Abortive transposition by a group II intron in yeast mitochondria

Group II intron homing in yeast mitochondria is initiated at active target sites by activities of intron-encoded ribonucleoprotein (RNP) particles, but is completed by competing recombination and repair mechanisms. Intron aI1 transposes in haploid cells at low frequency to target sites in mtDNA that resemble the exon 1-exon 2 (E1/E2) homing site. This study investigates a system in which aI1 can transpose in crosses (i.e., in trans). Surprisingly, replacing an inefficient transposition site with an active E1/E2 site supports <1% transposition of aI1. Instead, the ectopic site was mainly converted to the related sequence in donor mtDNA in a process we call "abortive transposition." Efficient abortive events depend on sequences in both E1 and E2, suggesting that most events result from cleavage of the target site by the intron RNP particles, gapping, and recombinational repair using homologous sequences in donor mtDNA. A donor strain that lacks RT activity carries out little abortive transposition, indicating that cDNA synthesis actually promotes abortive events. We also infer that some intermediates abort by ejecting the intron RNA from the DNA target by forward splicing. These experiments provide new insights to group II intron transposition and homing mechanisms in yeast mitochondria.     

The group II intron ribonucleoprotein precursor is a large, loosely packed structure

Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state.     

Multiple requirements for nematode spliced leader RNP function in trans-splicing.

The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.     

Characterization of the lactococcal group II intron target site in its native host

The Lactococcus lactis group II intron (Ll.ltrB) retrohomes into the ltrB gene at high efficiency. To date, the critical DNA bases recognized in vivo by the Ll.ltrB ribonucleoprotein (RNP) have been exclusively elucidated in Escherichia coli. However, recent evidence indicates host-dependant differences in Ll.ltrB mobility, raising the possibility of limitations of the current model for RNP-homing site recognition in the native L. lactis host. In this work, intron retargeting experiments in L. lactis have demonstrated that adherence to specific target site critical bases is not sufficient to predict success or failure of chromosomal invasion, as in E. coli. Accordingly, a quantitative real-time PCR (QPCR) assay was developed to test target site nucleotides previously demonstrated as critical for homing in E. coli, for relevance in its native host. This two-plasmid QPCR homing assay is highly sensitive and, unlike previous E. coli-based assays, resolves differential homing efficiencies in the absence of selection. As in E. coli, deviation from wild type at target site positions -23, -21, -20, -19, and +5 resulted in lower homing efficiencies in L. lactis. Furthermore, the same trends are observed when assaying select variants in Enterococcus faecalis. Our results suggest that these target site positions are critical in both E. coli and L. lactis.     

Quaternary arrangement of an active,native group II intron ribonucleoprotein complex revealed by small-angle X-ray scattering

The stable ribonucleoprotein (RNP) complex formed between the Lactococcus lactis group II intron and its self-encoded LtrA protein is essential for the intron’s genetic mobility. In this study, we report the biochemical, compositional, hydrodynamic and structural properties of active group II intron RNP particles (+A) isolated from its native host using a novel purification scheme. We employed small-angle X-ray scattering to determine the structural properties of these particles as they exist in solution. Using sucrose as a contrasting agent, we derived a two-phase quaternary model of the protein–RNA complex. This approach revealed that the spatial properties of the complex are largely defined by the RNA component, with the protein dimer located near the center of mass. A transfer RNA fusion engineered into domain II of the intron provided a distinct landmark consistent with this interpretation. Comparison of the derived +A RNP shape with that of the previously reported precursor intron (ΔA) particle extends previous findings that the loosely packed precursor RNP undergoes a dramatic conformational change as it compacts into its active form. Our results provide insights into the quaternary arrangement of these RNP complexes in solution, an important step to understanding the transition of the group II intron from the precursor to a species fully active for DNA invasion.     

Group II intron-based gene targeting reactions in eukaryotes

Background

Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors (“targetrons”) with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.

Methodology/Principal Findings

By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg²⁺ concentrations. By supplying additional Mg²⁺, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg²⁺-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.

Conclusions/Significance

Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.     

Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

H/ACA RNP complexes change uridines to pseudouridines in target non-coding RNAs in eukaryotes and archaea. H/ACA RNPs are comprised of a guide RNA and four essential proteins: Cbf5 (pseudouridine synthase), L7Ae, Gar1 and Nop10 in archaea. The guide RNA captures the target RNA via two antisense elements brought together to form a contiguous binding site within the pseudouridylation pocket (internal loop) of the guide RNA. Cbf5 and L7Ae interact independently with the guide RNA, and here we have examined the impacts of these proteins on the RNA in nucleotide protection assays. The results indicate that the interactions observed in a fully assembled H/ACA RNP are established in the sub-complexes, but also reveal a unique Cbf5–guide RNA interaction that is displaced by L7Ae. In addition, the results indicate that L7Ae binding at the kink (k)-turn of the guide RNA induces the formation of the upper stem, and thus also the pseudouridylation pocket. Our findings indicate that L7Ae is essential for formation of the substrate RNA binding site in the archaeal H/ACA RNP, and suggest that k-turn-binding proteins may remodel partner RNAs with important effects distant from the protein-binding site.     

Mutational analysis of simian virus 40 large T antigen DNA binding sites.

We have tested the effects of various mutations within SV40 T antigen DNA recognition sites I and II on specific T antigen binding using the DNase footprint technique. In addition, the replication of plasmid DNA templates carrying these T antigen binding site mutations was monitored by Southern analysis of transfected DNA in COS cells. Deletion mapping of site I sequences defined a central core of approximately 18 bp that is both necessary and sufficient for T antigen recognition; this region contains the site I contact nucleotides that were previously mapped using methylation-interference and methylation-protection experiments. A similar deletion analysis delineated sequences that impart specificity of binding to site II. We find that T antigen is capable of specific recognition of site II in the absence of site I sequences, indicating that binding to site II in vitro is not dependent on binding of T antigen at site I. Site II binding was not diminished by small deletion or substitution mutations that perturb the 27-bp palindrome central to binding site II, whereas extensive substitution of site II sequences completely eliminated specific site II binding. Analysis of the replication in COS7 cells of plasmids that contain these mutant origins revealed that sequences both at the late side of binding site I and within the site II palindrome are crucial for viral DNA replication, but are not involved in binding T antigen.     

Identification of a second DNA binding site in the human Rad52 protein

Rad52 plays essential roles in homology-dependent double-strand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the Rad52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that Rad52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that Rad52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.