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PubMed2004

Translational control of the rat angiotensin type 1a receptor by alternative splicing.

The rat angiotensin type 1a receptor (AT1aR) is comprised of three exons. Two transcripts are possible due to alternative splicing of exon 2 (E1,3 and E1,2,3). Both transcripts code for identical AT1aR proteins since they differ only in the length of their 5' leader sequence (5'LS). We investigated the functional differences of these two transcripts in stably transfected Chinese hamster ovary (CHO) cells and also determined the splice variant composition in rat tissues. E1,3 expressing cells exhibited 1.8-fold higher AT1R densities and five-fold higher levels of Ang II-stimulated inositol phosphate production compared to E1,2,3 expressing cells. No differences in E1,3 and E1,2,3 mRNA levels or mRNA stability were seen. In vitro translation assays revealed 1.8-fold higher AT1aR protein levels from E1,3 compared to E1,2,3 transcripts, suggesting exon 2 reduces functional AT1R expression by inhibiting translation. Deletion of 10 nucleotides in exon 2 increased translation of the mutated E1,2,3 transcript to levels which were indistinguishable from E1,3, suggesting that this loop region of a predicted hairpin contributes to the inhibitory RNA cis element within exon 2. Comparison of AT1aR exonic composition and AT1R densities in rat tissues suggests alternative splicing is regulated in a tissue-specific manner and contributes to tissue-specific differences in AT1R density.

Adrenal Cortex↗

PubMed1983

Attenuation and modulation of mRNA secondary structure in a feedback control system regulating SV40 gene expression.

Alternative secondary structures can be predicted for the initial 94 nucleotides synthesized from the major transcription initiation site of SV40 late RNA: a transcription-termination conformation results in the production of aborted RNA and a readthrough conformation leads to the synthesis of the primary SV40 late RNA. In the cytoplasm similar alternative conformations can be predicted for the initial nucleotides at the 5' ends o both the major 16S and 19S late mRNAs. In one of these alternative conformations the AUG initiation codon of the leader protein (agnoprotein) is sequestered and not available for ribosome binding. In the alternative conformation the same AUG is accessible for ribosome binding. We suggest that these mutually exclusive conformations are fundamental elements in a transcription and translation feedback control mechanism regulating the synthesis of 16S and 19S mRNA in the nucleus and the translation of their encoded proteins in the cytoplasm.

Feedback↗

PubMed1989

Rapid changes in the expression of a gene encoding a calcium-binding protein in Schistosoma mansoni.

Genes expressed in a stage-specific manner may help us understand the molecular events controlling the complex life cycle of schistosomes. cDNA and genomic clones encoding a calcium-binding protein (CaBP) were obtained from cercariae and their sequence determined. The encoded protein (69 amino acids long) shows clear resemblance to the domain structure and organization of CaBP molecules. It contains two typical calcium-binding loops, the distance between which is identical to the length conserved in other CaBP molecules. In addition, the schistosome CaBP shows Ca2+-dependent electrophoretic mobility (increased with Ca2+-ions and decreased with EGTA). Northern blots revealed expression of the CaBP gene in cercariae but not in sporocyst or worm (developmental stages preceding and following cercaria). The preferential expression of this CaBP in the cercaria raises questions as to what cercaria-specific function(s) it performs. The structure of the gene is similar to that in other eukaryotes, and one intron interrupts the coding sequence. The region of the cap site was determined, and there was no evidence of the spliced leader sequence found in the mRNAs of other parasites. The CaBP reveals a rapid change in gene expression, since the mRNA is missing in the parasite residing in infected snails, but is readily detected in cercariae 1 h after shedding. We identified other genes which are turned on (like the CaBP) or shut off within the short period of transition from cercariae in the snail to free-swimming cercariae.

Aging↗

PubMedDate not supplied

Multiple promoter and splicing mRNA variants of the epididymis-specific gene EP2.

The EP2 gene codes for a family of androgen-dependent, epididymis-specific secretory proteins. Using probes derived from human HE2 cDNA, a chimpanzee epididymal cDNA library was screened. Five variants of chimpanzee EP2 cDNA were identified. Variant 1 (EP2A) is the chimpanzee ortholog of HE2. Variant 2 (EP2B) has an alternative 5' end. Variant 3 (EP2C) has an alternative 3' end. Two additional variants were identified by reverse transcriptase-polymerase chain reaction analysis. Variant 4 (EP2D) and variant 5 (EP2E) appear to lack an exon, resulting in a shift in the open reading frame. Presumably, the 5 variants originate from the same gene and result from alternative promoters and alternative splicing. Each of the putative proteins encoded by these variant messages has a leader sequence characteristic for a secretory protein. After removal of the leader sequence, each of these proteins is predicted to consist of 1 or 2 out of 4 possible peptide modules. Two of these modules have no recognizable homology to known proteins. The other 2 modules have a distribution of cysteine residues characteristic for beta-defensins, a family of proteins with antimicrobial activity.

Alternative Splicing↗

PubMed1980

Polyoma virus DNA: Sequence from the late region that specifies the leader sequence for late mRNA and codes for VP2, VP3, and the N-terminus of VP1.

The DNA sequence of part of the late region of the polyoma virus genome is presented. This sequence of 1,348 nucleotide pairs encompasses the leader region for late mRNA and the coding sequence for the two minor capsid proteins VP2 and VP3. The coding sequence for the N-terminus of the major capsid protein overlaps the C-terminus of VP2/VP3 by 32 nucleotide pairs. From the DNA sequence the sizes and sequences of VP2 and VP3 could be predicted. Potential splicing signals for the processing of late mRNA's could be identified. Comparisons are made between the sequence of polyoma virus DNA and corresponding regions of simian virus 40 DNA.

Amino Acid Sequence↗

PubMed1997

The primary structure of Trypanosoma (Nannomonas) congolese variant surface glycoproteins.

The complete nucleotide sequences were determined for three transcripts each encoding a different variant surface glycoprotein (VSG) of Trypanosoma (Nannomonas) congolense. The nucleotide sequence was determined also for a transcript encoding a fourth VSG, but this was truncated. The data obtained confirm absence of the canonical polyadenylation signal, lack of conserved sequence elements in the 3' untranslated region, and heterogeneity in the spliced-leader acceptor site in the T. congolense VSG transcripts examined. A comparison of the amino acids deduced from the nucleotide sequences of the four VSGs and those of other VSGs published previously reveals a strong conservation of several structural domains, particularly cysteine residues located throughout most of the molecules. The majority of T. congolense VSGs analyzed in this study resemble most the N-terminal cysteine residue domain type B of T. brucei, characterized by a cysteine residue located toward the N-terminal end, a cluster of cysteine residues in the central region, and at least three cysteine residues between positions 250 and 300 of the molecules. One of the VSGs analyzed, ILNat3.3, did not fit into any of the classification schemes proposed for the VSGs so far studied, and thus may represent a different class of these surface molecules. Unlike VSGs of T. brucei, the T. congolense VSGs have no cysteine residues at the carboxy-terminal end. These data now make it possible to predict general primary structural features of T. congolense VSGs.

Amino Acid Sequence↗

PubMed1995

Transient expression mediated by the Trypanosoma cruzi rRNA promoter.

Plasmid constructs containing a putative Trypanosoma cruzi rRNA promoter and transcription start point upstream from the bacterial chloramphenicol acetyltransferase (CAT) reporter gene were transfected into cultured T. cruzi epimastigotes to verify the presence of a promoter activity. Constructs bearing the putative promoter and a 3' trans-splicing acceptor site in the proper orientation yielded approx. two orders of magnitude greater CAT expression than that previously observed with the T. cruzi spliced leader (SL) gene promoter. In contrast, similar constructs lacking the known 3' splice site yielded reduced but readily measurable expression suggesting that sequences near the promoter may function as cryptic 3' splice sites. A repeated sequence upstream from the putative basal rRNA promoter in a position analogous to rRNA gene enhancer elements in other eukaryotes did not enhance expression from the T. cruzi rRNA promoter. Finally, these constructs were functional in some but not all T. cruzi isolates, and were inactive in other kinetoplastid species, suggesting that the T. cruzi rRNA promoter may have a limited host range.

Animals↗

PubMed1987

Evolution of the primary and secondary structures of the E1a mRNAs of the adenovirus.

In this paper we investigate and compare (evolutionary) patterns in the primary and secondary structure of four homologous E1a mRNAs of the adenovirus. Our main results are as follows: (1) The similarity of the coding regions of the mRNA sequences reflects both similarity in function (i.e., oncogenicity) and evolutionary divergence. (2) The similarity of the leader and the trailer regions reflects host specificity (i.e., human or simian) and must therefore arise from convergence. (3) Minimal energy foldings of the mRNAs show similar secondary structures (in particular around the splice sites). The conservation of pre-mRNA secondary structure shows that mRNAs are subject to selection constraints in addition to those associated with proteins. (4) The conserved secondary (helical) structures consist of nonhomologous subsequences, i.e., shifts have occurred. The observed shifts near the splice sites seem to be the simplest way of dealing with the dual constraints.

Adenoviridae↗

PubMed1987

The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron.

The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message. Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon. A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection. Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides. The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns. One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron. The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.

DNA, Fungal↗

PubMed1995

Conserved alternative splicing in the 5'-untranslated region of the muscle-specific enolase gene. Primary structure of mRNAs, expression and influence of secondary structure on the translation efficiency.

We report here the isolation and characterization of cDNAs covering the 5'-end region of mouse and rat mRNAs that encode the beta or muscle-specific isoform of the glycolytic enzyme enolase. As previously determined for humans, two classes of beta-enolase transcripts with distinct sequences in their 5'-untranslated regions are present in both mouse and rat muscles. A mechanism of alternative splicing, conserved from mouse to man, generates the two forms of mRNA. Secondary-structure predictions indicated that, in all cases, a more stable secondary structure could exist in the 5' end of the message with the longer leader. In vitro transcripts containing defined human or mouse 5'-untranslated sequences were obtained by fusion of the different cDNA clones and tested for their relative translational efficiencies in rabbit reticulocyte lysates. Transcripts containing the human long and short leader sequences showed differences in the translational rate, suggesting a role for the 5'-untranslated region in the regulation of translation. No detectable difference was found between transcripts with the two distinct mouse leader sequences. In addition, both transcripts are bound to polysomes and are equally distributed along differently sized polysomes in C2C12 myogenic cells. The relative expression of the two spliced forms in developing and adult muscle tissues by means of reverse transcription and polymerase chain reaction did not show a stage-specific or a tissue-type-specific pattern. A putative functional role for the 5'-untranslated sequences of beta-enolase transcripts is discussed.

Alternative Splicing↗

PubMed2006

On the paucity of duplicated genes in Caenorhabditis elegans operons.

Spliced leader trans-splicing is an mRNA maturation process used by a small set of eukaryotes, including the nematode C. elegans, to cap the downstream genes of operons. We analyzed the frequency of duplication of operonic genes in C. elegans and confirmed that they are duplicated less often in the genome than monocistronic genes. Because operons account for about 15% of the genes in C. elegans, this lower duplication frequency might place a large constraint on the plasticity of the genome. Further analyses suggest that this paucity of duplicated genes results from operon organization hindering specific types of gene duplication.

Animals↗

PubMed2000

Isolation and molecular characterisation of the gene encoding the cytoplasmic ribosomal protein S28 in Prunus persica [L.]] Batsch.

RT-PCR was performed on peach (Prunus persica [L.] Batsch) RNA to isolate cDNAs corresponding to transcripts which are differentially expressed in leaves borne on basal and apical shoots. A gene was identified which was more highly expressed in the leaves of basal shoots, and codes for the cytoplasmic protein S28 present in the small ribosomal subunit. The 5' leader regions of RPS28 mRNAs were found to harbour 8-11 pyrimidine tracts, which suggested similarities to regulatory stretches that control the translation of mRNAs for ribosomal proteins in animals. The peach S28 is encoded by two intron-containing genes, which are both transcribed in mitotically active tissues such as developing leaves and roots. In situ hybridisation to shoot vegetative apices and the measurement of nucleus/nucleolus ratios indicated that RPS28 expression was confined to areas undergoing active cell division. The mature RPS28 mRNA was detected as a single species in actively dividing tissues such as apical tips, developing leaves, vegetative buds, stamens, developing fruits and roots. In contrast, accumulation of a precursor RNA, in the presence of the mature product, was found in fully expanded leaves and subtending stems, while only the precursor species was detected in several late-stage tissues. This phenomenon suggested that expression of the mature RNA is controlled at the level of splicing and turnover of the precursor RNA. This is similar to the mode of regulation of ribosomal protein genes in animals.

Amino Acid Sequence↗

PubMed2002

Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

5' Untranslated Regions↗

PubMed1993

Characterization of the Schistosoma mansoni gene encoding the glycolytic enzyme, triosephosphate isomerase.

The complete gene encoding Schistosoma mansoni triosephosphate isomerase (TPI) was isolated from a lambda phage genomic library on 2 overlapping clones. These genomic clones have been characterized by restriction mapping and DNA sequencing of the 5' flanking region, the exons, the intron boundaries and the polyadenylation addition site. S. mansoni TPI is encoded by 6 exons spanning a region of about 12 kb. The 5 introns are located at positions precisely analogous to those of mammalian TPI genes but one of the 6 mammalian TPI introns is missing in S. mansoni. We find no evidence of spliced leader involvement in TPI gene expression. The gene is preceded by at least 4 tandem copies of a 2.5-kb repetitive sequence. While the 12-kb size for the S. mansoni TPI gene is much larger than the 3-4 kb typical of mammalian TPI genes, the 42-bp first intron is unusually short. The transcription initiation site for the S. mansoni TPI gene is heterogeneous. Genomic Southern blot analysis suggests that TPI is expressed from a single copy gene.

Amino Acid Sequence↗

PubMed2003

Powdery mildew-induced Mla mRNAs are alternatively spliced and contain multiple upstream open reading frames.

In barley (Hordeum vulgare), the Mla13 powdery mildew resistance gene confers Rar1-dependent, AvrMla13-specific resistance to Blumeria graminis f. sp. hordei (Bgh). We have identified cDNA and genomic copies of Mla13 and used this coiled-coil nucleotide-binding site leucine-rich repeat protein-encoding gene as a model for the regulation of host resistance to obligate biotrophic fungi in cereals. We demonstrate quantitatively that a rapid increase in the accumulation of Mla transcripts and transcripts of the Mla-signaling genes, Rar1 and Sgt1, is triggered between 16 and 20 h post inoculation, the same time frame that haustoria of avirulent Bgh make contact with the host cell plasma membrane. An abundance of Mla13 cDNAs revealed five classes of transcript leader regions containing two alternatively spliced introns and up to three upstream open reading frames (uORFs). Alternative splicing of introns in the transcript leader region results in a different number of uORFs and variability in the size of uORF2. These results indicate that regulation of Mla transcript accumulation is not constitutive and that induction is coordinately controlled by recognition-specific factors. The sudden increase in specific transcript levels could account for the rapid defense response phenotype conferred by Mla6 and Mla13.

Alternative Splicing↗

PubMed1988

The Ds1 transposable element acts as an intron in the mutant allele Adh1-Fm335 and is spliced from the message.

The Ds-induced maize Adh1 allele Adh1-Fm335 retains its anaerobic regulation and normal transcription start site despite the presence of the 405 bp Ds element in the 5' untranslated leader region of the gene. The steady state level of Adh1-specific transcript is reduced to about 1% that of the progenitor or revertant alleles. Run-on transcription studies show that the reduced level of Adh1 specific mRNA is not attributable to a decreased transcription rate. S1 mapping indicates that the Ds element is spliced from the Adh1-Fm335 transcript using a donor site 14 bp into the Ds element and an acceptor site at the 3' junction of the Ds element with the flanking genome DNA.

Alcohol Dehydrogenase↗

PubMed1993

Resolution of the HLA-DRB6 puzzle: a case of grafting a de novo-generated exon on an existing gene.

HLA-DRB6, one of the human major histocompatibility complex genes, lacks exon 1, which normally codes for the leader and the first four amino acid residues of the mature protein. Because it also lacks the HLA promoter, it was surprising to find that the gene is transcribed at a low level in a chimpanzee B-lymphoblastoma cell line, in which the DRB6 homolog is truncated as in humans. The study designed to resolve the paradox has revealed that a retrovirus related to the mouse mammary tumor viruses was inserted into intron 1 of DRB6 > 23 million years ago. The insertion was either accompanied or followed by the deletion of exon 1 and the promoter region of DRB6. In the 3' long terminal repeat of the retrovirus, however, an open reading frame for a new exon arose, which codes for a sequence of mostly hydrophobic amino acid residues; the sequence could function as a leader for the truncated DRB6 gene. This new exon has a functional donor splice site at its 3' end, which enables it to be spliced in register with DRB6 exon 2. Upstream from the new exon is a promoter enabling transcription of the DRB6 gene. Besides providing an example of a de novo generation of an exon, the study suggests a potential mechanism for generating new genes through the replacement of old exons with newly generated ones.

Amino Acid Sequence↗

PubMed1993

Human immunodeficiency virus type 2 multiply spliced transcripts.

Viral transcripts, particularly those of the regulatory genes (e.g., rev) in lymphocytic cells chronically infected with human immunodeficiency virus type 2, consist of two types, differing in the structure of the leader sequence derived from the 5' long terminal repeat (LTR). Some transcripts undergo a specific splicing event within the 5' LTR, removing an intron consisting of a part of the R region whereas others do not. Because this spliced-out R region is a part of the trans-activation response element (TAR), it could influence trans-activator (Tat)-mediated trans-activation of viral gene expression. Moreover, this part of the R region is predicted to contain a stable secondary structure that could affect the efficiency of translation of the transcripts without this splicing. Thus, the 5' LTR splicing could have important consequences for virus replication, latency, and pathogenicity.

Base Sequence↗