Retropseudogenes derived from the human Ro/SS-A autoantigen-associated hY RNAs

We report the characterization in the human genome of 966 pseudogenes derived from the four human Y (hY) RNAs, components of the Ro/SS-A autoantigen. About 95% of the Y RNA pseudogenes are found in corresponding locations on the chimpanzee and human chromosomes. On the contrary, Y pseudogenes in mice are both infrequent and found in different genomic regions. In addition to this rodent/primate discrepancy, the conservation of hY pseudogenes relative to hY genes suggests that they occurred after rodent/primate divergence. Flanking regions of hY pseudogenes contain convincing evidence for involvement of the L1 retrotransposition machinery. Although Alu elements are found in close proximity to most hY pseudogenes, these are not chimeric retrogenes. Point mutations in hY RNA transcripts specifically affecting binding of Ro60 protein likely contributed to their selection for direct trans retrotransposition. This represents a novel requirement for the selection of specific RNAs for their genomic integration by the L1 retrotransposition machinery. Over 40% of the hY pseudogenes are found in intronic regions of protein-coding genes. Considering the functions of proteins known to bind subsets of hY RNAs, hY pseudogenes constitute a new class of L1-dependent non-autonomous retroelements, potentially involved in post-transcriptional regulation of gene expression

Trans-splicing of the Ll.LtrB group II intron in Lactococcus lactis

The Ll.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. Ll.LtrB interrupts the relaxase gene of three L. lactis conjugative elements. The relaxase enzyme recognizes the origin of transfer (oriT ) and initiates the intercellular transfer of its conjugative element. The splicing efficiency of Ll.LtrB from the relaxase transcript thus controls the conjugation level of its host element. Here, we used the level of sex factor conjugation as a read-out for Ll.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (107-fold detection range), we demonstrate that Ll.LtrB can trans-splice in L. lactis when fragmented at various positions such as: three different locations within domain IV, within domain I and within domain III. We also demonstrate that the intron-encoded protein, LtrA, is absolutely required for Ll.LtrB trans-splicing. Characteristic Y-branched trans-spliced introns and ligated exons are detected by RT-PCR from total RNA extracts of cells harbouring fragmented Ll.LtrB. The splicing/conjugation assay we developed constitutes the first model system to study group II intron trans-splicing in vivo. Although only previously observed in bacterial-derived organelles, we demonstrate that assembly and trans-splicing of a fragmented group II intron can take place efficiently in bacterial cells

Leishmania-Induced IRAK-1 Inactivation Is Mediated by SHP-1 Interacting with an Evolutionarily Conserved KTIM Motif

Parasites of the Leishmania genus can rapidly alter several macrophage (MØ) signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR) signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1). Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif). This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases (e.g. Erk1/2, IKKα/β) involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif

Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H)1 CD4(+) and CD8(+) T cells and a systemic LACK-specific T(H)1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H)1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H)1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania

First unravelling of the hidden and intricate evolutionary history of a bacterial group II intron family

Bacterial group II introns are large RNA enzymes that self-splice from primary transcripts. Following excision, they can invade various DNA target sites using RNA-based mobility pathways. As fast-evolving retromobile elements that move between genetic loci within and across species, their evolutionary history was proved difficult to study and infer. Here we identified several homologs of Ll.LtrB, the model group II intron from Lactococcus lactis, and traced back their evolutionary relationship through phylogenetic analyses. Our data demonstrate that the Ll.LtrB homologs in Lactococci originate from a single and recent lateral transfer event of Ef.PcfG from Enterococcus faecalis. We also show that these introns disseminated in Lactococci following recurrent episodes of independent mobility events in conjunction with occurences of lateral transfer. Our phylogenies identified additional lateral transfer events from the environmental clade of the more diverged Lactococci introns to a series of low GC gram-positive bacterial species including E. faecalis. We also determined that functional intron adaptation occurred early in Lactococci following Ef.PcfG acquisition from E. faecalis and that two of the more diverged Ll.LtrB homologs remain proficient mobile elements despite the significant number of mutations acquired. This study describes the first comprehensive evolutionary history of a bacterial group II intron family.Funding provided by: Natural Sciences and Engineering Research Council of CanadaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000038Award Number: 22782

Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron

The Ll.LtrB intron, from the low G+C gram-positive bacterium Lactococcus lactis, was the first bacterial group II intron shown to splice and mobilize in vivo. The detailed retrohoming and retrotransposition pathways of Ll.LtrB were studied in both L. lactis and Escherichia coli. This bacterial retroelement has many features that would make it a good gene delivery vector. Here we report that the mobility efficiency of Ll.LtrB expressing LtrA in trans is only slightly affected by the insertion of fragments <100 nucleotides within the loop region of domain IV. In contrast, Ll.LtrB mobility efficiency is drastically decreased by the insertion of foreign sequences >1 kb. We demonstrate that the inhibitory effect caused by the addition of expression cassettes on Ll.LtrB mobility efficiency is not sequence specific, and not due to the expression, or the toxicity, of the cargo genes. Using genetic screens, we demonstrate that in order to maintain intron mobility, the loop region of domain IV, more specifically domain IVb, is by far the best region to insert foreign sequences within Ll.LtrB. Poisoned primer extension and Northern blot analyses reveal that Ll.LtrB constructs harboring cargo sequences splice less efficiently, and show a significant reduction in lariat accumulation in L. lactis. This suggests that cargo-containing Ll.LtrB variants are less stable. These results reveal the potential, yet limitations, of the Ll.LtrB group II intron to be used as a gene delivery vector, and validate the random insertion approach described in this study to create cargo-containing Ll.LtrB variants that are mobile

The circle to lariat ratio of the Ll.LtrB group II intron from Lactococcus lactis is greatly influenced by a variety of biological determinants in vivo.

Bacterial group II introns mostly behave as versatile retromobile genetic elements going through distinct cycles of gain and loss. These large RNA molecules are also ribozymes splicing autocatalytically from their interrupted pre-mRNA transcripts by two different concurrent pathways, branching and circularization. These two splicing pathways were shown to release in bacterial cells significant amounts of branched intron lariats and perfect end-to-end intron circles respectively. On one hand, released intron lariats can invade new sites in RNA and/or DNA by reverse branching while released intron circles are dead end spliced products since they cannot reverse splice through circularization. The presence of two parallel and competing group II intron splicing pathways in bacteria led us to investigate the conditions that influence the overall circle to lariat ratio in vivo. Here we unveil that removing a prominent processing site within the Ll.LtrB group II intron, raising growth temperature of Lactococcus lactis host cells and increasing the expression level of the intron-interrupted gene all increased the relative amount of released intron circles compared to lariats. Strengthening and weakening the base pairing interaction between the intron and its upstream exon respectively increased and decreased the overall levels of released intron circles in comparison to lariats. Host environment was also found to impact the circle to lariat ratio of the Ll.LtrB and Ll.RlxA group II introns from L. lactis and the Ef.PcfG intron from Enterococcus faecalis. Overall, our data show that multiple factors significantly influence the balance between released intron circles and lariats in bacterial cells

Conjugative Transfer of the Lactococcus lactis Chromosomal Sex Factor Promotes Dissemination of the Ll.LtrB Group II Intron

The Ll.LtrB group II intron from the low-G+C gram-positive bacterium Lactococcus lactis was the first bacterial group II intron shown to splice and mobilize in vivo. This retroelement interrupts the relaxase gene (ltrB) of three L. lactis conjugative elements: plasmids pRS01 and pAH90 and the chromosomal sex factor. Conjugative transfer of a plasmid harboring a segment of the pRS01 conjugative plasmid including the Ll.LtrB intron allows dissemination of Ll.LtrB among L. lactis strains and lateral transfer of this retroelement from L. lactis to Enterococcus faecalis. Here we report the dissemination of the Ll.LtrB group II intron among L. lactis strains following conjugative transfer of the native chromosomally embedded L. lactis sex factor. We demonstrated that Ll.LtrB dissemination is highly variable and often more efficient from this integrative and conjugative element than from an engineered conjugative plasmid. Cotransfer among L. lactis strains of both Ll.LtrB-containing elements, the conjugative plasmid and the sex factor, was detected and shown to be synergistic. Moreover, following their concurrent transfer, both mobilizable elements supported the spread of their respective copies of the Ll.LtrB intron. Our findings explain the unusually high efficiency of Ll.LtrB mobility observed following conjugation of intron-containing plasmids

Gene Targeting in the Gram-Positive Bacterium Lactococcus lactis, Using Various Delta Ribozymes

The trans-acting antigenomic delta ribozyme, isolated from the human hepatitis delta virus, was shown to be highly stable and active in vitro, as well as in mammalian cell lines. However, the stability and gene-targeting competence of this small ribozyme have not been studied previously in bacterial cells. In this paper we describe the use of two variants of the trans-acting antigenomic delta ribozyme targeting the abundant EF-Tu mRNA in the industrially important gram-positive bacterium Lactococcus lactis. These two delta ribozyme variants were expressed at significant levels and were shown to be highly stable in vivo. The half-life of the EF-Tu mRNA was slightly but consistently reduced in the presence of the classical delta ribozymes (7 to 13%). In contrast, delta ribozymes harboring a specific on/off riboswitch (SOFA-delta ribozymes) targeting the same sites on the EF-Tu mRNA considerably reduced the half-life of this mRNA (22 to 47%). The rates of catalysis of the SOFA-delta ribozymes in L. lactis were similar to the rates determined in vitro, showing that this new generation of delta ribozymes was highly efficient in these bacterial cells. Clearly, SOFA-delta ribozymes appear to be an ideal means for development of gene inactivation systems in bacteria

Trans-splicing versatility of the Ll.LtrB group II intron

Group II introns are found in organelles, bacteria, and archaea. Some harbor an open reading frame (ORF) with reverse transcriptase, maturase, and occasionally endonuclease activities. Group II introns require the assistance of either intron-encoded or free-standing maturases to excise from primary RNA transcripts in vivo. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA sites by retrohoming or retrotransposition. Group II introns are also hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs—U1, U2, U4, U5, and U6) that are part of the spliceosome. The ability of some fragmented group II introns to undergo splicing in trans supports the theory that the snRNAs evolved from portions of group II introns. Here, we developed a Tn5-based genetic screen to explore the trans-splicing potential of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. Proficient trans-splicing variants of Ll.LtrB were selected using a highly sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support splicing in trans, showing that this intron is remarkably more tolerant to fragmentation than expected from the fragmentation sites uncovered within natural trans-splicing group II introns. This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo