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PubMed1995

Alternative leader sequences in insulin-like growth factor I mRNAs modulate translational efficiency and encode multiple signal peptides.

Rat insulin-like growth factor I (IGF-I) mRNAs contain multiple 5'-untranslated regions due to the use of leader exons transcribed from several transcription initiation sites and to alternative splicing within leader exon 1. Synthetic RNAs with 5'-ends corresponding to the use of exon 1 transcription initiation sites were translated in vitro into prepro-IGF-I peptides initiated at a Met-48 codon in exon 1 or a Met-22 codon in exon 3, and RNAs with a 5'-end corresponding to the major exon 2 transcription start site were translated into a prepro-IGF-I peptide initiated at a Met-32 codon in exon 2. All forms of prepro-IGF-I were processed by canine pancreatic microsomes, suggesting that all these prepeptides function as signal peptides. The translational efficiency of IGF-I RNAs was inversely proportional to the length of the 5'-untranslated region. Mutation of the first of three upstream AUG codons in exon 1, which potentially initiates a 14-amino acid open reading frame, did not affect prepro-IGF-I translation. The other two AUG codons are immediately followed by stop codons. The absence of both upstream AUG codons in a completely spliced exon 1-derived RNA enhanced the in vitro and in vivo translatability of this RNA as compared with the full-length RNA. Mutation of the downstream initiation codon in particular increased translational efficiency in vitro and in intact cells, suggesting that an inefficient reinitiation event at the Met-48 codon contributes to the poorer translation of IGF-I mRNAs in which these upstream AUGUGA motifs occur. We conclude that IGF-I mRNAs potentially encode multiple forms of preproIGF and that specific differences in their 5'-untranslated regions provide a molecular basis for translational control of IGF-I biosynthesis.

Animals↗
PubMed1994

Operons as a common form of chromosomal organization in C. elegans.

Although eukaryotic genes are usually transcribed individually, at least a few Caenorhabditis elegans genes appear to be transcribed polycistronically in clusters resembling bacterial operons. The spliced leader SL2 (ref. 2) is specific for trans-splicing to downstream genes in these operons. In addition, many C. elegans pre-mRNAs are trans-spliced to SL1 (ref. 3) near the 5' ends of pre-mRNAs. Because operons have not previously been found in higher eukaryotes, we have investigated how widespread they are in the C. elegans genome. We identified gene clusters using the extensive data generated by the genome project and tested seven for trans-splicing specificity. All were found to fit expectations for polycistronic transcription. In addition, we surveyed reported C. elegans genes for trans-splicing specificity. Both methods indicate that the pre-mRNAs of about 70% of C. elegans genes are trans-spliced and as many as a quarter are transcribed in operons.

Animals↗
PubMed1991

mRNA transcripts initiating within the human immunoglobulin mu heavy chain enhancer region contain a non-translatable exon and are extremely heterogeneous at the 5' end.

Transcription events are thought to precede gene rearrangement in the immunoglobulin (Ig) loci and may be the mechanism by which the various gene regions are made accessible for recombination. If this is the case, identification and characterization of transcripts from the Ig loci should permit a better understanding of the gene rearrangement process. We have isolated a 2.3 kb cDNA clone from the human pre-B cell line Nalm-1 that contains enhancer-specific sequences from the Ig heavy (H) chain gene locus. The 2.3 kb transcript initiated within the enhancer region and showed extreme 5' heterogeneity, with more than 50 initiation sites mapping near the Ig-specific octamer ATTTGCGT. Sequencing of the cDNA clone demonstrated that 644 nucleotides from the Ig enhancer region were incorporated as a leader exon spliced to the mu constant (Cmu) region. This leader exon contained many translation termination codons and may function to inhibit the translation of sterile Cmu polypeptides. Using an enhancer-derived probe, we detected two low-abundancy mRNA transcripts with sizes of 2.3 and 12 kb. Northern blot analysis suggested that the 12 kb transcript was the unspliced precursor mRNA of a VDJ rearrangement. The potential role of these enhancer-containing transcripts in the opening of the IgH chain gene for rearrangement and for class switching is discussed.

Amino Acid Sequence↗
PubMed2005

The origins of eukaryotic gene structure.

Most of the phenotypic diversity that we perceive in the natural world is directly attributable to the peculiar structure of the eukaryotic gene, which harbors numerous embellishments relative to the situation in prokaryotes. The most profound changes include introns that must be spliced out of precursor mRNAs, transcribed but untranslated leader and trailer sequences (untranslated regions), modular regulatory elements that drive patterns of gene expression, and expansive intergenic regions that harbor additional diffuse control mechanisms. Explaining the origins of these features is difficult because they each impose an intrinsic disadvantage by increasing the genic mutation rate to defective alleles. To address these issues, a general hypothesis for the emergence of eukaryotic gene structure is provided here. Extensive information on absolute population sizes, recombination rates, and mutation rates strongly supports the view that eukaryotes have reduced genetic effective population sizes relative to prokaryotes, with especially extreme reductions being the rule in multicellular lineages. The resultant increase in the power of random genetic drift appears to be sufficient to overwhelm the weak mutational disadvantages associated with most novel aspects of the eukaryotic gene, supporting the idea that most such changes are simple outcomes of semi-neutral processes rather than direct products of natural selection. However, by establishing an essentially permanent change in the population-genetic environment permissive to the genome-wide repatterning of gene structure, the eukaryotic condition also promoted a reliable resource from which natural selection could secondarily build novel forms of organismal complexity. Under this hypothesis, arguments based on molecular, cellular, and/or physiological constraints are insufficient to explain the disparities in gene, genomic, and phenotypic complexity between prokaryotes and eukaryotes.

Animals↗
PubMed1991

Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila.

Metamorphosis in Drosophila is triggered by a pulse of the steroid hormone ecdysone at the end of larval development. Ecdysone initiates a genetic hierarchy that can be visualized as a series of puffs in the larval salivary gland polytene chromosomes. The E74 gene is responsible for the early ecdysone-inducible puff at position 74EF and encodes two related DNA-binding proteins which appear to play a regulatory role in the hierarchy. Here we describe the spatial and temporal patterns of E74A RNA and protein expression at the onset of metamorphosis. We use in situ hybridization, antibody stains, and western and northern blot analyses to follow E74A expression from its initial appearance as nascent transcripts on the polytene chromosomes, to spliced mRNA, to post-translationally modified nuclear E74A protein. E74A is expressed in a wide variety of late-third instar tissues, suggesting that it plays a broad pleiotropic role in response to the hormone. In early prepupae, when the overall levels of E74A mRNA are decreasing, relatively high levels of E74A RNA persist in the gut, peripodial membranes of the imaginal discs, and proliferation centers of the brain. The spatial distribution of nuclear E74A protein correlates with the RNA distribution with the single exception that no E74A protein can be detected in the proliferation centers of the brain. There is also a temporal discrepancy between E74A mRNA and protein accumulation. The peak of E74A protein induced by the late larval ecdysone pulse follows the peak of E74A mRNA by approximately 2 h. This delay is not seen in 10 h prepupae, when the next pulse of ecdysone induces the simultaneous expression of E74A mRNA and protein. We discuss possible mechanisms for post-transcriptional regulation of E74A expression and suggest that the unusually long and complex 5' leader in the E74A mRNA may regulate its translation.

Animals↗
PubMed1987

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species.

The nucleotide sequences corresponding to bovine alpha S2- and beta-casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.

Animals↗
PubMed1994

The 3' region of c-src gene mRNA is entirely included in exon 12 and does not encode another protein.

We previously reported the isolation of PR2257, a novel replication defective avian sarcoma virus which transduced the entire c-src coding region together with about 900 bp of c-src 3' non coding sequences. This virus originated from a chicken sarcoma induced by inoculation of a transformation-defective Rous sarcoma virus carrying only replicative genes. The 5' end of PR2257 was formed by a splice junction between viral leader sequences and the first exon of c-src. To understand the mechanism of 3' recombination between viral and cellular sequences, we analyzed the genomic organization of the 3' region in chicken and quail src DNA. We report that this sequence is colinear with that of a chicken src cDNA. Therefore, exon 12, which encodes the last 68 amino acids of c-src, also contains all 3' non coding sequences present in c-src mRNA and consequently, appears to be the last and largest (about 2 kbp) exon of c-src. We also show that the 3' regions of chicken and quail c-src genes does not contain the additional open reading frame (orf) which was previously reported (Dorai et al. (1991) Mol. Cell. Biol. 11, 4165-4176), and that no other significant conserved open reading frames could be found in this region for both species. Therefore, this region of src does not code for another protein. Taken together, our results suggest that PR2257 was generated by recombination at the RNA level. However, because of the absence of introns in this region of c-src, we cannot formally rule out the possibility that this recombination took place at the DNA level.

Animals↗
PubMed1997

Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization.

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1996, and references therein; M. Laughrea and L. Jette, Biochemistry 35:9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the kissing-loop hairpin might affect the in vivo dimerization and encapsidation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molecular clone in order to produce viruses by transfection methods. The infectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, and the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchanged. There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splicing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are probably more concentrated in transfected cells than in cells infected by one virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization and encapsidation defects would have been manifested within cells infected by no more than one virus.

Animals↗
PubMed2002

The alternative transcript of CD79b is overexpressed in B-CLL and inhibits signaling for apoptosis.

The B-cell receptor (BCR) for antigen is composed of surface immunoglobulin (sIg), which provides antigen specificity, and a noncovalently associated signaling unit, the CD79a/b heterodimer. Defects in CD79 can influence both BCR expression and signaling and may explain why cells from certain malignancies, such as B-chronic lymphocytic leukemia (B-CLL), often express diminished and inactive BCR. Recently, an alternative transcript of CD79b (DeltaCD79b) has been reported that is up-regulated in B-CLL and may explain this diminished BCR expression. Here we assess the expression of DeltaCD79b in B-CLL and other lymphoid malignancies and investigate its function. High relative expression of DeltaCD79b was confirmed in most cases of B-CLL and found in 6 of 6 cases of splenic lymphomas with villous lymphocytes (SLVLs) and hairy cell leukemia. In a range of Burkitt lymphoma cell lines, expression of DeltaCD79b was relatively low but correlated inversely with the ability of the BCR to signal apoptosis when cross-linked by antibody (Ab). Interestingly, when Ramos-EHRB cells, which express low DeltaCD79b, were transfected with this transcript, they were transformed from being sensitive to anti-Fcmu-induced apoptosis to being highly resistant. Although DeltaCD79b was expressed as protein, its overexpression did not reduce the level of cell surface BCR. Finally, we showed that the inhibitory activity of DeltaCD79b depended on an intact leader sequence to ensure endoplasmic reticulum (ER) trafficking and a functional signaling immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail. These results point to DeltaCD79b being a powerful modulator of BCR signaling that may play an important role in normal and malignant B cells.

Alternative Splicing↗
PubMed1992

The Euglena gracilis rbcS gene contains introns with unusual borders.

We have recently shown that, in Euglena gracilis, leader sequences are transferred by trans-splicing to the vast majority of cytoplasmic mRNAs. Trans-splicing is involved in the maturation of the rbcS transcript, which encodes eight small subunits of the ribulose 1,5 bisphosphate carboxylase/oxygenase. In this report, we show that the Euglena rbcS gene introns are different from introns found in plant rbcS genes. In addition these introns do not have the conserved 5' and 3' border sequences found in introns of eucaryotic nuclear-encoded pre-mRNAs, and they do not present any homology with self-splicing introns of groups I and II. Secondary structure analyses show that the 5' and 3' ends of Euglena introns can base-pair, suggesting that an unusual splicing mechanism exists in Euglena.

Amino Acid Sequence↗
PubMed1998

Genomic structure of the human pre-T cell receptor alpha chain and expression of two mRNA isoforms.

The pre-TCR, which is minimally composed of the TCRbeta chain, the pre-Talpha chain, and the CD3 complex, regulates early T cell development. The pre-Talpha chain is a 33-kDa type I transmembrane glycoprotein with an extracellular part similar to the constant domain of the immunoglobulin supergene family. We have sequenced (11 kb) the human pTalpha gene, which like the murine pTalpha gene consists of four exons: exon 1 encodes the 5' untranslated region, the leader peptide and the first three amino acids of the mature protein, exon 2 the extracellular immunoglobulin (Ig)-like domain, exon 3 a 15-amino acid peptide including a cysteine required for heterodimerization with TCRbeta, exon 4 the transmembrane region, the cytoplasmic tail and the 3' untranslated sequence. The human pTalpha gene is located on chromosome 6p21.3, close to the HLA-A locus. Reverse transcription-PCR studies with human thymus and leukemic cells showed that alternative splicing produces a shorter pTalpha isoform, which lacks the Ig-like domain but contains the transmembrane elements and the extracytoplasmic cystein and which could thus permit pairing with TCRbeta chain and association with CD3 molecules. The conserved splice sites suggest a yet ill-defined biological function of the short pTalpha protein.

Humans↗
PubMed2000

Identification of an alternatively spliced seprase mRNA that encodes a novel intracellular isoform.

Seprase is a homodimeric 170-kDa integral membrane gelatinase that is related to the ectoenzyme dipeptidyl peptidase IV. We have identified an alternatively spliced seprase messenger from the human melanoma cell line LOX that encodes a novel truncated isoform, seprase-s. The splice variant mRNA is generated by an out-of-frame deletion of a 1223-base pair exonic region that encodes part of the cytoplasmic tail, transmembrane, and the membrane proximal-central regions of the extracellular domain (Val(5) through Ser(412)) of the seprase 97-kDa subunit (seprase-l). The seprase-s mRNA has an elongated 5' leader (548 nucleotides) that harbors at least two upstream open reading frames that inhibit seprase-s expression from a downstream major open reading frame. Deletion mutagenesis of the wild type splice variant cDNA confirms that initiation of the seprase-s coding sequence begins with an ATG codon that corresponds to Met(522) of seprase-l. The seprase-s open reading frame encodes a 239-amino acid polypeptide with an M(r) approximately 27,000 that precisely overlaps the carboxyl-terminal catalytic region of seprase-l.

Alternative Splicing↗
PubMed1988

The E5 gene of bovine papillomavirus type 1 is sufficient for complete oncogenic transformation of mouse fibroblasts.

The transforming 69% fragment of the bovine papillomavirus type-1 genome was inserted into a retrovirus vector, which also expresses G418 resistance, and the resulting construct was used for transfection of psi 2 cells. C127 cells infected with virus-containing medium from G418 resistant psi 2 clones were selected for G418 resistance and/or transformation. G418 resistant cells contained invariably a 4.4 kb provirus. The transformed cells, in contrast, contained either a 2.8 kb or a 5.4 kb provirus. Cells containing the 2.8 or the 5.4 kb proviruses were fully transformed according to several criteria: they had a transformed morphology, formed colonies in soft agar, contained disarranged F-actin cables and induced tumors when injected subcutaneously into nude mice. A molecular analysis of the 2.8 kb provirus showed that it contained only one complete BPV-1 gene, namely E5. Cells transformed by the 2.8 kb provirus contained colinear RNAs as well as subgenomic mRNAs, composed of a retrovirus leader sequence connected to an exon starting at nucleotide 3605 in the BPV-1 sequence. The only papillomavirus protein that can be expressed from these mRNAs is the E5 protein. The results suggest that the 44 amino acid long membrane protein which is encoded by the E5 gene of BPV-1, confers a fully transformed phenotype to immortalized mouse cells in the absence of other viral gene products or papillomavirus regulatory elements.

Actin Cytoskeleton↗
PubMed1997

Transcriptional analysis of walleye dermal sarcoma virus (WDSV).

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas of walleye that develop and regress on a seasonal basis. WDSV contains, in addition to gag, pol, and env, three open reading frames (ORFs) designated ORF A, ORF B, and ORF C. The polymerase chain reaction technique was used to amplify and clone cDNAs representing subgenomic viral mRNAs isolated from developing (fall) and regressing (spring) tumors. Nine different singly or multiply spliced viral transcripts were identified and all were found to utilize a common 5' leader sequence. This leader sequence is spliced to the pol/env junction or downstream of env to generate singly spliced transcripts. Multiply spliced transcripts contain the 5' leader, the pol/env junction, and sequences derived from the 3' end of the genome. One multiply spliced transcript was isolated with the potential to encode the full-length ORF A protein. In addition, WDSV produced mRNAs that utilize alternative splice acceptor sites which would allow synthesis of five variant forms of the ORF A protein. In contrast, the ORF B protein is postulated to arise from a singly spliced transcript with the potential to encode the entire open reading frame. Spliced subgenomic transcripts representing ORF C mRNAs were not identified, suggesting that ORF C may be encoded from the full-length viral genomic transcript. We estimate that at least a 100-fold lower amount of the accessory/regulatory subgenomic transcripts exists in developing vs regressing tumors. These results demonstrate that WDSV undergoes an elaborate pattern of mRNA splicing similar to that of other complex retroviruses.

Animals↗
PubMed1992

Conservation of gene organization and trans-splicing in the glyceraldehyde-3-phosphate dehydrogenase-encoding genes of Caenorhabditis briggsae.

The genes encoding body-wall-specific glyceraldehyde-3-phosphate dehydrogenase from Caenorhabditis briggsae were sequenced and compared to the homologous genes from Caenorhabditis elegans. The direct tandem organization of these genes, gpd-2 and gpd-3, and the size and location of the two introns in each gene are the same in C. elegans and C. briggsae. Primer-extension studies demonstrated that the two genes in C. briggsae are trans-splice differentially with the same splice leader (SL) RNAs as are observed in C. elegans. The gdp-2 gene is trans-spliced with SL1 while gdp-3 is trans-spliced with SL2. Significant sequence conservation was observed within the promoter regions of each species and may indicate those regions responsible for body-wall-muscle-specific gene expression and/or differential trans-splicing. Comparisons of the sequences suggest that the tandem repeat of the genes has been subjected to concerted evolution and that C. briggsae and C. elegans diverged much earlier than would be anticipated based on morphological similarities alone. Finally, an open reading frame found several hundred nucleotides upstream from gpd-2, in both species, appears to be homologous to the ATP synthase subunit, ATPase inhibitor protein, from bovine mitochondria.

Amino Acid Sequence↗
PubMed2001

Non-coding snoRNA host genes in Drosophila: expression strategies for modification guide snoRNAs.

Modification guide snoRNAs either are encoded within introns and co-transcribed with the host gene pre-mRNA or are independently transcribed as mono- or polycistronic units. Different eukaryotic kingdoms utilize these coding strategies to various degrees. Intron-encoded and polycistronic snoRNAs are released from primary transcripts as pre-snoRNAs by the spliceosome or by an RNase III-like activity, respectively. In the spliceosomal pathway, the resulting intron lariat is then linearized by a debranching activity. The leader and trailer sequences of pre-snoRNAs are removed by exonucleolytic activities. The majority of snoRNA host genes encode proteins involved in the synthesis, structure or function of the translational apparatus. Several vertebrate snoRNA host genes do not appear to code for functional proteins. We have identified two unusually compact box C/D multi-snoRNA host genes in D. melanogaster, dUHG1 and dUHG2, similar in their organization to the corresponding vertebrate non-protein-coding host genes. In dUHG1 and dUHG2, the snoRNA sequences are located within introns at a conserved distance of about 75 nucleotides upstream of the 3' splice sites. Both genes initiate transcription with TOP-like sequences that share unique features with previously reported Drosophila snoRNA host genes. Although the spliced dUHG RNAs are relatively stable, they exhibit little potential for protein coding.

Animals↗
PubMed2001

Complete 5' and 3' end maturation of group II intron-containing tRNA precursors.

Higher plant chloroplasts provide the only experimentally validated example of functional tRNA genes that are disrupted by group II introns. Here, precursor transcripts for tRNA(Gly)(UCC), tRNA(Val)(UAC), and tRNA(Ala)(UGC) were investigated for processing of 5' leader and 3' trailer sequences in vivo. Use of intron-specific primer pairs and inclusion of a barley chloroplast splicing mutant specifically allowed us to evaluate the potential effect of intervening sequences that disrupt tRNA secondary and tertiary structures. The data suggest that (1) neither integrity of the dihydrouridine nor the anticodon domain is required for the nucleotidyltransferase-mediated addition of 3'-terminal CCA; (2) interruption of these two structural elements by group II introns does not interfere with nucleotide-specific 5' maturation by RNase P; (3) processing intermediates of chloroplast tRNAs can be 3' polyadenylated; and (4) plastid DNA-encoded proteins are not required for 3' and 5' maturation of plastid tRNAs.

Base Pairing↗
PubMed1995

Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/PTB splicing factor.

Moloney murine leukemia virus (Mo-MuLV) genomic mRNA codes for two gag precursors by alternative initiations of translation. An AUG codon governs the synthesis of the retroviral capsid proteins precursor, whereas a CUG codon directs the synthesis of a glycosylated cell surface antigen, the gross cell surface antigen. Control of the relative synthesis of the two precursors is crucial for MuLV infectivity and pathology. Furthermore, the MuLV mRNA leader sequence is very long and should inhibit translation according to the classical scanning model. This suggests a different translation initiation mechanism allowing gag efficient expression. We demonstrate, by using bicistronic vectors expressed in COS-7 cells, that the Mo-MuLV mRNA leader drives translation initiation by internal ribosome entry. We have localized the internal ribosome entry site (IRES) between the two initiation codons. This 126 nucleotide long IRES implies an oligopyrimidine tract located 45 nucleotides upstream of AUG codon. UV cross-linking and affinity chromatography experiments show that the PTB/p57 splicing factor specifically interacts with this oligopyrimidine tract. The MuLV IRES controls alternative translation initiation by activating the capsid protein precursor expression. This gag translational enhancer could exist in other retroviruses.

Animals↗