PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “RNA Caps”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed1993

Structure of the mouse lysyl oxidase gene.

Lysyl oxidase, an extracellular enzyme involved in the maturation of collagen and elastin, also appears to function as a phenotypic suppressor of transformation by the ras gene product, p21. Genomic clones of the mouse lysyl oxidase gene have been isolated, analyzed, and sequenced. Lysyl oxidase appears to be a single-copy gene, organized into seven exons and six introns, and spans approximately 14 kb of the mouse genome. The gene encodes two messages, sized at about 4.8 and 3.8 kb, that differ in the length of the untranslated sequence at the 3' end of the gene. All of the 3' untranslated sequence and the polyadenylation signals are contained in exon VII; there is no evidence of alternate splicing. Primer extension and ribonuclease protection experiments revealed two sites of transcription initiation in a region with sequence motifs characteristic of a promoter, which was upstream and adjacent to the 5' untranslated sequence found in the cDNA.

3T3 Cells↗
PubMed1994

Requirement of 3'-terminal guanosine in (-)-stranded RNA for in vitro replication of cucumber mosaic virus satellite RNA by viral RNA-dependent RNA polymerase.

The 3' terminus of the (-) RNA strand in the replicative forms of several (+)-stranded RNA viruses possesses an unpaired guanosine with unknown function. This unpaired guanosine is also found at the 3' terminus of the (-) strand in the double-stranded form of two cucumoviral satellite RNAs. Using a cucumber mosaic virus (CMV) RNA-dependent RNA polymerase capable of replicating the satellite RNA in vitro, the 3'-terminal guanosine of the satellite (-) strand was shown to be an absolute requirement for satellite (+) strand synthesis. If genomic RNA synthesis of CMV and other members of the alphavirus-like superfamily that produce (-) strands terminating in an unpaired 3' guanosine follows a similar strategy, the work reported here would represent the first experimental support for the notion of 3'-terminal guanosine functioning as an essential recognition signal for viral replicases, enabling (+) strand RNA synthesis to be initiated internally from a (-) strand template.

Base Sequence↗
PubMed1998

Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism.

The variety of biogenesis pathways for small nucleolar RNAs (snoRNAs) reflects the diversity of their genomic organization. We have searched for yeast snoRNAs which are affected by the depletion of the yeast ortholog of bacterial RNase III, Rnt1. In a yeast strain inactivated for RNT1, almost half of the snoRNAs tested are depleted with significant accumulation of monocistronic or polycistronic precursors. snoRNAs from both major families of snoRNAs (C/D and H/ACA) are affected by RNT1 disruption. In vitro, recombinant Rnt1 specifically cleaves pre-snoRNA precursors in the absence of other factors, generating intermediates which require the action of other enzymes for processing to the mature snoRNA. Most Rnt1 cleavage sites fall within potentially double-stranded regions closed by tetraloops with a novel consensus sequence AGNN. These results demonstrate that biogenesis of a large number of snoRNAs from the two major families of snoRNAs requires a common RNA endonuclease and a putative conserved structural motif.

Base Sequence↗
PubMed2000

Expression of mitochondrial protein-coding genes in Tetrahymena pyriformis.

In the ciliate protozoon, Tetrahymena pyriformis, mitochondrial protein-coding genes are highly divergent in sequence, and in a number of cases they lack AUG initiation codons. We asked whether RNA editing might be acting to generate protein sequences that are more conventional than those inferred from the corresponding gene sequences, and/or to create standard AUG initiation codons where these are absent. However, comparison of genomic and cDNA sequences (the latter generated by reverse transcriptase sequencing of T. pyriformis mitochondrial mRNAs) yielded no evidence of mitochondrial RNA editing in this organism. To delineate the 5' ends of mitochondrial protein-coding transcripts, primer extension experiments were conducted. In all cases, 5' termini were found to map within a few nucleotides of potential initiation codons, indicating that T. pyriformis mitochondrial mRNAs have little or no 5' untranslated leader sequence. The pattern of strong primer extension stops suggested that both standard (AUG) and non-standard (AUU, AUA, GUG, UUG) initiation codons are utilized by the Tetrahymena mitochondrial translation system. We also investigated expression of the nad1 gene, which in both T. pyriformis and Paramecium aurelia is split into two portions that are encoded by and transcribed from different DNA strands. Northern hybridization analysis showed that the corresponding transcripts are not trans-spliced, implying that separate N-terminal and C-terminal portions of Nad1 are made in this system. Finally, in a search for primary transcripts, we isolated from a T. pyriformis mitochondrial fraction several small RNAs that were reproducibly labeled by incubation in the presence of [alpha-(32)P]GTP and guanylyltransferase. Partial sequence information revealed that none of these cappable RNAs is encoded in the T. pyriformis mitochondrial genome.

5' Untranslated Regions↗
PubMed1998

Cloning and expression of a P2y purinoceptor from the adult bovine corpus callosum.

We have isolated an ATP receptor clone by screening a bovine corpus callosum cDNA library. The clone includes one open reading frame encoding for a protein of 373 amino acid residues (42 kDa) which belongs to the G-protein-coupled receptor superfamily. In Xenopus oocytes, this clone expressed an ATP receptor that triggered an oscillatory current in response to ATP (EC50 approximately 20 microM). This current may have resulted from the activation of phospholipase C, the formation of inositol trisphosphate, and the release of Ca2+, which then opens Cl- channels. The order of potency for ATP receptor agonists was 2-MeSATP approximately ATP >> alpha, beta-MeATP > adenosine, and UTP was ineffective, a pharmacological profile consistent with that of a P2y purinoceptor. Northern blot analysis of mRNAs from various bovine brain tissues showed that the gene is expressed in the cerebellum, medulla, corpus callosum, hippocampus, superior colliculus, frontal cortex, and retina. In situ RT-PCR showed transcripts of the gene in many glial cells and endothelial cells of the corpus callosum. The cloned receptor may play an important role in neuron-glial signaling under normal and pathological conditions.

Adenosine Triphosphate↗
PubMed1997

In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5' untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers.

The 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) RNA forms a highly ordered secondary structure that has been implicated in controlling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5'UTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron, the chloramphenicol acetyltransferase gene, designed to block ribosomal scanning, the CVB3 5'UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (IRES). This finding was further supported by the data on predicted secondary structures within the 5'UTR. Of special note, analysis of various deletion mutants demonstrated that the IRES of CVB3 is located roughly at stem-loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (stem-loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings, in vitro translation inhibition assays using RNA fragments of the 5'UTR as inhibitor were performed. Both antisense and sense RNA segments transcribed from these two cis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking ribosome and initiation factor binding within the 5'UTR via specific hybridization to their viral RNA target sequences, while sense sequences may function by competing with viral RNA for ribosomes and/or translation initiation factors. These cis-acting translational elements may serve as potential targets for the antiviral action of oligomers.

Animals↗
PubMed1998

Identification of viral genes required for cell-to-cell movement of southern bean mosaic virus.

Inoculation of Vigna unguiculata (cowpea) with transcripts synthesized in vitro from a genome-length cDNA clone of the cowpea strain of southern bean mosaic virus (SBMV-C) resulted in a systemic SBMV-C infection of this host. Capped RNA was about five times more infectious than uncapped RNA as determined by a local lesion assay. The SBMV-C cDNA clone was also used for mutagenesis of the four SBMV-C open reading frames (ORFs). ORF1, ORF3, and coat protein (CP) mutants were not infectious in cowpea. Electroporation of cowpea protoplasts with mutant transcripts demonstrated that the ORF1, ORF3, and CP gene products were not required for SBMV-C RNA synthesis, and the ORF1 and ORF3 gene products were not required for SBMV-C assembly. From these results, it was concluded that the ORF1 and ORF3 proteins and the CP are required for SBMV-C cell-to-cell movement. One of the ORF3 mutants pSBMV2-UAA1833 contained a nonsense codon between the predicted -1 ribosomal frameshift site (SBMV-C nucleotides 1796-1802) and a potential ORF3 translation initiation codon at SBMV-C nucleotide 1895. The lack of infectivity of this mutant suggested that ORF3 was expressed by a -1 ribosomal frameshift in ORF2 rather than by initiation of translation at nucleotide 1895.

Electroporation↗
PubMed2003

A shine-dalgarno-like sequence mediates in vitro ribosomal internal entry and subsequent scanning for translation initiation of coxsackievirus B3 RNA.

Translation initiation of coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. However, the details of ribosome-template recognition and subsequent translation initiation are still poorly understood. In this study, we have provided evidence to support the hypothesis that 40S ribosomal subunits bind to CVB3 RNA via basepairing with 18S rRNA in a manner analogous to that of the Shine-Dalgarno (S-D) sequence in prokaryotic systems. We also identified a new site within both the 18S rRNA and the polpyrimidine-tract sequence of the IRES that allows them to form stronger sequence complementation. All these data were obtained from in vitro translation experiments using mutant RNAs containing either an antisense IRES core sequence at the original position or site-directed mutations or deletions in the polypyrimidine tract of the IRES. The mutations significantly reduced translation efficiency but did not abolish protein synthesis, suggesting that the S-D-like sequence is essential, but not sufficient for ribosome binding. To determine how ribosomes reach the initiation codon after internal entry, we created additional mutants: when the authentic initiation codon at nucleotide (nt) 742 was mutated, a 180-nt downstream in-frame AUG codon at nt 922 is able to produce a truncated smaller protein. When this mutation was introduced into the full-length cDNA of CVB3, the derived viruses were still infectious. However, their infectivity was much weaker than that of the wild-type CVB3. In addition, when a stable stem-loop was inserted upstream of the initiation codon in the bicistronic RNA, translation was strongly inhibited. These data suggest that ribosomes reach the initiation codon from the IRES likely by scanning along the viral RNA.

Base Sequence↗
PubMed1998

Regulation of mRNA 1 expression by the 5'-untranslated region (5'-UTR) of the coronavirus infectious bronchitis virus (IBV).

In this report, we show that expression of the coronavirus IBV mRNA1 is regulated by its 5'-UTR. Evidence presented demonstrates that the IBV sequence from nucleotide 1 to 1904 directs very inefficient synthesis of a product of approximately 43 kDa. Deletion of either the first 362 bp or the whole part of the 5'-UTR, however, dramatically increased the expression of the 43 kDa protein species. The mechanisms involved were investigated by two different approaches. Firstly, translation of the same construct in the presence of [3H]-leucine ruled out the possibility that initiation of small reading frames from non-AUG codons located in the 5'-UTR may compete with the authentic AUG initiation codon, and therefore inhibit the expression of ORF 1a. Secondly, expression and deletion analyses of a dicistronic construct showed that translation of the 43 kDa protein was initiated by ribosome internal entry mechanism. These studies suggest that a 'weak' ribosome internal entry signal is located in the 5'-UTR and is involved in the regulation of mRNA1 expression.

5' Untranslated Regions↗
PubMed2005

Advances in genome-wide protein expression using the wheat germ cell-free system.

In the current post-genomic era, cell-free translation platforms are gaining importance in structural as well as functional genomics. They are based on extracts prepared from Escherichia coli cells, wheat germ, or rabbit reticulocytes, and when programmed with any mRNA in the presence of energy sources and amino acids, can synthesize the respective protein in vitro. Among the cell-free systems, the wheat germ-based translation system is of special interest due to its eukaryotic nature and robustness. This chapter outlines the existing protein production platforms and their limitations, and describes the basic concept of the wheat germ-based cell-free system. It also demonstrates how the conventional wheat germ system can be improved by eliminating endogenous inhibitors, by using an expression vector specially designed for this system and polymerase chain reaction-directed protein synthesis directly from cDNAs in a bi-layer translation system. Finally, a robotic procedure for translation based on the wheat germ extract and bi-layer cell-free translation is described.

3' Untranslated Regions↗
PubMed1994

Tobacco cytochrome b5: cDNA isolation, expression analysis and in vitro protein targeting.

A full-length clone encoding cytochrome b5 has been isolated from a tobacco leaf cDNA library in lambda gt11 by PCR using degenerate primers. This cDNA encodes a protein of 139 residues which exhibits a high degree of homology to other cytochrome b5s, the message for which is expressed predominantly in developing seeds and in pigmented flower tissue. In the developing tobacco seed the mRNA is abundant at very early stages (< 10 days after flowering). Southern analysis indicated that more than one gene encodes cytochrome b5 in the tobacco genome. In vitro transcription and translation studies of the cDNA indicated that the protein inserts into the ER membrane by a non-SRP-mediated pathway and that the C-terminus of the protein is required for targeting and insertion.

Amino Acid Sequence↗
PubMed1996

Characterization of the potato mitochondrial transcription unit containing 'native' trnS (GCU), trnF (GAA) and trnP (UGG).

In order to identify the sequences promoting the expression of plant mitochondrial tRNA genes, we have characterized the trnS (GCU), trnF (GAA) and trnP (UGG) transcription unit of the potato mitochondrial genome. These three tRNA genes were shown to be co-transcribed as a 1800 nt long primary transcript. The transcription initiation site located 305 to 312 nt upstream of trnS is surrounded by a purine-rich region but does not contain the consensus motif proposed as a promoter element in dicotyledonous plants. Differential labelling of potato mitochondrial RNA with either guanylyltransferase or T4 polynucleotide kinase suggests that this site corresponds to the unique functional region responsible for the transcription of the three tRNA genes. The initiation site recently found upstream of Oenothera mitochondrial trnF does not seem to be used in potato mitochondria, although a very similar sequence is present 317 nt upstream of the corresponding potato gene. Major processing sites were identified at the 3' end of each tRNA gene. Another processing site, surrounded by a double hairpin structure, is located 498 nt downstream of trnP in stretch of 10 A residues. As judged from northern experiments, this region is close to the determination site of this transcription unit.

Base Sequence↗
PubMed1995

The rps3-rpl16-nad3-rps12 gene cluster in rice mitochondrial DNA is transcribed from alternative promoters.

The two gene clusters rps3-rpl16 and nad3-rps12 are separated from each other in the mitochondrial genome and are expressed as the individual transcription units in many plants. In rice mitochondrial DNA (mtDNA), the four genes rps3, rpl16, nad3 and rps12 are located within a region of 6 kbp. Northern-blot analysis revealed that a large transcript (6.6 kb) hybridized to both the rps3-rpl16 and the nad3-rps12 gene clusters. Using RT-PCR, we amplified a fragment of anticipated size (790 bp) from two primers that corresponded to sequences in the coding regions of rpl16 and nad3, demonstrating that at least two of the four genes, namely rpl16 and nad3, were co-transcribed. These results together indicated that all four genes, namely, rps3, rpl16, nad3 and rps12, were co-transcribed in rice mitochondria. Transcription initiation sites were determined by an in vitro capping/ribonuclease protection assay and primer extension analysis. Two initiation sites were identified in the rps3-rpl16-nad3-rps12 gene cluster: one was located upstream of rps3 and the other was located between rpl16 and nad3. This evidence indicates that the rps3-rpl16-nad3-rps12 gene cluster is transcribed from two alternative promoters.

Base Sequence↗