIP16). The immune precipitate contained a labeled protein with the apparent molcular weight of αTIF, indicating that at least one mRNA packaged in virions was intact and capable of being translated. The basis for the apparent selectivity in the packaging of the viral RNAs packaged in virions is unknown.

scholarly journals The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

Retrovirology ◽  
2010 ◽  
Vol 7 (1) ◽  
pp. 40 ◽  
Author(s):  
Jinwoo Ahn ◽  
In-Ja L Byeon ◽  
Sanjeewa Dharmasena ◽  
Kelly Huber ◽  
Jason Concel ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Transcription In Vitro ◽  
Hiv 1

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

1997 ◽  
Vol 44 (1) ◽  
pp. 69-77 ◽  
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.

scholarly journals Unr Is Required In Vivo for Efficient Initiation of Translation from the Internal Ribosome Entry Sites of both Rhinovirus and Poliovirus

2003 ◽  
Vol 77 (6) ◽  
pp. 3353-3359 ◽  
Author(s):  
Oréda Boussadia ◽  
Michael Niepmann ◽  
Laurent Créancier ◽  
Anne-Catherine Prats ◽  
François Dautry ◽  
...  
Keyword(s):  
Rna Binding ◽  
Es Cells ◽  
Internal Ribosomal Entry Site ◽  
Embryonic Stem ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Initiation Of Translation ◽  
Mouth Disease Virus

ABSTRACT Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (−/+) or both (−/−) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr +/+, unr +/−, and unr −/− cell lines. Translation directed by the HRV IRES was severely impaired in unr −/− cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr −/− cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.

scholarly journals Identification of minimal sequences of the Rhopalosiphum padi virus 5′ untranslated region required for internal initiation of protein synthesis in mammalian, plant and insect translation systems

2007 ◽  
Vol 88 (5) ◽  
pp. 1583-1588 ◽  
Author(s):  
Elisabetta Groppelli ◽  
Graham J. Belsham ◽  
Lisa O. Roberts
Keyword(s):  
Untranslated Region ◽  
Rhopalosiphum Padi ◽  
Open Reading Frames ◽  
Distinct Region ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Internal Initiation ◽  
Minimal Sequences ◽  
Translation Systems

Rhopalosiphum padi virus (RhPV) is a member of the family Dicistroviridae. The genomes of viruses in this family contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The RhPV 5′ IRES is functional in mammalian, insect and plant translation systems and can form 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and eIF1. Large regions of the 5′ untranslated region (UTR) can be deleted without affecting initiation-complex formation. The minimal sequences required for directing internal initiation in mammalian (rabbit reticulocyte lysate), plant (wheatgerm extract) and insect (Sf21 cells) translation systems have now been defined. A fragment (nt 426–579) from the 3′ portion of the 5′ UTR can direct translation in each of these translation systems. In addition, a distinct region (nt 300–429) is also active. Thus, unstructured regions within the 5′ UTR seem to be critical for IRES function.

The biogenesis of the cyanellae of Cyanophora paradoxa . III. In vitro synthesis of cyanellar polypeptides using separated cytoplasmic and cyanellar RNA

1989 ◽  
Vol 238 (1290) ◽  
pp. 89-102 ◽  
Keyword(s):  
Protein A ◽  
D1 Protein ◽  
Subcellular Fractionation ◽  
Coupling Factor ◽  
Cyanophora Paradoxa ◽  
In Vitro Synthesis ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Precursor Molecules

RNA from Cyanophora paradoxa was separated into cytoplasmic and cyanellar fractions by using a combination of subcellular fractionation and oligo-dT chromatography. In vitro translation of the separated cyto­plasmic and cyanellar RNAs in a rabbit reticulocyte lysate system in the presence of [ 35 S]methionine resulted in the incorporation of radiolabel into electrophoretically distinct sets of polypeptides. Monospecific and polyspecific antibodies that react with cyanellar polypeptides were used to probe the in vitro translation products by indirect immunoprecipitation by using Staphylococcus protein A conjugated to Sepharose beads. The results indicate that linker polypeptide L1 of the phycobilisome, the γ subunit of coupling factor CF1, and subunit II of PS I are syn­thesized in the cytoplasm as precursor molecules that are 5–8 kDa larger than their mature sizes. Antibodies directed against the psb A gene prod­uct (the D1 protein) precipitated a polypeptide found in the translation products of the cyanellar RNA-directed reactions, which is about 1.5 kDa larger than the mature protein.

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