Insertion of group II intron retroelements after intrinsic transcriptional terminators

Abstract

Mobile DNAs use many mechanisms to minimize damage to their hosts. Here we show that a subclass of group II introns avoids host damage by inserting directly after transcriptional terminator motifs in bacterial genomes (stem-loops followed by Ts). This property contrasts with the site-specific behavior of most group II introns, which insert into homing site sequences. Reconstituted ribonucleoprotein particles of the Bacillus halodurans intron B.h.I1 are shown to reverse-splice into DNA targets in vitro but require the DNA to be single-stranded and fold into a stem-loop analogous to the RNA structure that forms during transcription termination. Recognition of this DNA stem-loop motif accounts for in vivo target specificity. Insertion after terminators is a previously unrecognized strategy for a selfish DNA because it prevents interruption of coding sequences and restricts expression of the mobile DNA after integration.reverse transcriptase ͉ ribozyme ͉ mobile DNA ͉ bacteria R eduction of host damage is a basic principle of survival for selfish DNAs. The most common mechanisms involve suppression of transcription and translation, and integration into comparatively innocuous sites (1-4). Among retroelements, for example, R2 elements insert site-specifically into defined sequences within rDNA arrays while leaving most rDNAs intact (5). LINE1 elements have a less stringent preference for the sequence TTTTA, which results in integrations into gene-poor regions (6). Other retroelements target genomic loci through protein-protein interactions, to insert at pol III promoters (7), into telomeric elements (8), or into heterochromatin regions (9).Group II intron retroelements avoid host damage in two ways. First, the introns splice out of interrupted sequence at the RNA level to reconstitute functional coding sequence. Second, the introns insert site-specifically into compatible targets through retrohoming, thereby limiting potential targets. The mechanism of retrohoming is well characterized and is carried out by a ribonucleoprotein (RNP) complex composed of excised intron lariat and intron-encoded protein (IEP). The process is initiated by reverse splicing of lariat RNA into the top strand of a DNA target. The bottom strand is cleaved by the endonuclease domain of the IEP, and the integrated intron is reverse transcribed by the IEP, using the cleaved DNA as a primer. Repair processes complete the insertion steps (10-12).Normally, retrohoming of group II introns is highly sitespecific because of an Ϸ20-to 35-bp target sequence. Thirteen positions of the DNA target are recognized by base pairings between the intron RNA and the DNA exons via the interactions IBS1-EBS1, IBS2-EBS2 (intron and exon binding sites 1 and 2; 6 bp each), and either ␦-␦Ј (IIA introns; 1 bp) or IBS3-EBS3 (IIB, IIC introns; 1 bp). Additional target-specificity comes from IEP recognition of upstream exon DNA sequences Ϫ23 to Ϫ1 and downstream exon sequences ϩ4 to ϩ9 (11,13,14).In contrast to such site-specificity, introns of the phylogenetic subclass ''b...

Group II introns in Eubacteria and Archaea: ORF-less introns and new varieties

“…Overall, slightly more than half of the introns in our database are found in predicted intergenic regions, with approximately half of these belonging to class C. This is expected as class C introns target transcriptional terminators (Granlund et al 2001;Dai and Zimmerly 2002;Robart et al 2007). The remainder of introns are found in mobile DNA ORFs (19%), hypothetical genes (18%), or housekeeping genes (8%).…”

“…At a much lower frequency, introns are able to invade noncognate sites through retrotransposition (Cousineau et al 2000;Martínez-Abarca and Toro 2000b;Ichiyanagi et al 2002). A third substrate specificity is seen for a phylogenetic subclass (class C, below) in which the introns insert after diverse intrinsic terminator motifs (Granlund et al 2001;Dai and Zimmerly 2002;Robart et al 2007). …”

“…The most well-established example is class C, whose introns insert directly after terminator motifs (Granlund et al 2001;Dai and Zimmerly 2002;Robart et al 2007). Interestingly, within class C is a lineage of eight introns with a different insertion pattern: they insert into the inverted repeats of attC sites of integrons rather than the inverted repeats of terminators ( Fig.…”

“…In addition, each ORF phylogenetic class is associated with a distinct RNA secondary structure (Toor et al 2001;Ferat et al 2003;Toro 2003). For example, classes ML and CL have IIA and IIB secondary structures, respectively (Michel et al 1989), while class C introns have IIC structures that exhibit a number of unique features (Martínez-Abarca and Toro 2000a;Granlund et al 2001;Toor et al 2001;Robart et al 2007).…”

Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani

“…IBS1-EBS1 (intron/exon-binding site 1) is a 6-bp interaction between EBS1 in D1 of the intron and IBS1 of the 5 ′ exon , while IBS2-EBS2 occurs between another sequence in D1 (EBS2) and the sequence immediately upstream of IBS1 in the exon (IBS2). IIC introns have only a 4-bp IBS1-EBS1 pairing and no IBS2-EBS2 pairing (Robart et al 2007). In nearly all group II introns, the IBS1 sequence is located directly upstream of the 5 ′ GUGYG start of the intron, with no spacer between them.…”