A mixed group II/group III twintron in the Euglena gracilis chloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution.

The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre-mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts.     

Chloroplast gene for Mr 32000 polypeptide of photosystem II in Euglena gracilis is interrupted by four introns with conserved boundary sequences

The gene for the Mr 32000 herbicide binding polypeptide of photosystem II has previously been mapped to the 5 kbp EcoRI fragment Eco I of Euglena gracilis chloroplast DNA. The nucleotide sequence of 3324 bp of Eco I, containing the psbA locus, has been determined. This locus encodes a polypeptide of 345 amino acids which is co-linear with, and has 86% derived amino acid sequence homology to sequences derived from four higher plants chloroplast psbA loci. The Euglena psbA gene contains four introns of size 435, 443, 434, and 617 bp. The four introns have conserved boundary sequences of the type previously described in the Euglena chloroplast gene (rbcL) for the large subunit of ribulose-1,5-bisphosphate carboxylase (Koller et al., Cell 36, 545-553, 1984).     

The enigma of the gene coding for ribosomal protein S12 in the chloroplasts of Nicotiana.

A 2.9 kbp region from within the inverted repeat of Nicotiana chloroplast DNA hybridized with a chloroplast DNA fragment from Euglena containing the complete rps12 gene coding for ribosomal protein S12. Nucleotide sequencing within this region revealed the existance of two rps12 coding stretches interrupted by 540 bp having class II intron structure. Joining and decoding the exon regions produced a sequence of 85 amino acids colinear and 81% homologous to the S12 protein of Euglena chloroplasts and E. coli, starting from amino acid residue 38 to the stop codon. Immediately upstream of codon 38, conserved intron sequences were located. However, the 5' 37 codon of Nicotiana chloroplast rps12 could not be identified by electron microscopy of RNA-DNA hybrids within a DNA region extending 4000 bp upstream of codon 38, nor by computer search of a completely sequenced region extending for more than 9000 bp upstream of this codon. In E. coli, alteration in rps12 codons 42 or 87 causes streptomycin resistance. However, the nucleotide sequence of the identified rps12 exons in two Nicotiana chloroplast mutants resistant to streptomycin were found to be identical to that of wild type.     

Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants

The excised introns of pre-mRNAs and intron-containing splicing intermediates are in a lariat configuration in which the 5' end of the intron is linked by a 2'-5' phosphodiester bond (RNA branch) to a single adenosine residue near the 3' end of the intron. To determine the role of the specific sequence surrounding the RNA branch, we have mutated the branch point sequence of the human beta-globin IVS1. Pre-mRNAs lacking the authentic branch point sequence are accurately spliced in vitro; processing of the mutant pre-mRNAs generates RNA lariats due to the activation of cryptic branch points within IVS1. The cryptic branch points always occur at adenosine residues, but the sequences surrounding the branched nucleotide vary. Regardless of the type of mutation or the sequences remaining within IVS1, the cryptic branch points are 22 to 37 nucleotides upstream of the 3' splice site. These results suggest that RNA branch point selection is primarily based on a mechanism that measures the distance from the 3' splice site.     

Chloroplast group III twintron excision utilizing multiple 5''- and 3''-splice sites.

The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.     

The Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene. I. Complete DNA sequence and analysis of the nine intervening sequences

The nucleotide sequence of 6225 base pairs (bp) of Euglena gracilis chloroplast DNA including the complete DNA sequence of the chloroplast-encoded ribulose-1,5-bisphosphate carboxylase large subunit gene along with the flanking DNA sequences is presented. The gene is greater than 5.5 kilobase pairs in length and is organized as 10 exons coding for 475 amino acids, separated by 9 introns. The exons range in size from 45 to 438 bp, while the introns range in size from 382 to 568 bp. The introns have highly conserved boundary sequences with the consensus, 5'-N GTGTGGATTT...(intron)...TTAATTTTAT N-3'. The introns are 82-85 mol% AT, with a pronounced T greater than A greater than G greater than C base bias in the RNA-like strand. They do not appear to encode any polypeptides. In addition, the introns have a conserved sequence 30-50 bp from their 3'-ends with the consensus, 5'-TACAGTTTGAAAATGA-3'. The 5'-TACA sequence bears some homology to the 5'-end of the TACTAACA sequence found in a similar location in yeast nuclear mRNA introns. The conserved sequences of the Euglena rbcL introns may be indicative of a splicing mechanism similar to that of eucaryotic nuclear mRNA introns and group II mitochondrial introns.     

A group III twintron encoding a maturase-like gene excises through lariat intermediates.

The 1605 bp intron 4 of the Euglena gracilis chloroplast psbC gene was characterized as a group III twintron composed of an internal 1503 nt group III intron with an open reading frame of 1374 nt (ycf13, 458 amino acids), and an external group III intron of 102 nt. Twintron excision proceeds by a sequential splicing pathway. The splicing of the internal and external group III introns occurs via lariat intermediates. Branch sites were mapped by primer extension RNA sequencing. The unpaired adenosines in domains VI of the internal and external introns are covalently linked to the 5' nucleotide of the intron via 2'-5' phosphodiester bonds. This bond is susceptible to hydrolysis by the debranching activity of the HeLa nuclear S100 fraction. The internal intron and presumptive ycf13 mRNA accumulates primarily as a linear RNA, although a lariat precursor can also be detected. The ycf13 gene encodes a maturase-like protein that may be involved in group III intron metabolism.     

A complex twintron is excised as four individual introns.

Twintrons are introns-within-introns excised by sequential splicing reactions. A new type of complex twintron comprised of four individual group III introns has been characterized. The external intron is interrupted by an internal intron containing two additional introns. This 434 nt complex twintron within a Euglena gracilis chloroplast ribosomal protein gene is excised by four sequential splicing reactions. Two of the splicing reactions utilize multiple 5'- and/or 3'-splice sites. These findings are evidence that introns with multiple active splice sites can be formed by the repeated insertion of introns into existing introns.     

A self-splicing RNA excises an intron lariat

We have investigated the in vitro self-splicing of a class II mitochondrial intron. A model pre-mRNA containing intron 5 gamma of the oxi 3 gene of yeast mitochondrial DNA undergoes an efficient intramolecular rearrangement reaction in vitro. This reaction proceeds under conditions distinct from those optimal for self-splicing of class I introns, such as the Tetrahymena nuclear rRNA intron. Intron 5 gamma is excised as a nonlinear RNA indistinguishable from the in vivo excised intron product by gel electrophoresis and primer extension analysis. Studies of the in vitro excised intron product strongly indicate that it is a branched RNA with a circular component joined by a linkage other than a 3'-5' phosphodiester. Two other products, the spliced exons and the broken form of the lariat, were also characterized. These results show that the class II intron products are similar to those of nuclear pre-mRNA splicing.     

Nine introns with conserved boundary sequences in the Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene

The single, chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Euglena gracilis is found to contain nine intervening sequences. The intervening sequences were identified by heteroduplex analysis between Euglena rbcL and the non-intron-containing rbcL from Spinacea oleracea, by electron microscopy of Euglena rbcL DNA-mRNA hybrids, and by cloning, restriction endonuclease analysis, and partial DNA sequencing. The identification, locus, and coding properties for six of ten exons was confirmed by partial DNA sequence analysis. Each of the nine introns in the approximately 6.5 kb rbcL locus is approximately 0.5 kb in length. The DNA sequence of five 3'-intron/5'-exon and four 3'-exon/5'-intron boundaries are highly conserved. A proposed consensus sequence is (formula; see text) These conserved sequences could play a role in an mRNA splicing mechanism in chloroplasts analogous to that in eucaryotic nuclei.