RESUMO
The 4,144 nt Euglena gracilis chloroplast psbC intron 2 has been characterized as a single, cis-spliced 593 nt group II intron interrupted by an open reading frame of 758 codons in the loop region of domain IV. The 2,277 nt coding region of orf 758 is interrupted by two additional group II introns of 369 nt and 352 nt. Another 553 nt group II intron is located in the 5' untranslated leader region of orf 758. Because the psbC intron 2 orf encodes a maturase-like protein that has reverse transcriptase domains and a domain X characteristic of group II intron-encoded proteins, the locus has been designated mat2. The psbC intron 2 is the first member of a new category of twintron, characterized by introns within a gene within another intron. A potential role of psbC intron 2 as a "founder" intron involved in the spread of introns to new sites in the plastid genome of the Euglenophycae is discussed.
Assuntos
Cloroplastos/genética , Euglena gracilis/genética , Íntrons , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Cloroplastos/metabolismo , Primers do DNA/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Euglena gracilis/metabolismo , Genoma , Modelos Moleculares , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Protozoário/química , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A novel gene, roaA (ribosomal operon-associated gene), encoding a potential RNA-binding protein has been identified in the rpl23 ribosomal protein operon of the Euglena gracilis chloroplast genome. The roaA gene is interrupted by one group III and three group II introns. Introns 1 and 2 of roaA can be interpreted as a twintron, formed from the insertion of a group II intron into the 5'splice site of a group III intron. Alternative splicing of the group III intron results in two distinct transcripts encoding proteins of 516 and 514 amino acids. Group III introns may play a role in the generation of alternatively spliced transcripts.
Assuntos
Processamento Alternativo , Euglena gracilis/genética , Genes de Plantas , Proteínas de Protozoários , RNA Mensageiro/análise , Proteínas de Ligação a RNA/genética , Animais , Sequência de Bases , Cloroplastos/ultraestrutura , DNA Complementar , DNA de Plantas , Euglena gracilis/metabolismo , Éxons , Íntrons , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas de Plantas/metabolismo , RNA de PlantasRESUMO
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.
Assuntos
Euglena gracilis/genética , Íntrons , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Animais , Sequência de Bases , Northern Blotting , Cloroplastos/química , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , RNA/química , Precursores de RNA/análise , Precursores de RNA/química , Splicing de RNA , RNA Mensageiro/análise , RNA Mensageiro/química , Ribonuclease H/metabolismo , Análise de SequênciaRESUMO
A novel ribosomal protein operon in the Euglena gracilis chloroplast genome was characterized. It encodes the genes for ribosomal proteins S4 and S11 (rps4 and rps11). The coding region of the rps11 gene is interrupted by two introns of 107 and 100 bp. The introns belong to a distinct class known as group III introns. The major transcript from this operon was characterized as a fully spliced dicistronic rps4-rps11 mRNA by RNA blot analysis, primer extension sequencing, and cDNA cloning and sequencing. An additional 95 nucleotide (nt) group III intron was identified in the 123 nt rps4-rps11 intercistronic region. The identification of the intercistronic intron between the rps4 and rps11 genes was unexpected. Other RNA transcripts from regions of the genome that could potentially contain intercistronic introns were re-examined and two other intercistronic, group III introns were found. These are located in a large ribosomal protein operon between the genes for the ribosomal proteins L23 and L2, and between L14 and L5. There are at least 50 group III introns in the E. gracilis chloroplast genome. All but 6 are found in genes encoding protein components of the transcriptional and translational apparatus. The distribution of group III introns and the unusual location of intercistronic group III introns may reflect some aspect of gene expression, or provide some insight into the mechanism of their splicing.
