Intercistronic group III introns in polycistronic ribosomal protein operons of chloroplasts

Abstract

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.

The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif

“…An alignment of representative sequences of ribosomal protein S4 from bacteria and chloroplasts. The abbreviations for each are as follows: Bstearo, B.stearothermophilus (S.E.Gerchmann and V.Ramakrishnan, unpublished); Bsubtili, B.subtilis (Grundy and Henkin, 1990); Ecoli, E.coli (Thomas et al ., 1987); Haemoph, Haemophilus influenza (Fleischmann et al ., 1995); SpinChl, spinach chloroplast (Ben Tahar et al ., 1986); TobacChl, tobacco chloroplast (Shinozaki et al ., 1986); Marchan, Marchantia polymorpha chloroplast (Ohyama et al ., 1986); Euglena, Euglena chloroplast (Stevenson et al ., 1991); Chlamydo, Chlamydomonas reinhardtii chloroplast (Randolph‐Anderson et al ., 1995). For clarity, 31 residues at the C‐terminus of the C.reinhardtii chloroplast sequence, which are unique to this species, have been omitted.…”

The Chloroplast Genome of Euglena mutabilis—Cluster Arrangement, Intron Analysis, and Intrageneric Trends

“…The intercistronic rps 11‐ rps 4 intron of E. mutabilis was found at the same position as in E. gracilis and also shared the same consensus boundary sequence of 5′‐TTGTG (Stevenson et al. ).…”

A complex twintron is excised as four individual introns

“…Perhaps the correct mRNA reading frame is recognized by a mechanism which suppresses mis-splicing of exons. Since the mRNA readine fIame is established during translation, the chloroplast 70S ribosome may play a role in the splicing of group III introns (19). The structure of group III introns or the difference in nucleotide composition between introns and exons may be recognized as signals that distinouish exons from introns.…”

“…The results of our studies of RNA processing in Euglena gracilis chloroplasts have several implications concerning intron biology. Both group II and group III introns are likely to be mobile genetic elements which insert into not only coding regions but other introns, twintrons, intercistronic spacers (19), and perhaps any RNA. Insertion of introns into existing introns is a mechanism for the assembly of introns with multiple 5'and 3 '-splice sites.…”