Twintrons: Introns-within-introns in the chloroplast genes of Euglena gracilis.

Abstract

The chloroplast genes of Euglena gracilis contain more than 100 introns. A comparison of intron content and position among plastid and prokaryote genes has led to the hypothesis that introns have been inserted into chloroplast genes during evolution. Several Euglena loci contain unusual introns. These introns have been characterized by direct primer extension cDNA sequencing, cDNA cloning and sequencing, and northern hybridization. The psbF locus has a 1042 nt intron that appears to be one group II intron inserted into domain V of another group II intron. It was determined that a 618 nt internal intron is first excised from the 1042 nt intron, resulting in a partially spliced pre-mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. The term "twintron" was used to define this new genetic element. Splicing of the internal and external introns occurs via lariat intermediates. The splicing of the 409 nt intron of the rps3 gene was also examined. This intron is a "mixed" twintron, composed of a 311 nt group II intron internal to a 98 nt group III intron. The splicing of four additional introns with mean lengths twice typical group III introns, three within the rpoC1 gene and one within the rpl16 gene, was analyzed. The 1604 nt intron in the psbC gene, which encodes orf458, was also examined. These introns are group III twintrons. Orf458 is encoded within the internal group III intron of the psbC twintron. Splicing of internal introns in three of the five group III twintrons involves multiple 5'- and/or 3'-splice sites. Excised group III introns accumulate as lariat RNAs. Twintrons represent evidence for intron insertion during gene evolution. One possible mechanism for twintron formation is by intron transposition. The disruption of functional domains by internal introns may necessitate a sequential in vivo splicing pathway, requiring excision of internal introns prior to excision of external introns. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.