Methylation of rRNA as a host defense against rampant group II intron retrotransposition.

BACKGROUND: Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. RESULTS: To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. CONCLUSIONS: Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting.

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns.

Interaction between conjugative and retrotransposable elements in horizontal gene transfer.

Mobile genetic elements either encode their own mobilization machineries or hijack them from other mobile elements. Multiple classes of mobile elements often coexist within genomes and it is unclear whether they have the capacity to functionally interact and even collaborate. We investigate the possibility that molecular machineries of disparate mobile elements may functionally interact, using the example of a retrotransposon, in the form of a mobile group II intron, found on a conjugative plasmid pRS01 in Lactococcus lactis. This intron resides within the pRS01 ltrB gene encoding relaxase, the enzyme required for nicking the transfer origin (oriT) for conjugal transmission of the plasmid into a recipient cell. Here, we show that relaxase stimulates both the frequency and diversity of retrotransposition events using a retromobility indicator gene (RIG), and by developing a high-throughput genomic retrotransposition detection system called RIG-Seq. We demonstrate that LtrB relaxase not only nicks ssDNA of its cognate oriT in a sequence- and strand-specific manner, but also possesses weak off-target activity. Together, the data support a model in which the two different mobile elements, one using an RNA-based mechanism, the other using DNA-based transfer, do functionally interact. Intron splicing facilitates relaxase expression required for conjugation, whereas relaxase introduces spurious nicks in recipient DNA that stimulate both the frequency of intron mobility and the density of events. We hypothesize that this functional interaction between the mobile elements would promote horizontal conjugal gene transfer while stimulating intron dissemination in the donor and recipient cells.

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-ribosome association protects intron RNA from degradation.

The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility.

Redox-responsive zinc finger fidelity switch in homing endonuclease and intron promiscuity in oxidative stress.

It is well understood how mobile introns home to allelic sites, but how they are stimulated to transpose to ectopic locations on an evolutionary timescale is unclear. Here we show that a group I intron can move to degenerate sites under oxidizing conditions. The phage T4 td intron endonuclease, I-TevI, is responsible for this infidelity. We demonstrate that I-TevI, which promotes mobility and is subject to autorepression and translational control, is also regulated posttranslationally by a redox mechanism. Redox regulation is exercised by a zinc finger (ZF) in a linker that connects the catalytic domain of I-TevI to the DNA binding domain. Four cysteines coordinate Zn(2+) in the ZF, which ensures that I-TevI cleaves its DNA substrate at a fixed distance, 23-25 nucleotides upstream of the intron insertion site. We show that the fidelity of I-TevI cleavage is controlled by redox-responsive Zn(2+) cycling. When the ZF is mutated, or after exposure of the wild-type I-TevI to H(2)O(2), intron homing to degenerate sites is increased, likely because of indiscriminate DNA cleavage. These results suggest a mechanism for rapid intron dispersal, joining recent descriptions of the activation of biomolecular processes by oxidative stress through cysteine chemistry.

Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Tuberculosis (TB) is a major global health problem, infecting millions of people each year. The causative agent of TB, Mycobacterium tuberculosis, is one of the world's most ancient and successful pathogens. However, until recently, no work on small regulatory RNAs had been performed in this organism. Regulatory RNAs are found in all three domains of life, and have already been shown to regulate virulence in well-known pathogens, such as Staphylococcus aureus and Vibrio cholera. Here we report the discovery of 34 novel small RNAs (sRNAs) in the TB-complex M. bovis BCG, using a combination of experimental and computational approaches. Putative homologues of many of these sRNAs were also identified in M. tuberculosis and/or M. smegmatis. Those sRNAs that are also expressed in the non-pathogenic M. smegmatis could be functioning to regulate conserved cellular functions. In contrast, those sRNAs identified specifically in M. tuberculosis could be functioning in mediation of virulence, thus rendering them potential targets for novel antimycobacterials. Various features and regulatory aspects of some of these sRNAs are discussed.

Global regulators orchestrate group II intron retromobility.

Group II introns are hypothesized to share common ancestry with both nuclear spliceosomal introns and retrotransposons, which collectively occupy the majority of genome space in higher eukaryotes. These phylogenetically diverse introns are mobile retroelements that move through an RNA intermediate. Disruption of Escherichia coli genes encoding enzymes that catalyze synthesis of global regulators cAMP and ppGpp inhibits group II intron retromobility. These small molecules program genetic transitions between nutrient excess and starvation. Accordingly, we demonstrated that glucose depletion of wild-type cells and cAMP supplementation of mutants stimulated retromobility. Likewise, amino acid starvation, which induces the alarmone ppGpp, activated retromobility. In both cases, retrotransposition to ectopic sites was favored over retrohoming. Interestingly, these stimulatory effects are mediated at the level of the DNA target, rather than of expression of the retroelement. Thereby, during metabolic stress, cAMP and ppGpp control group II intron movement in concert with the cell's global genetic circuitry, stimulating genetic diversity.

Homing endonuclease I-TevIII: dimerization as a means to a double-strand break.

Homing endonucleases are unusual enzymes, capable of recognizing lengthy DNA sequences and cleaving site-specifically within genomes. Many homing endonucleases are encoded within group I introns, and such enzymes promote the mobility reactions of these introns. Phage T4 has three group I introns, within the td, nrdB and nrdD genes. The td and nrdD introns are mobile, whereas the nrdB intron is not. Phage RB3 is a close relative of T4 and has a lengthier nrdB intron. Here, we describe I-TevIII, the H-N-H endonuclease encoded by the RB3 nrdB intron. In contrast to previous reports, we demonstrate that this intron is mobile, and that this mobility is dependent on I-TevIII, which generates 2-nt 3' extensions. The enzyme has a distinct catalytic domain, which contains the H-N-H motif, and DNA-binding domain, which contains two zinc fingers required for interaction with the DNA substrate. Most importantly, I-TevIII, unlike the H-N-H endonucleases described so far, makes a double-strand break on the DNA homing site by acting as a dimer. Through deletion analysis, the dimerization interface was mapped to the DNA-binding domain. The unusual propensity of I-TevIII to dimerize to achieve cleavage of both DNA strands underscores the versatility of the H-N-H enzyme family.

Visualization of a group II intron in the 23S rRNA of a stable ribosome.

Thousands of introns have been localized to rRNA genes throughout the three domains of life. The consequences of the presence of either a spliced or an unspliced intron in a rRNA for ribosome assembly and packaging are largely unknown. To help address these questions, and to begin an intron imaging study, we selected a member of the self-splicing group II intron family, which is hypothesized to be the progenitor not only of spliceosomal introns but also of non-LTR retrotransposons. We cloned the self-splicing group II Ll.LtrB intron from Lactococcus lactis into L. lactis 23S rRNA. The 2,492-nt Ll.LtrB intron comprises a catalytic core and an ORF, which encodes a protein, LtrA. LtrA forms a ribonucleoprotein (RNP) complex with the intron RNA to mediate splicing and mobility. The chimeric 23S-intron RNA was shown to be splicing proficient in its native host in the presence of LtrA. Furthermore, a low-resolution cryo-EM reconstruction of the L. lactis ribosome fused to the intron-LtrA RNP of a splicing-defective Ll.LtrB intron was obtained. The image revealed the intron as a large, well defined structure. The activity and structural integrity of the intron indicate not only that it can coexist with the ribosome but also that its presence permits the assembly of a stable ribosome. Additionally, we view our results as a proof of principle that ribosome chimeras may be generally useful for studying a wide variety of structured RNAs and RNP complexes that are not amenable to NMR, crystallographic, or single-particle cryo-EM methodologies.

Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming.

Retrohoming of group II introns occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Host repair enzymes are predicted to complete this process. Here, we screened a battery of Escherichia coli mutants defective in host functions that are potentially involved in retrohoming of the Lactococcus lactis Ll.LtrB intron. We found strong (greater than threefold) effects for several enzymes, including nucleases directed against RNA and DNA, replicative and repair polymerases, and DNA ligase. A model including the presumptive roles of these enzymes in resection of DNA, degradation of the intron RNA template, traversion of RNA-DNA junctions, and second-strand DNA synthesis is described. The completion of retrohoming is viewed as a DNA repair process, with features that may be shared by other non-LTR retroelements.

The small noncoding DsrA RNA is an acid resistance regulator in Escherichia coli.

DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (sigmas), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.

Retrotransposition of the Ll.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets.

Catalytic group II introns are mobile retroelements that invade cognate intronless genes via retrohoming, where the introns reverse splice into double-stranded DNA (dsDNA) targets. They can also retrotranspose to ectopic sites at low frequencies. Whereas our previous studies with a bacterial intron, Ll.LtrB, supported frequent use of RNA targets during retrotransposition, recent experiments with a retrotransposition indicator gene indicate that DNA, rather than RNA, is a prominent target, with both dsDNA and single-stranded DNA (ssDNA) as possibilities. Thus retrotransposition occurs in both transcriptional sense and antisense orientations of target genes, and is largely independent of homologous DNA recombination and of the endonuclease function of the intron-encoded protein, LtrA. Models based on both dsDNA and ssDNA targeting are presented. Interestingly, retrotransposition is biased toward the template for lagging-strand DNA synthesis, which suggests the possibility of the replication folk as a source of ssDNA. Consistent with some use of ssDNA targets, many retrotransposition sites lack nucleotides critical for the unwinding of target duplex DNA. Moreover, in vitro the intron reverse spliced into ssDNA more efficiently than dsDNA substrates for some of the retrotransposition sites. Furthermore, many bacterial group II introns reside on the lagging-strand template, hinting at a role for DNA replication in intron dispersal in nature.