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PubMed1984

Structural comparison of yeast ribosomal protein genes.

The primary structure of the genes encoding the yeast ribosomal proteins L17a and L25 was determined, as well as the positions of the 5'- and 3'-termini of the corresponding mRNAs. Comparison of the gene sequences to those obtained for various other yeast ribosomal protein genes revealed several similarities. In all split genes the intron is located near the 5'-side of the amino acid coding region. Among the introns a clear pattern of sequence conservation can be observed. In particular the intron-exon boundaries and a region close to the 3'-splice site show sequence homology. Conserved sequences were also found in the leader and trailer regions of the ribosomal protein mRNAs. The 5'-flanking regions of the yeast ribosomal protein genes appeared to contain sequence elements that many but not all ribosomal protein genes have in common, and therefore may be implicated in the coordinate expression of these genes. The amino acid coding sequences of the ribosomal protein genes show a biased codon usage. Like most yeast ribosomal protein molecules, L17a and L25 are particularly basic at their N-terminus.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of the gene for the murine T cell differentiation antigen and immunoglobulin-related molecule, Lyt-2.

We present here the sequence of the 5310 base pair Hind III-cleaved genomic DNA segment that includes the gene for the Lyt-2, a murine differentiation antigen expressed on most immature T lymphocytes as well as the cytotoxic suppressor T cell subset. We also present the complete intron/exon structure of Lyt-2. There are five exons: a fused leader and immunoglobulin variable region like exon, a hinge region exon, a transmembrane exon and two alternatively spliced intracytoplasmic exons (alternative splicing of these exons yields the 38 kDa alpha and 34 kDa alpha' Lyt-2 polypeptides). The promotor region contains a "TATA" box and sequences homologous to the putative immunoglobulin transcriptional control elements cd/pd. S1 protection analysis reveals that thymocytes, T cells from lymph nodes, and a Lyt-2 transfectant obtained by introduction of total genomic DNA have the same initiation site. In the 3' region, there is a polyadenylation signal sequence after a 700 bp long 3' untranslated region.

Amino Acid Sequence↗
PubMed1995

Proteins binding to the leader of the 6.0 kb mRNA of human insulin-like growth factor 2 influence translation.

The leader of the 6.0 kb human insulin-like growth factor 2 (IGF-2) mRNA, leader 3, has been reported to partially repress translation. In the regulation of this phenomenon, RNA-binding proteins may play a role. Using UV-irradiation crosslinking, we found specific binding of four proteins (57, 43, 37 and 36 kDa) to this leader. Binding of these proteins to RNA proved to be highly sensitive to the potassium chloride concentration in the buffer solution, each protein having its own optimum. The 57 kDa protein was indistinguishable by size, binding properties and immunoprecipitation from the polypyrimidine tract binding protein (PTB), first described as a nuclear protein binding to the polypyrimidine tracts (PPTs) in introns. Cross-competition experiments showed that leader 3 has a much higher affinity for this 57 kDa protein than the PPT on which PTB was originally characterized. By competition with different fragments of leader 3, we were able to localize the binding of the 57 kDa protein to a 162 nt RNA fragment (AsnI-PvuII) in the 3'-part of the leader. When placed before a chloramphenicol acetyltransferase (CAT) open reading frame, this RNA fragment stimulated translation in reticulocyte lysate 3-fold, while other fragments of leader 3 repressed translation. The efficient translation directed by the 162 nt AsnI-PvuII fragment fused to CAT could be repressed by adding free AsnI-PvuII RNA fragment, indicating that the high translation efficiency of the AsnI-PvuII-CAT synthetic mRNA was due to the binding of protein and not to the structure of the RNA itself.

Base Sequence↗
PubMed1994

Defective processing of human adenovirus 2 late transcription unit mRNAs during abortive infections in monkey cells.

Growth of human adenoviruses is severely restricted in monkey cells. We examined the synthesis of mRNAs from the Ad2 late transcription unit (LTU) in abortively infected monkey cells at late times in infection. All L2, L3, and L5 mRNAs were absent or drastically reduced in abortive infections. Most L1 and L4 mRNAs were also greatly decreased in abortive infections; however, a single large messenger was produced from each of the L1 and L4 families, at levels approaching those found in productive infections. The pattern of i-leader containing mRNAs was also changed in abortive infections. These defects could be corrected in monkey cells by the presence of SV40 T antigen or an altered adenoviral DNA binding protein. These defects in late gene expression in abortive infections could not be attributed to differences in transcription along the LTU or levels of DNA replication. In abortively infected cells, nuclear levels of L5 mRNA were decreased 2 to 6 fold, while cytoplasmic levels were decreased over 200-fold. These findings imply a general defect in processing of viral mRNAs, most likely due to defective splicing and/or transport, during abortive infections.

Adenoviruses, Human↗
PubMed2001

Trans-spliced leader addition to mRNAs in a cnidarian.

A search of databases with the sequence from the 5' untranslated region of a Hydra cDNA clone encoding a receptor protein-tyrosine kinase revealed that a number of Hydra cDNAs contain one of two different sequences at their 5' ends. This finding suggested the possibility that mRNAs in Hydra receive leader sequences by trans-splicing. This hypothesis was confirmed by the finding that the leader sequences are transcribed as parts of small RNAs encoded by genes located in the 5S rRNA clusters of Hydra. The two spliced leader (SL) RNAs (SL-A and -B) contain splice donor dinucleotides at the predicted positions, and genes that receive SLs contain splice acceptor dinucleotides at the predicted positions. Both of the SL RNAs are bound by antibody against trimethylguanosine, suggesting that they contain a trimethylguanosine cap. The predicted secondary structures of the Hydra SL RNAs show significant differences from the structures predicted for the SLs of other organisms. Messenger RNAs have been identified that can receive either SL-A or -B, although the impact of the two different SLs on the function of the mRNA is unknown. The presence and features of SL addition in the phylum Cnidaria raise interesting questions regarding the evolution of this process.

Animals↗
PubMed1991

Modulation of alternative splicing of adenoviral E1A transcripts: factors involved in the early-to-late transition.

The E1A pre-mRNA of adenovirus is spliced into three mRNA species (13S, 12S, and 9S mRNAs) by the use of three alternative 5'-splice sites. The 13S and 9S mRNAs predominate during the early and late periods of infection, respectively. With HeLa nuclear extracts isolated in early and late periods of infection, we were able to reproduce a 13S-9S modulation that resembles that occurring in infected cells. An in vitro analysis of the cis-acting parameters involved in the 13S-9S switch indicates that the 13S mRNA splicing inhibition is one of the first events of the late period and leads to the subsequent stimulation of the 9S mRNA reaction. The new abilities of the late nuclear extract for the 9S mRNA reaction were also confirmed by analyzing splicing of a major late transcript containing leaders 1 and 2 separated by the wild-type intervening sequence (IVS) of 1021 nucleotides. Complementation experiments show that the trans-acting factor(s) are micrococcal nuclease sensitive. They were partially characterized by induction experiments, and we show that the primary factors responsible for the 13S-9S modulation in vitro are viral RNAs of high molecular weight that accumulate late in infection. We postulate that the splicing modulation of E1A pre-mRNA results from an indirect mode of action for these viral RNAs, based on a sequestration of common splicing factors that are not present in vast excess in HeLa cells.

Adenoviridae↗
PubMed2001

Introns and their positions affect the translational activity of mRNA in plant cells.

In an attempt to further increase transgene expression levels in plants over and above the enhancement obtained with a 5' untranslated leader intron, three different maize introns were inserted at three different positions within the coding sequence of the luciferase reporter gene. Constructs were transformed into maize (Black Mexican Sweet) cells and protoplasts, and their activity determined. Although all introns tested were correctly spliced, only one of them in a particular position was able to enhance gene expression. Correct splicing sites were used for intron removal and the quantity of luciferase mRNA produced did not differ significantly. These data indicate that both the position and the sequence of an intron have marked effects on expression levels, suggesting that nuclear processing of the pre-mRNA determines final expression levels through the structure of the mRNP.

Gene Expression Regulation, Plant↗
PubMed1991

Characterization of mRNAs and coding potential of the PET54 gene from Saccharomyces cerevisiae.

The nuclear PET54 gene in yeast controls expression of two mitochondrial genes: COX1 at the level of pre-mRNA splicing and COX3 at the level of mRNA translation. Two size classes (1.6 and 1.1 kb) of transcripts that contain the PET54 coding region are produced in vivo. Relative to the majority of yeast mRNAs analyzed so far, the 5' untranslated leader region of the 1.6 kb transcript is unusually long (254 bases), while that for the major 1.1 kb transcript is unusually short (1 base). The majority of each class of PET54 mRNA was associated with polysomes in vivo. The possibility that two polypeptides are produced in vivo from the 1.1 kb PET54 mRNA was raised by the work of Sedman et al. [J. Virol. 64: 453-457, 1990], which showed that translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' end of the mRNA. However, two sensitive assays for production of a second polypeptide, which is predicted to be 22 kD, were employed and no second polypeptide was detected. Furthermore, a nonsense mutation introduced near the beginning of the PET54 open reading frame abolished both COX1 and COX3 gene expression. These results indicate that the PET54 gene encodes predominantly a single functional polypeptide that is employed for expression of both the COX1 and COX3 genes of mitochondrial DNA.

Base Sequence↗
PubMed1982

gamma Heavy chain disease in man: cDNA sequence supports partial gene deletion model.

Human gamma heavy chain disease (HCD) is characterized by the presence in serum of a short monoclonal Ig gamma chain unattached to light chains. Although most HCD proteins have internal deletions, in some the defect is NH2-terminal. The OMM gamma 3 HCD serum protein is of the latter type, having undergone an extensive NH2-terminal deletion with a sequence starting within the hinge. A cell line synthesizing the OMM protein has enabled us to study the biogenesis of the abnormal molecule. In vitro translation of isolated mRNA yields a protein containing a hydrophobic NH2-terminal leader sequence. In the intact cell, the precursor molecule is processed normally to yield a protein with an NH2-terminal sequence homologous to the beginning of the variable (V) region. The nucleotide sequence of cDNA prepared from the OMM mRNA encodes a 19-amino acid leader followed by the first 15 residues of the V region. An extensive internal deletion encompasses the remainder of the V and the entire CH1 domain. Immediately following the short V region, there is information in the cDNA for the entire normal hinge. The primary synthetic product is thus an internally deleted molecule that undergoes postsynthetic degradation to yield the NH2-terminally deleted serum protein. The structure of the OMM mRNA suggests that the protein abnormality results from a partial gene deletion rather than defective splicing.

Amino Acid Sequence↗
PubMed1996

Construction of portable intron cassettes for the delivery and expression of foreign genes.

The use of viral vectors to deliver foreign genes offers some promise of generating new and more efficacious vaccines. However, the insertion of foreign genes into viral genomes often results in the insertional mutagenesis of one or more genes that adversely affect replication. In an attempt to overcome this problem, we constructed two portable intron cassettes. The cassettes were derived from the adenovirus late leader 1 intron and were cloned into either the chloramphenicol acetyltransferase (CAT) gene or the LacZ gene of Escherichia coli. The intron cassettes were transfected into chicken embryo fibroblasts (CEFs) and the cell lysates were later assayed for either beta-galactosidase (beta-Gal) or CAT activity. The first intron cassette (type A) contained flanking adenovirus exon sequences. Consequently, the flanking adenovirus exon sequences remained in the spliced transcript. With the type A intron inserted in the correct orientation for splicing, CAT activity was not diminished. However, in the reverse orientation, no CAT activity could be detected. The second intron cassette (type B) had the splice donor and splice acceptor sites converted to the blunt-end restriction endonuclease sites Pml I and Pvu II, respectively. The blunt-end restriction endonuclease sites enabled the portable intron to be removed from the flanking adenovirus exon sequences and inserted into any blunt-end restriction endonuclease site in the recipient gene. After splicing, no adenovirus exon sequences remained in the recipient gene's RNA transcript. To demonstrate its usefulness, an insertion cassette was made by cloning the E. coli LacZ gene into a multiple cloning site within the type B intron. The insertion cassette was then cloned into a Pvu II site in the middle of the CAT gene. Following transfection in CEFs, high levels of both CAT and beta-Gal were detected, demonstrating that both genes were properly transcribed and translated.

Adenoviridae↗
PubMed1993

Functional analysis of a C. elegans trans-splice acceptor.

The rol-6 gene is trans-spliced to the 22 nt leader, SL1, 173 nt downstream of the transcription start. We have analyzed splicing in transformants carrying extrachromosomal arrays of rol-6 with mutations in the trans-splice acceptor site. This site is a close match to the consensus, UUUCAG, that is highly conserved in both trans-splice and intron acceptor sites in C. elegans. When the trans-splice site was inactivated by mutating the perfectly-conserved AG, trans-splicing still occurred, but at a cryptic site 20 nt upstream. We tested the frequency with which splicing switched from the normal site to the cryptic site when the pyrimidines at this site were changed to A's. Since most C. elegans 3' splice sites lack an obvious polypyrimidine tract, we hypothesized that these four pyrimidines might play this role, and indeed mutation of these bases caused splicing to switch to the cryptic site. We also demonstrated that a major reason the downstream site is normally favored is because it occurs at a boundary between A+U rich and non-A+U rich RNA. When the RNA between the two splice sites was made less A+U rich, splicing occurred preferentially at the upstream site.

Amino Acid Sequence↗
PubMed1996

Molecular defects in TCRBV genes preclude thymic selection and limit the expressed TCR repertoire.

A prerequisite for the assembly of a functional TCR is the rearrangement of gene segments to result in in-frame transcripts that can vary in length across the CDR3 region. Selection for in-frame 3-bp spaced rearrangements is observed for functional TCRB genes in thymocyte DNA and mRNA transcripts from PBMC. Previous analyses of the expressed human TCRBV gene repertoire have suggested that BV10S1 and BV19S1 gene segments may be expressed at very low levels or not at all in some individuals. CDR3 size analysis for BV10 and BV19 transcripts and thymic DNA rearrangements revealed no such selection of in-frame 3-bp spaced rearrangements. Comparison of the BV19 leader intron sequence with consensus 5'-splice signal sequences suggested that the mature mRNA for this gene would contain the unspliced leader intron. Sequencing of BV19 transcripts from PBMC confirmed that the intron was not spliced, resulting in a predicted translation product that terminates prematurely. Both genomic DNA and mRNA were analyzed for the BV10 gene. The leader sequence contained a single extra base, which would result in a shift in the V region reading frame upon conventional mRNA splicing. This gene is predicted to be nonfunctional due to the presence of a stop codon in the V gene segment just after the splice signal. A splice variant that uses an alternative 3'-splice site further downstream in the V region was also detected. This variant is predicted to be nonfunctional due to the presence of an in-frame stop codon in the V region. These processing defects are sufficient to abrogate positive selection. Therefore, the conclusions drawn from previous studies of the expressed T cell repertoire in normal and disease states based on the presumed functional status of these two genes need to be reassessed.

Base Sequence↗
PubMed1990

Sequences of three VSG mRNAs expressed in a mixed population of Trypanosoma brucei rhodesiense.

A cDNA library was constructed from a mixed population of bloodstream Trypanosoma brucei rhodesiense expressing at least three different VSGs, one of which is immunologically similar to a VSG present during the metacyclic stage. Complete sequence determinations of three full length VSG cDNAs showed that the 5' spliced leader sequence is located 20-26 nucleotides upstream of the start codons of each of the three VSG mRNAs. Two of the VSGs (472 and 487 aa) are members of one C-terminal isotype group while the other, metacyclic-like, VSG (517 aa) is a member of the other C-terminal group. Since VSGs of different sequences have similar three-dimensional structures, comparison of many different VSGs sequences should lead to a more detailed understanding of the relationship between primary and tertiary structures of proteins in general. GenBank accession numbers: M33823, M33824, and M33825.

Amino Acid Sequence↗
PubMed2006

Genomic organization and expression pattern of scapinin (PHACTR3) in mouse and human.

Scapinin has been found to bind to cytoplasmic actin and is also a putative regulatory subunit of protein phosphatase-1 (PP1). It is found attached to the nuclear matrix-intermediate filament (NM-IF) and is down-regulated by differentiation of tumor cells. We have analyzed the genomic structure and tissue-specific expression pattern of both the human scapinin gene (PHACTR3) and the orthologous mouse gene. Both genes showed a highly conserved complex genomic organization with four different leader exons. Alternative splicing of exon 5 was found to be limited to human and variable polyadenylation in mouse transcripts only. In both species expression seems to occur predominantly in the brain. By Northern blot analysis two major transcripts in human and three transcripts in mouse were detected. Expression analysis in the mouse revealed a tissue-specific complex transcription pattern in the brain and a specific pattern was observed during prenatal development. Based on the transcriptional data we therefore assume scapinin to have a distinct biological function in the mammalian brain.

Alternative Splicing↗
PubMed2002

Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene: alternative promoter usage and splicing yield transcripts exhibiting differential translational efficiency.

Peroxisome proliferator-activated receptor (PPAR) beta/delta is ubiquitously expressed, but the level of expression differs markedly between different cell types. In order to determine the molecular mechanisms governing PPARbeta/delta gene expression, we have isolated and characterized the mouse gene encoding PPARbeta/delta. The gene spans approx. 41 kb and comprises 11 exons of which the six exons located in the 3'-end of the gene are included in all transcripts. Primer-extension and 5'-rapid amplification of cDNA ends experiments revealed the presence of multiple transcription start points and splice variants, originating from the use of at least four different promoters. One of these transcription start points was found to be used predominantly in all tissues examined. Initiation from this major transcription start point gives rise to a transcript with a 548 nt 5'-untranslated leader containing eight upstream AUG codons. We show that the presence of the 548 nt leader resulted in a low translational efficiency of the corresponding PPARbeta/delta mRNA and propose, based on structural features of the 5'-untranslated region, that translational initiation may be mediated via an internal ribosome entry site-dependent mechanism.

3T3 Cells↗
PubMed1990

Genomic organization of mouse Fc gamma receptor genes.

We have isolated and characterized the gene coding for the mouse Fc receptor that is termed Fc gamma RIIa. The gene contains five exons and spans approximately 9 kilobases. Unlike most members of the immunoglobulin gene superfamily, this gene utilizes multiple exons to encode its leader peptide. The first exon encodes the hydrophobic region of the signal sequence; the second exon, which contains only 21 base pairs, encodes a segment of the signal peptidase recognition site; and the beginning of the third exon encodes the predicted site of peptidase cleavage. The third and fourth exons each code for immunoglobulin-like extracellular domains. The fifth exon encodes the hydrophobic transmembrane domain and the cytoplasmic tail. Partial characterization of the Fc gamma RIIb gene indicates that it also contains multiple leader exons, including a 21-base-pair exon and two exons coding for homologous immunoglobulin-like extracellular domains. However, the Fc gamma RIIb gene uses four exons to encode its intracytoplasmic region. Analysis using contour-clamped homogeneous electric field (CHEF) gels indicates that the Fc gamma RIIa and Fc gamma RIIb genes are linked within 160 kilobases on mouse chromosome 1.

Amino Acid Sequence↗
PubMedDate not supplied

Organization and chromosomal localization of the human interleukin 5 receptor alpha-chain gene.

The gene for the hIL-5R alpha subunit, which is present in a single copy in the human genome, has been analysed in detail. It is located on chromosome 3 in the region 3p26. The gene organization reflects its relationship to the cytokine/haematopoietin receptor superfamily. Three introns are located in the 5' untranslated region. The subsequent exons determine the functional domains of the hIL-5R alpha protein: the signal peptide, three fibronectin type III-like (FN-like) modules, each built up by two exons, the membrane anchor and two exons forming the cytoplasmic tail, the first of which contains the proline cluster region. In addition, a specific exon generating a soluble isoform is located before the membrane anchor exon. This specific exon contains an in frame TAA stop codon, followed by a polyadenylation signal. Hence, a normal splicing event leads to a soluble IL-5R alpha variant, whereas alternative splicing is required for cell membrane anchoring. A second area of alternative splicing is found in the 5' leader sequence, and possibly relates to the presence of short open reading frames preceding the main ATG. All intron-exon junctions meet the GT-AG rule. The gene structures of all cytokine/haematopoietin receptors documented so far have also been compared with respect to intron phasing. This shows that all introns between the FN-III-like modules are of the +1 type, but in addition, splice sites within the Cys-module and WS-WS-module are invariably of the +2 and 0 type, respectively.

Amino Acid Sequence↗
PubMed2001

A small upstream open reading frame causes inhibition of human major vault protein expression from a ubiquitous mRNA splice variant.

Overexpression of the major vault protein (MVP) has been linked to a multidrug resistance (MDR) phenotype. We describe a ubiquitously expressed MVP mRNA splice variant (long (L)-MVP) differing from the regular isoform (short (S)-MVP) within the 5'-leader. Only L-MVP mRNA contains a small upstream open reading frame which was proven to inhibit in vitro and in vivo MVP expression in cis. L-MVP represented an almost constant portion of total MVP mRNA in diverse normal tissues, but was more variable in malignant cell types. MDR sublines with altered MVP expression displayed changed S-MVP/L-MVP ratios as compared to their drug-sensitive counterparts. Our results suggest alternative splicing as one mechanism for regulation of MVP expression.

5' Untranslated Regions↗