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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↗
PubMed2003

Sequences within both the 5' untranslated region and the gag gene are important for efficient encapsidation of Mason-Pfizer monkey virus RNA.

It has previously been shown that the 5' untranslated leader region (UTR), including about 495 bp of the gag gene, is sufficient for the efficient encapsidation and propagation of Mason-Pfizer monkey virus (MPMV) based retroviral vectors. In addition, a deletion upstream of the major splice donor, SD, has been shown to adversely affect MPMV RNA packaging. However, the precise sequence requirement for the encapsidation of MPMV genomic RNA within the 5' UTR and gag remains largely unknown. In this study, we have used a systematic deletion analysis of the 5' UTR and gag gene to define the cis-acting sequences responsible for efficient MPMV RNA packaging. Using an in vivo packaging and transduction assay, our results reveal that the MPMV packaging signal is primarily found within the first 30 bp immediately downstream of the primer binding site. However, its function is dependent upon the presence of the last 23 bp of the 5' UTR and approximately the first 100 bp of the gag gene. Thus, sequences that affect MPMV RNA packaging seem to reside both upstream and downstream of the major splice donor with the downstream region responsible for the efficient functioning of the upstream primary packaging determinant.

5' Untranslated Regions↗
PubMed1993

Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains.

The 5' region of HIV-1 RNA contains functional elements involved in key steps of the retroviral cycle, such as genomic RNA transcription, splicing, translation, dimerization or initiation of reverse transcription. In the present work, we investigated the conformation of the first 500 nucleotides covering the RNA leader and the 5' gag coding sequences of HIV-MAL, using chemical probing. We provide detailed information on almost each nucleotide at one of their Watson-Crick positions and on position N-7 of purines. Experiments were conducted on two in vitro transcribed RNA fragments (1 to 707 and 1 to 311). A secondary structure model was derived by combining the experimental data, computer predictions and sequence comparison. Under conditions favoring dimerization (high salt concentration), HIV-1 RNA folds into independent structural domains that can be related to defined functional regions. The first domain corresponds to TAR forming a stable stem-loop. Intrinsic structural features are found to stabilize the TAR hairpin loop. The second domain (nucleotides 56 to 299) contains the PBS sequence, which is located in a stable subdomain constrained by a four stem junction (nucleotides 139 to 218). Although the MAL isolate has an insertion near the PBS, probably resulting from the duplication of a 23-nucleotide sequence, the structural organization of this subdomain is conserved in all other HIV-1 isolates. The third domain (nucleotides 300 to 404) contains the splice donor site, packaging and dimerization elements and the AUG initiation codon of gag. A major result is the structural versatility of this region. Two mutually exclusive structures, both equally in agreement with probing data, could modulate the different functions involving this domain. The reduced accessibility of the gag translational initiation site possibly accounts for the low efficiency of the in vitro translation of the dimer. Finally, the 5' gag coding sequences form a metastable domain.

Animals↗
PubMed1990

Transcription vectors that facilitate the identification and mapping of RNA splice sites in genomic DNA.

Two transcription vectors were constructed that can identify the splice sites at exon-intron boundaries of inserted DNA fragments possessing the complementary splice site. One vector contains the 5' splice donor site and flanking exon-intron sequences from the 3' end of the adenovirus first late leader. The other vector contains the 3' splice acceptor site and the branch acceptor site, plus the flanking exon-intron sequences from the 5' end of the adenovirus second late leader. Both vectors contain a multiple cloning site for insertion of DNA fragments. DNA fragments supplying the complementary splice site, including the adjacent exon and intron sequences, were inserted into the vectors. The vectors were used as templates for the synthesis of chimeric RNA transcripts that were spliced in in vitro splicing extracts. Chimeric transcripts from the vectors containing complementary splice site boundary regions from the human growth hormone gene were accurately spliced in vitro. A splice site from a human growth hormone intron that is not normally spliced in vitro was spliced when paired with an adenovirus splice site. These vectors can be used to identify splice sites and to determine the lengths of exons and their attached introns within a DNA fragment of unknown coding content.

Base Sequence↗
PubMed1996

Nematode spliced leaders--ubiquity, evolution and utility.

Most nematode messenger RNAs (mRNAs) have at their 5' end a common 22 nucleotide leader sequence, the trans-spliced leader or SL1. The presence of this leader on some but not all mRNAs raises several questions: What is the role of the spliced leader in mRNA maturation, stability and translation? Why do some genes have a spliced leader and others not? What is the evolutionary origin of this trans-splicing mechanism? Recently, additional trans-spliced leaders (SL2, 3, 4, 5) have been described. What role do these variants play in nematode gene expression? While definitive answers to these questions remain elusive, it is clear that the spliced leader will significantly facilitate the cloning and sequence analysis of most nematode mRNAs.

Animals↗
PubMed1990

Polyomavirus late pre-mRNA processing: DNA replication-associated changes in leader exon multiplicity suggest a role for leader-to-leader splicing in the early-late switch.

Polyomavirus late mRNAs contain at their 5' ends multiple, tandem repeats of a 57-base noncoding sequence, the late leader, whose sequence appears only once in the viral genome. Pre-mRNA molecules are processed by a pathway that includes the splicing of late leader exons to each other in giant, multigenome-length precursors which are the result of inefficient transcription termination. We have devised a method involving reverse transcription and the polymerase chain reaction to determine the number of tandem late leader units on polyomavirus late RNA molecules. Using this technique, we have shown that each class of late viral mRNA (mVP1, mVP2, and mVP3) consists of molecules with between 1 and 12 tandem leader units at their 5' ends. Importantly, single-leader RNAs are underrepresented in both the cytoplasm and the nucleus, suggesting that single-leader primary transcripts are preferentially degraded in the nucleus. In addition, the average number of leaders on late RNAs increases in the presence of DNA replication. Taken together with previous work from our laboratory, the results presented here are consistent with a model for the control of late gene expression at the level of RNA splicing and stability which is in turn controlled by the efficiency of transcription termination.

Animals↗
PubMed1995

Orphon spliced-leader sequences form part of a repetitive element in Angiostrongylus cantonensis.

In nematodes, the 22 nucleotide (nt) spliced leader (SL) is normally encoded by a multi-copy, tandemly reiterated SL gene and is trans-spliced from SL-RNA onto the 5' end of a subset of mRNAs. We have found that the SL is also encoded at multiple (> 100) orphon genomic sites in the parasitic nematode Angiostrongylus cantonensis. At these sites the sequence forms part of a 198 bp repetitive element (designated con-198). Transcription from two genomic elements that contain the con-198 sequence has been characterised. At one element (G-2) an approximately 850 nt RNA with an internal SL is transcribed. At the other (G-1), transcription takes place 3 kb downstream of the con-198 sequence.

Angiostrongylus cantonensis↗
PubMed2003

New Otx2 mRNA isoforms expressed in the mouse brain.

The mouse Otx2 gene is essential throughout head and brain development, from anterior-posterior polarity determination and neuroectoderm induction to post-natal sensory organ maturation. These numerous activities must rely on a very finely tuned regulation of expression. In order to understand the molecular control of the Otx2 gene, we set out to isolate its promoter. During this quest, we identified three remote transcription start sites, two defining two new upstream exons and one mapping within the previously reported first exon. The three transcripts differed in their 5' non-coding region but encoded the same protein. The transcription start nucleotides of each mRNA species have been mapped by RNase protection assays and by an RNA circularization technique. We have demonstrated that they are all used and linked to functional promoters. In addition to leader versatility, we also detected alternative splicing within the coding sequence that gives rise to a new protein endowed with an 8 amino-acid insertion upstream of the homeodomain. Combined analysis of the relative abundance of Otx2 mRNA isoforms in representative tissues and in situ hybridization studies revealed distinct spatial and temporal, although partially overlapping, expression patterns of the mRNA isoforms. These findings provide new clues to a better understanding of the relationships between Otx2 gene architecture and its complex regulatory requirements.

5' Untranslated Regions↗
PubMed2003

Evidence for posttranscriptional regulation of the multi K homology domain protein vigilin by a small peptide encoded in the 5' leader sequence.

Vigilin, a K homology (KH) protein has been found in all eukaryotic species studied. It has a unique structure of 14-15 consecutively arranged KH domains which apparently mediate RNA-protein binding. Cloning and sequencing of the mouse vigilin cDNA confirmed that the amino acid sequences of vertebrate vigilins are highly conserved and contain conserved sequence motifs of nuclear import and export sequences. The human and murine vigilin mRNAs carry two alternatively spliced 5' exons. In the 5' leader region of one of the splice variants, variant 1A, we found an upstream open reading frame (uORF) highly conserved between mouse and human. Here we present for the first time evidence that a 13 amino acid long peptide encoded by this uORF is an inhibitor of vigilin expression operating on a posttranscriptional level. We propose that the two structurally different 5' leader sequences of the human vigilin mRNA are involved in the regulation of vigilin biosynthesis.

5' Untranslated Regions↗
PubMed1993

Gene structure and alternative splicing of XFG 5-1, a X. laevis Zn finger protein with RNA homopolymer binding activity.

We describe the fine-structure of the Xenopus laevis XFG 5-1 gene which codes for an RNA homopolymer binding Zn finger protein of the FAR (Finger Associated Repeat) subfamily. The gene contains six exons, i.e., a leader exon (I), four exons (II-V), each of them encoding one individual copy of the FAR repeat, and one exon (VI) encoding the linker as well as the complete multifinger-region of the corresponding protein. Isolation and characterization of distinct cDNAs revealed that primary transcripts are alternatively spliced, thereby leading either to mRNAs containing different copy numbers of the FAR repeat or, by utilization of an alternative splice acceptor site in front of exon VI, to an extension of the linker region between the FAR repeats and the multifinger domain. We also describe the fine-structure of a closely related gene, termed XFG 5-2, which is located downstream to the XFG 5-1 gene. The general structural organization in both genes is identical, but point mutations should give rise to a XFG 5-2 protein with a different number of Zn finger units.

Alternative Splicing↗
PubMed1989

The gene for the rat mast cell high affinity IgE receptor alpha chain. Structure and alternative mRNA splicing patterns.

We have isolated and characterized the gene coding for the alpha subunit of the rat mast cell high affinity IgE receptor. The gene comprises five exons and four introns and spans approximately 6.6 kilobases. The leader sequence is encoded by two exons, the second of which is only 21 base pairs long. The third and fourth exons each code for repeating immunoglobulin-like extracellular domains. A hydrophobic transmembrane domain and positively charged cytoplasmic tail are encoded by a single final exon. The sequence of the 5'-flanking region was determined, and the major transcription initiation site was mapped. The structure of the gene suggests that two minor species of RNA produced by rat basophilic leukemia cells are due to alternative splicing. In one case, the small second exon is removed entirely, and, in the other, 163 base pairs at the 3' end of the fourth exon are removed. The latter species of RNA suggests the possibility of a secreted form of the alpha chain of the receptor. A third variant species of RNA, lacking a leader sequence, may be due to use of an alternative transcription initiation site.

Animals↗
PubMed1979

Messenger RNA for the Ad2 DNA binding protein: DNA sequences encoding the first leader and heterogenity at the mRNA 5' end.

During the early stage of Ad2 infection of human cells, RNA is transcribed from five separate transcription units. Early region II encodes the mRNA for a 72K single-stranded DNA binding protein (DBP) which functions in DNA replication. This report describes the structure of the first leader of the DBP mRNA and the flanking sequences in the DNA. The leader, labeled in vivo with 32P, was isolated by DNA filter hybridization to the viral restriction fragment Eco RI F, and its RNAase T1 and RNAase A oligonucleotides were analyzed by RNA fingerprinting techniques. Comparison of this RNA sequence information with the DNA sequence of Eco RI F has located a 68 nucleotide region of the Hae III C subfragment at coordinate 75.1 that encodes the leader. This position is near the coordinate to which nascent chain analysis and ultraviolet transcription mapping have mapped an RNA initiation site, or promoter, for the DBP mRNA. The DNA sequence that overlaps the leader on the 3' side contains a donor sequence for splicing this leader to a second downstream leader. The splicing sequence shows a seven base homology with the comparable structure of the Ad2 major late leader, and a mouse globin mRNA splicing sequence. The DNA sequence upstream from the cap, the region oof the potential promoter site does not, however, contain a "TA-TAAA"-type homology of the sort noted by D. Hogness, M. Goldberg and R. Lifton (personal communication) for many cellular transcription units, and by other investigations for the Ad2 major late transcription unit. Also, the leader is found with two distinct capped 5' termini, 7meGpppA and 7meGpppG, which are encoded at adjacent positions in the DNA and thus are from mRNAs which are staggered by one nucleotide in length at the 5' end. The staggering at the 5' terminus and the lack of the upstream homolgy distinguish the DBP mRNA from many viral and cellular messenger. In both these respects, however, the DBP mRNA resembles the late messengers of SV40 and polyoma viruses. In this paper, we discuss the implications of these findings for the mechanism of specifying mRNA 5' ends.

Adenoviruses, Human↗
PubMed1998

Molecular cloning, functional expression, and characterization of pyruvate dehydrogenase kinase from anaerobic muscle of the parasitic nematode Ascaris suum.

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum. A cDNA coding for an A. suum pyruvate dehydrogenase kinase (APDK) has been cloned and sequenced from poly(A)+ RNA isolated from adult A. suum muscle.2 APDK exhibited significant sequence identity to mammalian PDKs. Nucleotide sequence analysis of the APDK cDNA revealed a 22-nucleotide spliced leader, characteristic of many nematode mRNAs, a 5'-UTR of 6 nucleotides, an open reading frame of 1197 nucleotides, and a 3'-UTR of 101 nucleotides that included a putative polyadenylation signal. The open reading frame predicted a protein of 399 amino acids with a molecular weight of 45,402 that included a putative 18-aminoacid leader peptide. Recombinant APDK (rAPDK) was functionally expressed in Escherichia coli with a his tag at its N-terminus and purified to apparent homogeneity on Ni-NTA-agarose. Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the presence of APDK-deficient adult A. suum muscle PDC presumably by the binding of APDK to the dihydrolipoyl transacetylase (E2) core of the complex. After binding to the core, rAPDK activity was stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios within the same ranges as observed for the native APDK. Immunoblotting suggested that native APDK focused as a series of 43-kDa spots (pI 6.1-6.8) on two-dimensional gels of the purified adult A. suum muscle PDC.

Amino Acid Sequence↗
PubMed1983

Alternative splicing pathways exist in the formation of adenoviral late messenger RNAs.

Total rapidly labeled RNA was extracted from nuclei of HeLa cells late after infection with adenovirus type 2. Most of this nuclear RNA is transcribed from the major late transcription unit (16.2 to 100.0 map units (m.u.)). To study the cleavage reactions that are involved in RNA splicing, we used the S1 nuclease mapping technique with HindIII B (16.8 to 31.5 m.u.) and XhoI F (15.5 to 22.4 m.u.) restriction fragments as DNA probes. The S1 mapping data showed that both intron 1 (16.3 to 19.1 m.u.) and intron 2 (19.4 to 26.2 m.u.) can be excised in more than one step. Kinetic labeling and pulse--chase experiments demonstrated that certain cleavage sites in the RNA are used within three minutes after the start of transcription, while other cleavages occur only after a significant time-lag. The use of 5.6-dichloro-1-beta-D-ribofuranosylbenzimidazole enabled us to label the nuclear RNA exclusively from the 5' end. Such an approach showed that the first cleavages are introduced in the nascent RNA before the RNA polymerase has passed more than 2000 nucleotides beyond the cleavage site. The chronology of the appearance of processing intermediates that results from cleavage of the RNA indicates that preferentially intron 1 is removed before intron 2. This is an agreement with the finding that leader 1 is ligated to leader 2 before ligation of leader 2 to leader 3 takes place. However, we have found that alternative splicing pathways exist in the excision of introns 1 and 2, demonstrating that cleavage in intron 2 may occur before intron 1 is attacked by the splicing enzymes.

Adenoviruses, Human↗
PubMed2004

Molecular dissection of the rDNA array and of the 5S rDNA gene in Meloidogyne artiellia: phylogenetic and diagnostic implications.

The sequence of a 13.423 nucleotide genomic fragment has been determined for the plant parasitic nematode Meloidogyne artiellia. It contains an entire rDNA cluster, the bordering intergenic regions and portions of the flanking coding regions. The sequence analysis of the rDNA repeats suggests homogeneity in M. artiellia, thus providing a further indication of the usefulness of these genes for the diagnostic identification of this species. The comparison of the secondary structures of the internal transcribed spacer 2 region in several Meloidogyne species indicates that RNA folding predictions can be used as a tool of potential diagnostic relevance. The other ribosomal gene, 5S rDNA, has been demonstrated to be functional and located near the trans-spliced leader sequences, in the same arrangement found in the distantly related nematode Caenorhabditis elegans but never in other Meloidogyne thus providing species-specific markers for the identification of several Thylenchida parasitic nematodes.

Animals↗
PubMed2005

The silent KIR3DP1 gene (CD158c) is transcribed and might encode a secreted receptor in a minority of humans, in whom the KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1 genes are duplicated.

Killer-cell Ig-like receptors (KIR) are structurally and functionally diverse, and enable human NK cells to survey the expression of individual HLA class I molecules, often altered in infections and tumors. Multiple events of non-reciprocal recombination have contributed to the rapid diversification of KIR. We show that approximately 4.5% of the individuals of a Caucasoid population bear a recombinant allele of KIR3DP1, officially designed KIR3DP1*004, that associates tightly with gene duplications of KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1. The KIR3DP1 gene is normally silent, but the recombinant allele carries a novel promoter sequence and, as a consequence, is transcribed in all tested individuals. Messenger RNA of KIR3DP1*004 is made up of six exons; of these, exons 1-5 are similar to, and spliced like, those encoding the leader peptide and Ig-domains of KIR3D. By contrast, exon 6 is homologous to no other human KIR sequence, but only to possible homologs in chimpanzees and rhesus macaques, and encodes a short hydrophilic tail. The putative KIR3DP1*004 product, like those of the related genes LAIR-2 and LILRA3/ILT6/LIR4, is predicted to be secreted to the extracellular medium rather than anchored to the cell membrane.

Alleles↗
PubMed2000

mRNA trans-splicing in the human parasitic cestode Echinococcus multilocularis.

An identical 36-nucleotide exon was identified at the 5' termini of different mRNAs from the cestode Echinococcus multilocularis. We provide evidence that this exon constitutes a new spliced leader (SL) that is obviously trans-spliced to echinococcal pre-mRNAs, donated by a non-polyadenylated, trimethylguanosine-capped SL-RNA of 104 nucleotides. Sequence comparisons indicated that cestode and trematode SLs are likely to be derived from a common ancestor gene. No conservation was, however, observed concerning the spectrum of mRNAs that is trans-spliced in cestodes and trematodes, indicating that trans-splicing of a particular flatworm mRNA is not correlated with the function of the encoded protein. We also show that the echinococcal gene elp, encoding a member of the ezrin/radixin/moesin protein family, is expressed via two alternative transcripts, spliced either cis or trans at an identical splice acceptor site. This was accompanied by the formation of different elp primary transcripts, harboring a complete or a truncated upstream intron, which supports the hypothesis that alternative cis/trans-splicing depends on the presence or absence of an upstream splice donor site. A putative SL gene was also identified on chromosomal DNA of Echinococcus granulosus, indicating widespread utilization of trans-splicing in the genus.

Alternative Splicing↗