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 am ino acids), and an external group III intron of 102 nt. Twintron excis ion proceeds by a sequential splicing pathway. The splicing of the int ernal and external group III introns occurs via lariat intermediates. Branch sites were mapped by primer extension RNA sequencing. The unpai red adenosines in domains VI of the internal and external introns are covalently linked to the 5' nucleotide of the intron via 2'-5' phospho diester bonds. This bond is susceptible to hydrolysis by the debranchi ng activity of the HeLa nuclear S100 fraction. The internal intron and presumptive ycfl3 mRNA accumulates primarily as a linear RNA, althoug h a lariat precursor can also be detected. The ycfl3 gene encodes a ma turase-like protein that may be involved in group III intron metabolis m.