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PubMed1988

Vestiges of lost introns in the thermal stability map of DNA.

The absence of introns in prokaryotic genes has been explained by intron loss on various bases. Here we report another piece of evidence on intron loss, which was found in the thermal stability map of DNA. We calculated the local melting temperature of cDNA sequences and found that (i) gaps in thermal stability tend to occur near intron positions with a statistical significance, and (ii) one-third of the gaps far from intron positions can be assigned to lost introns. From these results we conclude that the gaps of thermal stability in protein coding regions are the vestiges of lost introns.

Animals↗

PubMed1990

The role of introns in evolution.

What are the roles of 'classical' introns in the evolution of nuclear genes, and what was the origin of these introns? Exon shuffling has been important in the evolution of cell surface and extracellular proteins, but the evidence for it in respect of intracellular proteins is weak. Intron distributions imply that some introns have been removed while others have been inserted in the course of evolution: ancestral patterns of introns may thus have been obscured. Recent evidence on the self-splicing and reverse-splicing abilities of Group II introns supports the hypothesis that these could have been the ancestors of classical introns.

Animals↗

PubMed1994

Sequencing of eleven introns in genomic DNA encoding rat glucagon receptor and multiple alternative splicing of its mRNA.

We used the PCR (polymerase chain reaction) to amplify fragments of glucagon receptor DNA from genomic DNA. Sequencing of the subcloned fragments demonstrated that genomic DNA encoding the glucagon receptor spans over 12 exons interrupted by 11 introns. The introns were located mainly at the 5' end and in the core domain of the glucagon receptor CDS totalling 23 kb. Intron positions were similar to the positions of introns in growth hormone-releasing hormone receptor and parathyroid hormone receptor, two receptors belonging to the same receptor family as the glucagon receptor. This high number of introns might be the cause of the mRNA polymorphism observed at the 5' end: when PCR was performed on cDNA using primers amplifying the central or 3' end cDNA fragments, a single band corresponding to the cloned cDNA was observed. In contrast, if primers amplifying cDNA fragments corresponding to nucleotides -8 to 680 of CDS were used, cDNA fragments of approximately 500 bp, 600 bp, 700 bp, 800 bp and 900 bp were specifically and reproducibly amplified. Sequencing of these fragments showed either incomplete intron removal or splicing at alternative positions. Two of these sequenced variants were translatable in putative glucagon receptor variants: (1) unsplicing of intron III (81 bp) gave an additional 27 amino acid sequence after Lys91 in the N-terminal domain of the receptor. In the liver, where the normal CDS represented about one third of the mRNA molecules, this mRNA variant represented 18% of total mRNA forms; (2) a 21 bp deletion in exon V giving rise to a putative deletion of 7 amino acids in glucagon receptor (delta 64-84 CDS) was also relatively abundant in the liver (10%). The observed polymorphism of the glucagon receptor mRNA may contribute to the regulation of glucagon receptor expression and perhaps to the heterogeneity of these receptors.

Alternative Splicing↗

PubMed1989

The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing.

Plant cells do not in general process the introns of transcripts expressed from introduced vertebrate genes. By studying the processing of model introns in transfected plant protoplasts, we have investigated the special requirements for intron recognition by plant cells. Our results indicate that the requirements for intron recognition in plants are different from those of both metazoa and yeast. A synthetic intron of arbitrary sequence but incorporating splice site consensus sequences and a high proportion of U and A nucleotides, a characteristic feature of plant introns, was efficiently spliced in protoplasts. We have studied the effects of various sequence alterations and conclude that AU-rich sequences are necessary for intron recognition. In addition, we find that the criteria for branch site selection are relaxed, as they are in vertebrates, but a polypyrimidine tract is not necessary.

Base Sequence↗

PubMed1990

A self-splicing group I intron in the DNA polymerase gene of Bacillus subtilis bacteriophage SPO1.

We report a self-splicing intron in bacteriophage SPO1, whose host is the gram-positive Bacillus subtilis. The intron contains all the conserved features of primary sequence and secondary structure previously described for the group IA introns of eukaryotic organelles and the gram-negative bacteriophage T4. The SPO1 intron contains an open reading frame of 522 nucleotides. As in the T4 introns, this open reading frame begins in a region that is looped out of the secondary structure, but ends in a highly conserved region of the intron core. The exons encode SPO1 DNA polymerase, which is highly similar to E. coli DNA polymerase I. The demonstration of self-splicing introns in viruses of both gram-positive and gram-negative eubacteria lends further evidence for their early origin in evolution.

Amino Acid Sequence↗

PubMed1994

Presence of regulatory sequences within intron 4 of human and murine c-myb genes.

The molecular mechanisms that modulate c-myb mRNA transcription in hematopoietic cells appear to involve intron regulatory sequences. We have characterized the fourth of ten introns from both human and murine c-myb genes in regard to nucleotide sequence and specific protein binding. For this approach complete genomic c-myb intron 4 fragments were isolated from mouse and human DNA using PCR amplification with flanking exon-primers derived from the mouse gene. Comparison of the obtained sequences revealed strong homology between the two species. Using crude nuclear protein extracts from mouse and human myb expressing cells (70Z/3B; Molt4) and gel shift experiments we found specific protein interaction for both introns and to determine the protein binding site in detail, we performed DNase I footprinting. Our results indicate that the binding factor is absent in control cell lines without c-myb transcriptional activity, suggesting a possible positive regulatory function of the DNA-protein complex. To confirm these findings we introduced the human c-myb intron 4 DNA sequence into the EcoRI site of the pCAT-Promoter plasmid and transfected Molt4 cells with this chimeric construct. The transient expression studies revealed that intron 4 sequences possess enhancer activity. Thus, we have demonstrated that intron 4 sequences can be important for the regulation of c-myb proto-oncogene expression.

Animals↗

PubMed1989

Comparative and functional anatomy of group II catalytic introns--a review.

The 70 published sequences of group II introns from fungal and plant mitochondria and plant chloroplasts are analyzed for conservation of primary sequence, secondary structure and three-dimensional base pairings. Emphasis is put on structural elements with known or suspected functional significance with respect to self-splicing: the exon-binding and intron-binding sites, the bulging A residue involved in lariat formation, structural domain V and two isolated base pairs, one of them involving the last intron nucleotide and the other one, the first nt of the 3' exon. Separate sections are devoted to the 29 group II-like introns from Euglena chloroplasts and to the possible relationship of catalytic group II introns to nuclear premessenger introns. Alignments of all available sequences of group II introns are provided in the APPENDIX.

Animals↗

PubMed1990

Structural and functional analysis of the first intron of the human alpha 2(I) collagen-encoding gene.

The nucleotide (nt) sequence of the first intron of the human alpha 2(I) collagen-encoding gene (COL1A2) has been determined from its 5' terminus (nt 207) to nt 2045 with respect to the transcription start point. Although the first intron contains elements known to function in transcriptional regulation of other genes (two AP1-binding sites and an alternating GT stretch), comparison of this sequence with that of the mouse COL1A2 first intron revealed a low degree of nt sequence identity and very few common DNA-protein binding motifs. In keeping with this structural analysis, the human intron was found to inhibit COL1A2 promoter activity in transfection experiments, whereas a strong enhancer was reported to be present in the first intron of mouse COL1A2 [Rossi and deCrombrugghe, Proc. Natl. Acad. Sci. USA 84 (1987) 5590-5594]. We conclude that the high degree of nt sequence conservation existing in the promoter and first exons of human, mouse and chicken COL1A2 does not extend to the first introns of these genes but that the promoter activity of COL1A2 is strongly influenced by the presence of the first intron.

Animals↗

PubMed1992

Characterization of the rRNA-encoding genes and transcripts, and a group-I self-splicing intron in Pneumocystis carinii.

Although Pneumocystis carinii is the most common opportunistic pathogen infecting individuals with AIDS, very little is known of the basic biology of the organism. We have examined the ribosomal RNA (rRNA) and the DNA encoding it (rDNA) in P. carinii in an attempt to clarify its taxonomic position and to begin to study its genetic processes. Electrophoretic analysis showed that the sizes of the P. carinii rRNAs are quite similar to the sizes of the corresponding rRNAs from Saccharomyces cerevisiae. Direct sequence analysis of approx. 60% of the 18S small subunit-rRNA (Ss-rRNA) confirmed that its sequence is similar to that of yeast-like fungi and that a putative group-I intron previously observed in the 18S rDNA is, in fact, excised from the mature rRNA. PCR analysis of the intron in P. carinii genomic DNA showed that each of the multiple rDNA genes bears the group-I intron and in vitro transcripts of the intron autocatalytically excise from the rRNA primary transcript in the presence of GTP. Finally, analogues of GTP inhibit the self-splicing reaction, indicating that the guanosine-binding site of the intron closely resembles that of other well-characterized group-I introns. Since no group-I introns have been found in higher eukaryotes, this self-splicing process represents a viable target for chemotherapy of P. carinii pneumonia (PCP).

Antifungal Agents↗

PubMed1995

A group-I intron in the mitochondrial large-subunit ribosomal RNA-encoding gene of Dictyostelium discoideum: same site localization in alga and in vitro self-splicing.

A 547-bp group-I intron belonging to subgroup IA1 was found near the 3' end of the large subunit ribosomal RNA-encoding gene (LSUrRNA) in the mitochondrial (mt) DNA of the cellular slime mold Dictyostelium discoideum. This intron was inserted in a highly conserved stretch within the sequence that encodes the peptidyl transferase center domain V in the corresponding region of the Escherichia coli LSUrRNA. Interestingly, the insertion site of the intron is the same as that of the So.LSU.2 intron of the green alga, Scenedesmus obliquus, mt DNA and the Pw.LSU.2 intron of the colorless alga, Prototheca wickerhamii, mt DNA. The intron could self-splice in vitro at a concentration higher than 20 mM MgCl2. Polymerase chain reaction analysis showed the possible existence of an intron similar to that of D. discoideum LSUrRNA in another cellular slime mold, Polysphondylium pallidum (CK-8), but not in D. mucoroides (Dm7 and Dm11).

Animals↗

PubMed2003

Detection of potential positive regulatory motifs of transcription in yeast introns by comparative analysis of oligonucleotide frequencies.

We conducted a comparative statistical analysis of tetra- through hexanucleotide frequencies in two sets of introns of yeast genes. The first set consisted of introns of genes that have transcription rates higher than 30 mRNAs/h while the second set contained introns of genes whose transcription rates were lower than or equal to 10 mRNAs/h. Some oligonucleotides whose occurrence frequencies in the first set of introns are significantly higher than those in the second set of introns were detected. The frequencies of occurrence of most of these detected oligonucleotides are also significantly higher than those in the exons flanking the introns of the first set. Interestingly some of these detected oligonucleotides are the same as well known "signature" sequences of transcriptional regulatory elements. This could imply the existence of potential positive regulatory motifs of transcription in yeast introns.

Databases, Factual↗

PubMed2005

Systematic screening of nuclear encoded proteins involved in the splicing metabolism of group II introns in yeast mitochondria.

Studies of yeast, algae and plants have provided genetic and biochemical evidence that the splicing reaction of organellar localized group II introns either depends on proteins encoded by the introns themselves ('maturases') or encoded by other genes of the host organisms. However, only a few of those proteins have been identified to date and characterized in more detail. In order to find new nuclear encoded proteins that assist group II splicing, we screened a complete knockout library of Saccharomyces cerevisiae strain BY4741 consisting of 4878 viable haploid clones. The strain contains a rho+ mitochondrial genome with a set of 13 introns including the three group II introns (aI1, aI2, aI5gamma) in the gene encoding cytochrome-c-oxidase subunit 1 (COX1) and the single group II intron (bI1) in the gene encoding cytochrome b (CYTB). In our screen and initial molecular analysis, we focus on intron aI5gamma, the last intron in the COX1 gene.

Cell Nucleus↗

PubMed2006

Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity.

The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.

Adenosine Triphosphate↗

PubMed2006

The receptor for branch-site docking within a group II intron active site.

The distinguishing feature of group II introns, and the property that links them with spliceosomal catalysis, is their ability to undergo splicing through branching. In this reaction, the 2'-hydroxyl group of a specific adenosine within intron domain 6 serves as the nucleophile for attack on the 5' splice site. We know less about branching than any other feature of group II intron catalysis, largely because the receptor structure for activating the branch site is unknown. Here, we identify the intronic region that binds the branch site of a group IIB intron. Located in domain 1, close to receptors for intron domain 5 and both splice sites, we demonstrate that the branch-site receptor is a functional element required for transesterification. Furthermore, we show that crosslinked branch sites can carry out both steps of splicing, suggesting that the conformational state of the intron core is set early and that it persists throughout the entire splicing process.

Binding Sites↗

PubMed2004

A non-consensus branch point plays an important role in determining the stability of the 2-kb LAT intron during acute and latent infections of herpes simplex virus type-1.

Herpes simplex virus type 1 (HSV-1) establishes lifelong latent infection in sensory neurons of the peripheral nervous system. During HSV latency, the latency-associated transcripts (LATs) are the only viral transcripts abundantly expressed. The most abundant form of LATs is a 2-kb stable intron spliced from a primary transcript (mLAT). It has been previously reported that a non-consensus branch point influences the stability of the intron (in vitro) in cells transfected with plasmid constructs (J. Virol. 71 (1997) 5849; J. Virol. 71 (1997) 4199). However, it is unknown whether this branch point is important in determining LAT stability in vivo (in the context of virus). To study the role of this stable intron in HSV-1 infection, we have constructed a mutant virus KOS-CONS in which the branch point has been mutated to consensus branch point nucleotides. The accumulation of the 2-kb intron in KOS-CONS-infected cells was greatly reduced. The LAT intron was not detectable in KOS-CONS-infected mouse trigeminal ganglia (TG) during acute and latent phase infection by Northern blot analysis. Replication of the KOS-CONS and the wild-type KOS viruses on Vero cells was determined to be similar, as was the level of HSV-1 DNA in mouse trigeminal ganglia during acute and latent phase infection. Using the mouse TG explant model, the reactivation pattern of both viruses was shown to be similar. Our data suggest that the unique branch point plays a significant role in determining the stability of LAT intron in vivo, but that the stability of the intron does not appear to affect HSV-1 replication, the establishment of latency, or viral reactivation.

Acute Disease↗

PubMed2006

The stable 2-kb LAT intron of herpes simplex stimulates the expression of heat shock proteins and protects cells from stress.

During latency of herpes simplex virus type 1 (HSV-1), the major viral transcript that is detected is the latency-associated transcript (LAT) 2-kb intron. This intron is excised from a larger (approximately 10 kb) primary transcript. Infection of cells with HSV expressing LATs showed that their presence increased accumulation of Hsp70 protein specifically during cold shock. Transfection of cells with a plasmid, expressing the 2-kb LAT intron, showed increased translation compared to cells transfected with plasmids deleted in regions of the intron. Cold shock of cells expressing the intron resulted in an up-regulation of Hsp70 protein, but not Hsp70 mRNA. Furthermore, these cells showed increased cell viability. Using plasmid deletion mutants in the LAT gene sequence, we have shown that the effect requires full-length LAT intron. These findings show that a function of the stable 2-kb LAT intron is to protect cells from cold-induced stress and that this may be mediated via Hsp70.

Cold Temperature↗

PubMed2003

Mobility of the Sinorhizobium meliloti group II intron RmInt1 occurs by reverse splicing into DNA, but requires an unknown reverse transcriptase priming mechanism.

The mobile group II introns characterized to date encode ribonucleoprotein complexes that promote mobility by a major retrohoming mechanism in which the intron RNA reverse splices directly into the sense strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase/maturase cleaves the antisense strand and uses it as primer for reverse transcription of the inserted intron RNA. Here, we show that the Sinorhizobium meliloti group II intron RmInt1, which encodes a protein lacking a DNA endonuclease domain, similarly uses both the intron RNA and an intron-encoded protein with reverse transcriptase and maturase activities for mobility. However, while RmInt1 reverse splices into both single-stranded and double-stranded DNA target sites, it is unable to carry out site-specific antisense-strand cleavage due to the lack of a DNA endonuclease domain. Our results suggest that RmInt1 mobility involves reverse splicing into double-stranded or single-stranded DNA target sites, but due to the lack of DNA endonuclease function, it requires an alternate means of procuring a primer for target DNA-primed reverse transcription.

Base Sequence↗

PubMed1989

Self-splicing introns as a source for transposable genetic elements.

Previous theories have suggested that some introns with the ability to self-splice are derived from transposable elements. However, an interpretation is given here that suggests retrotransposons and retroviruses (transposable elements which move via RNA intermediates) have evolved from self-splicing introns. This is based on the involvement of RNA intermediates, the ancestral nature of the self-splicing reaction, and the assumed presence of introns in an RNA world. Conserved sequences within the introns, essential for splicing, and their wide phylogenetic distribution also make it unlikely that they are descended from transposable elements. Mitochondrial plasmids of Neurospora species containing features of both introns and retrotransposons have a central role in the resolution of the problem and are considered here to support the view that introns are, or have been, sources of mobile elements. The possibility of other transposable elements arising from introns is also considered.

Animals↗