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At least 415 records · Page 23Linked to original sources
PubMed1995

SL1 trans-splicing specified by AU-rich synthetic RNA inserted at the 5' end of Caenorhabditis elegans pre-mRNA.

In Caenorhabditis elegans, pre-mRNAs of many genes are trans-spliced to one of two spliced leaders, SL1 or SL2. Some of those that receive exclusively SL1 have been characterized as having at their 5' ends outrons, AU-rich sequences similar to introns followed by conventional 3' splice sites. Comparison of outrons from many different SL1-specific C. elegans genes has not revealed the presence of any consensus sequence that might encode SL1-specificity. In order to determine what parameters influence the splicing of SL1, we performed in vivo experiments with synthetic splice sites. Synthetic AU-rich RNA, 51 nt or longer, placed upstream of a consensus 3' splice site resulted in efficient trans-splicing. With all sequences tested, this trans-splicing was specifically to SL1. Thus, no information beyond the presence of AU-rich RNA at least as long as the minimum-length C. elegans intron, followed by a 3' splice site, is required to specify trans-splicing or for strict SL1 specificity.

Adenine↗
PubMed1999

RNA diversity has profound effects on the translation of neuronal nitric oxide synthase.

A comprehensive analysis of the structure of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) mRNA species revealed NOS1 to be the most structurally diverse human gene described to date in terms of promoter usage. Nine unique exon 1 variants are variously used for transcript initiation in diverse tissues, and each is expressed from a unique 5'-flanking region. The dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5'-mRNA leader sequences. Elaborate splicing patterns that involve alternatively spliced leader exons and exon skipping have been superimposed on this diversity. Highly structured nNOS mRNA 5'-untranslated regions, which have profound effects on translation both in vitro and in cells, contain cis RNA elements that modulate translational efficiency in response to changes in cellular phenotype.

5' Untranslated Regions↗
PubMed1999

Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif.

The unique catalytic characteristics of adenosine kinase (Adk) and its stage-specific differential activity pattern have made this enzyme a prospective target for chemotherapeutic manipulation in the purine-auxotrophic parasitic protozoan Leishmania donovani. However, nothing is known about the structure of the parasite Adk. We report here the cloning of its gene and the characterization of the gene product. The encoded protein, consisting of 345 amino acid residues with a calculated molecular mass of 37173 Da, shares limited but significant similarity with sugar kinases and inosine-guanosine kinase of microbial origin, supporting the notion that these enzymes might have the same ancestral origin. The identity of the parasite enzyme with the corresponding enzyme from two other sources so far described was only 40%. Furthermore, 5' RNA mapping studies indicated that the Adk gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon (spliced leader) occurring at nt -160 from the predicted translation initiation site. The biochemical properties of the recombinant enzyme were similar to those of the enzyme isolated from leishmanial cells. The intrinsic tryptophan fluorescence of the enzyme was substrate-sensitive. On the basis of a multiple protein-alignment sequence comparison and ATP-induced fluorescence quenching in the presence or the absence of KI and acrylamide, the docking site for ATP has been provisionally identified and shown to have marked divergence from the consensus P-loop motif reported for ATP- or GTP-binding proteins from other sources.

Acrylamide↗
PubMed1999

Caenorhabditis elegans mRNAs that encode a protein similar to ADARs derive from an operon containing six genes.

The Caenorhabditis elegans T20H4.4 open reading frame (GenBank accession no. U00037) predicted by Genefinder encodes a 367 amino acid protein that is 32-35% identical to the C-terminal domain of adenosine deaminases that act on RNA. We show that T20H4.4 cDNAs (GenBank accession no. AF051275) encode a larger 495 amino acid protein that is extended at its N-terminus to include a single double-stranded RNA-binding motif, and that T20H4.4 occupies the second position in a six-gene operon (5'-T20H4.5, T20H4.4, R151.8A, R151.8B, R151.7, R151.6-3'). Ten different spliced-leader (SL) sequences were found attached to T20H4.4 mRNAs, including SL1, SL2 and eight SL2-like leaders that include two new variants. Characterization of cDNAs derived from all six genes confirmed the essential features of C.elegans operons: intercistronic distances in the range of 104-257 nt between the upstream polyadenylation sites and the downstream trans -splice sites; SL2, or SL2-like leaders, attached to the downstream mRNAs. Polycistronic mRNA fragments revealed a 5'-untranslated region (5'-UTR) >705 nt. The 5'-UTR is removed in mature mRNAs from the first gene (T20H4.5) and replaced primarily by SL1, and to a lesser extent by SL2. Our study provides new information regarding operons and how they are processed.

5' Untranslated Regions↗
PubMed1992

The variant human isovaleryl-CoA dehydrogenase gene responsible for type II isovaleric acidemia determines an RNA splicing error, leading to the deletion of the entire second coding exon and the production of a truncated precursor protein that interacts poorly with mitochondrial import receptors.

Isovaleryl-CoA dehydrogenase (IVD) is a mitochondrial enzyme involved in leucine metabolism. Previous studies of fibroblasts from patients with isovaleric acidemia (IVA), an inherited defect in IVD, have revealed that IVD precursor protein produced by type II IVA cells is 3 kDa smaller than normal and is processed inefficiently to a mature form which is also 3 kDa smaller than normal. Using the polymerase chain reaction, we have identified a 90-base pair deletion encompassing bases 145-234 in type II IVD cDNA. This deletion is caused by an error in RNA splicing and predicts the in-frame deletion of 30 amino acids beginning with leucine 20 of the mature IVD. The rate of leader peptide cleavage by purified mitochondrial leader peptidases was similar for the variant and normal precursor IVDs expressed in vitro, and radiosequencing confirmed that both mature proteins contain identical amino termini. In vitro import studies showed that the efficiency of overall mitochondrial import of type II variant IVD precursor was approximately 30% of normal, as was its binding to the mitochondrial surface. Unlike its normal counterpart, the bound variant IVD precursor was readily released. These data suggest that binding of the variant protein to mitochondrial membrane receptors per se is hindered, resulting in the inefficient mitochondrial processing.

Acid-Base Equilibrium↗
PubMed1993

Complete variable region deletion in a mu heavy chain disease protein (ROUL). Correlation with light chain secretion.

In a patient affected with chronic lymphocytic leukemia with lymphocyte surface mu and kappa determinants and vacuolated bone marrow plasma cells, the serum contained polymers of a truncated mu chain and normal-sized kappa chains. These light chains were present as monomers and covalent dimers in studies performed under dissociating conditions, but they were linked by non-covalent bridges to a portion of the serum short mu chains. The patient's urine contained a kappa type Bence-Jones protein. Study of a messenger RNA and complementary DNA from blood cells showed the abnormal mu chain to lack the entire variable region, likely due to a direct splicing of the leader peptide exon onto the CH1 exon. The production of light chains, a rare event in heavy chain diseases, appears to correlate with the occurrence of a heavy chain deletion restricted to the variable domain, likely because the non-covalently linked light chains allow these unusual heavy chains to be secreted.

Amino Acid Sequence↗
PubMed1993

Promoter, spliced leader, and coding sequence for BICP4, the largest of the immediate-early proteins of bovine herpesvirus 1.

We report the complete nucleotide sequence of the bovine herpesvirus 1 (BHV-1) immediate-early gene encoding BICP4, the homolog of the ICP4 protein of herpes simplex virus. Combined with previous mapping studies, the sequence analysis revealed that the transcript for BICP4 consisted of a noncoding leader RNA (exon 1; 0.35 kb) separated by an intron (0.46 kb) from the main body (exon 2; 4.1 kb). The open reading frame for BICP4 (1343 amino acid residues) started 27 nt after the splice site and extended across exon 2 for most of its length, BICP4 contained two domains of high homology (regions 2 and 4), which had been recognized earlier to be most conserved in the ICP4 homologs of alpha-herpesviruses and to be functionally important. These domains were flanked by three regions of lower but still discernible homology. Unique features of BICP4 were two large clusters of glutamic acid residues near the end of region 3, and the displacement of a polyserine tract to region 5, which in all other ICP4 homologs residues near the end of region 1. Transient expression assays showed that BICP4 repressed its own promoter and activated other herpes-virus genes. The 8.1-kb sequence summarized here completes analysis of the inverted repeats of the BHV-1 genome; it includes a segment (2.5 kb) upstream of the BICP4 gene apparently devoid of coding sequences but containing numerous scattered transcription signals.

Alphaherpesvirinae↗
PubMed1979

Electron microscopic mapping of RNA transcribed from the late region of polyoma virus DNA.

The polyoma virus (Py) RNA species transcribed from the L DNA strand of the "late" region of the Py genome in Py-infected mouse cells have been mapped by hybridization with specific fragments of Py DNA followed by electron microscopic visualization of the hybrids. Total cellular polyadenylated Py-specific RNA molecules having an S value in the range of 16S to 20S were purified by oligodeoxythymidylic acidcellulose column chromatography, preparative hybridization with Py DNA, and sucrose gradient centrifugation. Cytoplasmic Py-specific RNA was similarily purified, except that it was not fractionated by sucrose gradient centrifugation. Hybrids of these RNA molecules and Py DNA fragments were spread for electron microscopy by either the cytochrome c technique or the bacteriophage T4 gene 32 protein method. The polyadenylic acid at the 3'-end of the RNA in the hybrids was identified by labeling with simian virus 40 DNA circles to which polybromodeoxyuridylic acid tails had been covalently attached. These experiments revealed the presence of three L DNA strand transcripts in both RNA preparations. Two of these RNA molecules were found to be spliced from chains transcribed from two noncontiguous parts of the late region. The third molecule either is a continuous transcript of the entire late region or contains a splicing feature which is too small to be reliably observed by the electron microscope methods used. The 5'-ends of the three RNA species map within a region extending from 68 to 70 map units on the Py restriction endonuclease map. Each of the two spliced molecules contains a 5'-terminal leader sequence transcribed from a DNA segment with an estimated length of 60 to 110 nuvleotides. The 3'-ends of the leaders map at 66.7 +/- 1.0 and 66.4 +/- 0.50 map units. In these molecules the 5'-ends of the other part (the main body) map at 59.4 +/- 0.90 and 49.4 +/- 2.0 map units, respectively. The 3'-termini of all three RNA species map at 24 to 25 map units.

DNA, Viral↗
PubMed1995

RNA trans-splicing in flatworms. Analysis of trans-spliced mRNAs and genes in the human parasite, Schistosoma mansoni.

Characteristics of trans-splicing in Schistosoma mansoni were examined to explore the significance and determinants of spliced leader (SL) addition in flatworms. Only a small subset of mRNAs acquire the SL. Analysis of 30 trans-spliced mRNAs and four genes revealed no discernable patterns or common characteristics in the genes, mRNAs, or their encoded proteins that might explain the functional significance of SL addition. While the mRNA encoding the glycolytic enzyme enolase is trans-spliced, mRNAs encoding four other glycolytic enzymes are not, indicating trans-splicing is not prevalent throughout this metabolic pathway. Although the 3' end of flatworm SLs contribute an AUG to mRNAs, the SL AUG does not typically serve to provide a methionine for translation initiation of reading frames in recipient mRNAs. SL RNA expression exhibits no apparent sex, tissue, or cell specificity. Trans-spliced genes undergo both cis- and trans-splicing, and the sequence contexts for these respective acceptor sites are very similar. These results suggest trans-splicing in flatworms is most likely associated either with some property conferred on recipient mRNAs by SL addition or related to some characteristic of the primary transcripts or transcription of trans-spliced genes.

Animals↗
PubMed2006

Sm core variation in spliceosomal small nuclear ribonucleoproteins from Trypanosoma brucei.

Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP-specific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification-tagging and in vitro protein-protein interaction assays. Using in vitro reconstitutions with canonical and U2-specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA-specific Sm sites.

Amino Acid Sequence↗
PubMed1995

Equine arteritis virus subgenomic RNA transcription: UV inactivation and translation inhibition studies.

The expression of the genetic information of equine arteritis virus (EAV), an arterivirus, involves the synthesis of six subgenomic (sg) mRNAs. These are 5' and 3' coterminal since they are composed of a leader and a body sequence, which are identical to the 5' and 3' ends of the genome, respectively. Previously, it has been suggested that cis-splicing of a genome-length precursor RNA is involved in their synthesis. This was reevaluated in a comparative analysis of the sg RNA synthesis of EAV, the coronavirus mouse hepatitis virus (MHV), and the alphavirus Sindbis virus. UV transcription mapping showed that the majority of the EAV sg RNAs made at later stages of infection is not derived from a genome-length precursor. However, complete independence of sg RNA synthesis from that of genomic RNA was never observed during the course of infection. The possibility that this resulted from UV irradiation-induced effects on the synthesis of the viral replicase was investigated by inhibiting translation using cycloheximide. For EAV, ongoing protein synthesis was found to be more important for the synthesis of sg RNA than for that of genomic RNA. In general, MHV transcription was extremely sensitive to translation inhibition, whereas EAV genomic RNA synthesis became independent of de novo protein synthesis late in infection.

Animals↗
PubMed1990

Plant UsnRNA genes.

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Cloning, Molecular↗
PubMed1997

Expression of functional elements inserted into the 35S promoter region of infectious cauliflower mosaic virus replicons.

We describe experiments directed towards development of cauliflower mosaic virus (CaMV) replicons for propagation of functional elements during infection of plants. Modifications and inserts were introduced into replaceable domains associated with the 35S promoter. The 35S enhancer (-208 to -56) was found to potentiate promoter activity when in reverse orientation sufficient to establish systemic infection. However, replacement of the 35S enhancer with that from the nos promoter caused loss of infectivity. A 31 bp oligonucleotide containing a polypurine tract specifying initiation of CaMV plus strand DNA synthesis was inserted into a 35S enhancer deletion mutant and propagated in plants. Analysis of progeny DNA showed the presence of an additional discontinuity at its new location in the 35S enhancer, indicating that the artificial primer had functioned correctly in an ectopic site. An intron and flanking sequences from the RNA leader of the Arabidopsis phytoene desaturase (pds) gene, when inserted into the 35S enhancer in forward orientation was very efficiently spliced during infection. The CaMV replicon carrying the pds gene fragment produced unusual infection characteristics, with plants showing early symptoms and then recovering. We conclude that infectious CaMV replicons can be used to carry a variety of elements that target both viral and host functions.

Arabidopsis↗
PubMed1989

Molecular characterization of the Trypanosoma brucei RNA polymerase I and III largest subunit genes.

We have sequenced a 7.1-kilobase fragment of the Trypanosoma brucei RNA polymerase (pol) I largest subunit gene. The 6.9-kilobase transcript from this gene is of roughly equal abundance in bloodstream- and procyclic-form trypanosomes and has a 39-nucleotide trans-spliced leader 290 nucleotides upsteam of the putative initiation codon. The 1781-amino acid trypanosome pol I polypeptide is considerably less similar to its yeast homolog than the trypanosome pol II and pol III polypeptides are to their counterparts in yeast. The yeast and trypanosome pol I polypeptides are distinguished from pol II and pol III largest subunits by two inserts, 100-200 amino acids in length, which are found in the nonconserved portions of the polypeptides. We also report our sequence of the trypanosome pol III largest subunit gene, which agrees with that reported by Köck et al. (Köck, J., Evers, R., and Cornelissen, A.W.C.A. (1988) Nucleic Acids Res. 16, 8753-8772) except for 13 nucleotide differences. Four spliced leader addition sites between 60 and 99 nucleotides upstream of the AUG initiation codon were observed for the 5.4-kilobase transcript from this gene. Four short stretches of amino acid homology between the yeast and trypanosome pol III largest subunit polypeptides were identified which appear to be specific to this class of RNA polymerase; only one short pol I-specific sequence was identified in our comparison of the yeast and trypanosome pol I largest subunit polypeptides.

Amino Acid Sequence↗
PubMed2006

Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIVmac239.

BACKGROUND: The 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIVmac239) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). We have constructed a series of deletions in the SIVmac239 leader sequence in order to determine the involvement of this region in both the packaging and dimerization of viral genomic RNA. We also assessed the impact of these deletions upon viral infectiousness, replication kinetics and gene expression in cell lines and monkey peripheral blood mononuclear cells (PBMC). RESULTS: Regions on both sides of the major splice donor (SD) were found to be necessary for the efficiency and specificity of viral genome packaging. However, stem-loop1 is critical for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the initiation site of SIV-Gag have additive effects on RNA packaging and contribute to a lesser degree to RNA dimerization. The targeted disruption of structures on both sides of the SD also severely impacts viral infectiousness, gene expression and replication in both CEMx174 cells and rhesus PBMC. CONCLUSION: In the leader region of SIVmac239, stem-loop1 functions as the primary determinant for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the translational initiation site of SIV-Gag are classified as secondary determinants and play a role in dimerization. Collectively, these data signify a linkage between the primary encapsidation determinant of SIVmac239 and RNA dimerization.

5' Untranslated Regions↗
PubMed2005

Extensive variation in the 5'-UTR of Dicer mRNAs influences translational efficiency.

The Dicer enzyme is a key component of the RNA interference pathway and also responsible for the processing of micro RNAs, non-coding RNA molecules which regulate the activity of mRNAs by antisense base pairing. Little is known about the structure and regulation of human Dicer mRNA. A comprehensive characterization of Dicer 5'-untranslated region (5'-UTR) RNA structure revealed important diversity within human Dicer mRNA transcripts. Three exon 1 variants were defined, some of which exhibited very restricted patterns of tissue distribution. A number of alternatively spliced 5'-leader exons were also noted, revealing the potential for complex post-transcriptional regulation. Surprisingly, this diversity all occurred within the 5'-UTR of Dicer mRNAs and did not affect the coding region. The Dicer mRNA 5'-UTR variants had profound effects on translational efficiency both in vitro and in transiently transfected cells. A number of major Dicer RNA species are inefficient substrates for the translational machinery.

5' Untranslated Regions↗
PubMed1999

Acanthocheilonema viteae: characterization of a molt-associated excretory/secretory 18-kDa protein.

Post-invasive third-stage larvae (pL3) of Acanthocheilonema viteae were labeled with [(35)S]-methionine in vivo, and proteins released into the culture supernatant before and during the third molt were analyzed. The molting supernatant (MSN) contained abundant proteins of 14, 18, 29, and 36 kDa. The 14- and 29-kDa proteins were exclusively found in the MSN, while the 18- and 36-kDa proteins were also produced by nonmolting pL3, albeit in much lower quantities. The cDNA for the most abundant protein in the MSN, an 18-kDa protein (Av18), was isolated by polymerase chain reaction (PCR) with reverse transcribed (RT) RNA of pL3, using information of the protein sequence. The Av18 full-length cDNA of 583 base pairs contained the 5' spliced leader sequence of nematodes, an open reading frame of 427 base pairs, and a poly(A) tail in typical distance to a polyadenylation signal. The deduced amino acid sequence encodes for a protein with a calculated size of 15.8 kDa. The N-terminus starts with a hydrophobic signal sequence and a predicted cleavage site after amino acid 20. The Av18 protein showed homologies to the deduced amino acid sequence of the larval transcripts Bm-alt-1 and alt-2 of Brugia malayi and to the Dirofilaria immitis proteins Di20/22 as well as to the Onchocerca volvulus proteins Ov-alt-1 and Ov-alt-2. Av18 is present in all parasite stages within the mammalian host, as determined by immunoblot with sera against the Escherichia coli-expressed protein and RT-PCR experiments. However, it was released into culture medium only by L3 and adult female worms. In female worms Av18 was localized in the cuticular region as demonstrated by immunofluorescent antibody tests using cryosections.

Amino Acid Sequence↗