Antibacterial immune competence of honey bees (Apis mellifera) is adapted to different life stages and environmental risks.

The development of all honey bee castes proceeds through three different life stages all of which encounter microbial infections to a various extent. We have examined the immune strength of honey bees across all developmental stages with emphasis on the temporal expression of cellular and humoral immune responses upon artificial challenge with viable Escherichia coli bacteria. We employed a broad array of methods to investigate defence strategies of infected individuals: (a) fate of bacteria in the haemocoel; (b) nodule formation and (c) induction of antimicrobial peptides (AMPs). Newly emerged adult worker bees and drones were able to activate efficiently all examined immune reactions. The number of viable bacteria circulating in the haemocoel of infected bees declined rapidly by more than two orders of magnitude within the first 4-6 h post-injection (p.i.), coinciding with the occurrence of melanised nodules. Antimicrobial activity, on the other hand, became detectable only after the initial bacterial clearance. These two temporal patterns of defence reactions very likely represent the constitutive cellular and the induced humoral immune response. A unique feature of honey bees is that a fraction of worker bees survives the winter season in a cluster mostly engaged in thermoregulation. We show here that the overall immune strength of winter bees matches that of young summer bees although nodulation reactions are not initiated at all. As expected, high doses of injected viable E.coli bacteria caused no mortality in larvae or adults of each age. However, drone and worker pupae succumbed to challenge with E.coli even at low doses, accompanied by a premature darkening of the pupal body. In contrast to larvae and adults, we observed no fast clearance of viable bacteria and no induction of AMPs but a rapid proliferation of E.coli bacteria in the haemocoel of bee pupae ultimately leading to their death.

Honey bee drones maintain humoral immune competence throughout all life stages in the absence of vitellogenin production.

Drones are haploid male individuals whose major social function in honey bee colonies is to produce sperm and mate with a queen. In spite of their limited tasks, the vitality of drones is of utmost importance for the next generation. The immune competence of drones - as compared to worker bees - is largely unexplored. Hence, we studied humoral and cellular immune reactions of in vitro reared drone larvae and adult drones of different age upon artificial bacterial infection. Haemolymph samples were collected after aseptic and septic injury and subsequently employed for (1) the identification of immune-responsive peptides and/or proteins by qualitative proteomic analyses in combination with mass spectrometry and (2) the detection of antimicrobial activity by inhibition-zone assays. Drone larvae and adult drones responded with a strong humoral immune reaction upon bacterial challenge, as validated by the expression of small antimicrobial peptides. Young adult drones exhibited a broader spectrum of defence reactions than drone larvae. Distinct polypeptides including peptidoglycan recognition protein-S2 and lysozyme 2 were upregulated in immunized adult drones. Moreover, a pronounced nodulation reaction was observed in young drones upon bacterial challenge. Prophenoloxidase zymogen is present at an almost constant level in non-infected adult drones throughout the entire lifespan. All observed immune reactions in drones were expressed in the absence of significant amounts of vitellogenin. We conclude that drones - like worker bees - have the potential to activate multiple elements of the innate immune response.

Infection of honey bees with acute bee paralysis virus does not trigger humoral or cellular immune responses.

We have studied the responses of honey bees at different life stages (Apis mellifera) to controlled infection with acute bee paralysis virus and have identified the haemolymph of infected larvae and adult worker bees as the compartment where massive propagation of ABPV occurs. Insects respond with a broad spectrum of induced innate immune reactions to bacterial infections, whereas defence mechanisms based on RNA interference play a major role in antiviral immunity. In this study, we have determined that honey bee larvae and adult workers do not produce a humoral immune reaction upon artificial infection with ABPV, in contrast to control individuals challenged with Escherichia coli. ABPV-infected bees produced neither elevated levels of specific antimicrobial peptides (AMPs), such as hymenoptaecin and defensin, nor any general antimicrobial activity, as revealed by inhibition-zone assays. Additionally, adult bees did not generate melanised nodules upon ABPV infection, an important cellular immune function activated by bacteria and viruses in some insects. Challenge of bees with both ABPV and E. coli showed that innate humoral and cellular immune reactions are induced in mixed infections, albeit at a reduced level.

Inactivation of a non-enveloped RNA virus by artificial ribonucleases: honey bees and acute bee paralysis virus as a new experimental model for in vivo antiviral activity assessment.

RNA-containing viruses represent a global threat to the health and wellbeing of humans and animals. Hence, the discovery of new approaches for the design of novel vaccines and antiviral compounds attains high attention. Here we describe the potential of artificial ribonucleases (aRNases), low molecular weight compounds capable to cleave phosphodiester bonds in RNA under mild conditions, to act as antiviral compounds via destroying the genome of non-enveloped RNA viruses, and the potential of utilizing honey bee larvae and adult bees (Apis mellifera) as a novel experimental system for the screening of new antiviral compounds. Pre-incubation of an Acute bee paralysis virus (ABPV) suspension with aRNases D3-12, K-D-1 or Dp12F6 in a concentration-dependent manner increased the survival rate of bee larvae and adult bees subsequently infected with these preparations, whereas incubation of the virus with aRNases ABL3C3 or L2-3 had no effect at all. The results of RT-PCR analysis of viral RNA isolated from aRNase-treated virus particles confirmed that virus inactivation occurs via degradation of viral genomic RNA: dose-dependent inactivation of ABPV correlates well with the cleavage of viral RNA. Electron microscopy analysis revealed that the morphology of ABPV particles inactivated by aRNases remains unaffected as compared to control virus preparations. Altogether the obtained results clearly demonstrate the potential of aRNases as a new virus inactivation agents and bee larvae/ABPV as a new in vivo system for the screening of antiviral compounds.

Branchiostoma floridae has separate healing and sealing enzymes for 5'-phosphate RNA ligation.

Animal cells have two tRNA splicing pathways: (i) a 5'-P ligation mechanism, where the 5'-phosphate of the 3' tRNA half becomes the junction phosphate of the new phosphodiester linkage, and (ii) a 3'-P ligation process, in which the 3'-phosphate of the 5' tRNA half turns into the junction phosphate. Although both activities are known to exist in animals, in almost three decades of investigation, neither of the two RNA ligases has been identified. Here we describe a gene from the chordate Branchiostoma floridae that encodes an RNA ligase (Bf RNL) with a strict requirement for RNA substrates with a 2'-phosphate terminus for the ligation of RNAs with 5'-phosphate and 3'-hydroxyl ends. Unlike the yeast and plant tRNA ligases involved in tRNA splicing, Bf RNL lacks healing activities and requires the action of a polynucleotide kinase (PNK) and a cyclic phosphodiesterase (CDPase) in trans. The activities of these two enzymes were identified in a single B. floridae protein (Bf PNK/CPDase). The combined activities of Bf RNL and Bf PNK/CPDase are sufficient for the joining of tRNA splicing intermediates in vitro, and for the functional complementation of a tRNA ligase-deficient Saccharomyces cerevisiae strain in vivo. Hence, these two proteins constitute the 5'-P RNA ligation pathway in an animal organism.

Dual functions of yeast tRNA ligase in the unfolded protein response: unconventional cytoplasmic splicing of HAC1 pre-mRNA is not sufficient to release translational attenuation.

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5' UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

Immune-related proteins induced in the hemolymph after aseptic and septic injury differ in honey bee worker larvae and adults.

We have employed the proteomic approach in combination with mass spectrometry to study the immune response of honey bee workers at different developmental stages. Analysis of the hemolymph proteins of noninfected, mock-infected and immune-challenged individuals by polyacrylamide gel electrophoresis showed differences in the protein profiles. We present evidence that in vitro reared honey bee larvae respond with a prominent humoral reaction to aseptic and septic injury as documented by the transient synthesis of the three antimicrobial peptides (AMPs) hymenoptaecin, defensin1, and abaecin. In contrast, young adult worker bees react with a broader spectrum of immune reactions that include the activation of prophenoloxidase and humoral immune responses. At least seven proteins appeared consistently in the hemolymph of immune-challenged bees, three of which are identical to the AMPs induced also in larvae. The other four, i.e., phenoloxidase (PO), peptidoglycan recognition protein-S2, carboxylesterase (CE), and an Apis-specific protein not assigned to any function (HP30), are induced specifically in adult bees and, with the exception of PO, are not expressed after aseptic injury. Structural features of CE and HP30, such as classical leucine zipper motifs, together with their strong simultaneous induction upon challenge with bacteria suggest an important role of the two novel bee-specific immune proteins in response to microbial infections.

Plant pre-tRNA splicing enzymes are targeted to multiple cellular compartments.

Splicing of precursor tRNAs in plants requires the concerted action of three enzymes: an endonuclease to cleave the intron at the two splice sites, an RNA ligase for joining the resulting tRNA halves and a 2'-phosphotransferase to remove the 2'-phosphate from the splice junction. Pre-tRNA splicing has been demonstrated to occur exclusively in the nucleus of vertebrates and in the cytoplasm of budding yeast cells, respectively. We have investigated the subcellular localization of plant splicing enzymes fused to GFP by their transient expression in Allium epidermal and Vicia guard cells. Our results show that all three classes of splicing enzymes derived from Arabidopsis and Oryza are localized in the nucleus, suggesting that plant pre-tRNA splicing takes place preferentially in the nucleus. Moreover, two of the splicing enzymes, i.e., tRNA ligase and 2'-phosphotransferase, contain chloroplast transit signals at their N-termini and are predominantly targeted to chloroplasts and proplastids, respectively. The putative transit sequences are effective also in the heterologous context fused directly to GFP. Chloroplast genomes do not encode intron-containing tRNA genes of the nuclear type and consequently tRNA ligase and 2'-phosphotransferase are not required for classical pre-tRNA splicing in these organelles but they may play a role in tRNA repair and/or splicing of atypical group II introns. Additionally, 2'-phosphotransferase-GFP fusion protein has been found to be associated with mitochondria, as confirmed by colocalization studies with MitoTracker Red. In vivo analyses with mutated constructs suggest that alternative initiation of translation is one way utilized by tRNA splicing enzymes for differential targeting.

Structure-function analysis of the kinase-CPD domain of yeast tRNA ligase (Trl1) and requirements for complementation of tRNA splicing by a plant Trl1 homolog.

Trl1 is an essential 827 amino acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two domains--an N-terminal ligase component and a C-terminal 5'-kinase/2',3'-cyclic phosphodiesterase (CPD) component--that can function in tRNA splicing in vivo when expressed as separate polypeptides. To understand the structural requirements for the kinase-CPD domain, we performed an alanine scan of 30 amino acids that are conserved in Trl1 homologs from other fungi. We thereby identified four residues (Arg463, His515, Thr675 and Glu741) as essential for activity in vivo. Structure-function relationships at these positions, and at four essential or conditionally essential residues defined previously (Asp425, Arg511, His673 and His777), were clarified by introducing conservative substitutions. Biochemical analysis showed that lethal mutations of Asp425, Arg463, Arg511 and His515 in the kinase module abolished polynucleotide kinase activity in vitro. We report that a recently cloned 1104 amino acid Arabidopsis RNA ligase functions in lieu of yeast Trl1 in vivo and identify essential side chains in the ligase, kinase and CPD modules of the plant enzyme. The plant ligase, like yeast Trl1 but unlike T4 RNA ligase 1, requires a 2'-PO4 end for tRNA splicing in vivo.

Plant 7SL RNA genes belong to type 4 of RNA polymerase III- dependent genes that are composed of mixed promoters.

The genes transcribed by RNA polymerase III (pol III) display a great diversity in terms of promoter structure and are placed in four groups accordingly. Type 3 subset of pol III genes has promoter elements which reside entirely upstream of the coding region of the gene whereas type 4 consists of genes with mixed promoters that enclose intra- and extragenic regulatory sequences. Plant 7SL RNA genes have been previously classified as type 3 of pol III genes requiring an upstream sequence element and a canonical TATA box for transcriptional activity in transfected plant protoplasts. We have identified two novel functional control regions within the coding region of an Arabidopsis 7SL RNA gene (At7SL-1) that resemble tRNA gene-specific A and B boxes with respect to sequence and position. Single and multiple nucleotide substitutions in either of these regions resulted in a pronounced reduction of transcription activity in tobacco nuclear extract that was not caused by a decreased stability as shown by decay kinetics of wild type and mutant RNA transcripts. These findings suggest that plant 7SL RNA genes should be actually placed in type 4 of pol III-transcribed genes. As a consequence of substantially different upstream promoters utilized by plant and human pol III, in vitro transcription of 7SL RNA genes in heterologous systems is severely impaired. A chimeric human 7SL RNA gene that contains the 5' flanking region up to position -300 of At7SL-1 is yet transcribed with a reduced efficiency in tobacco extract when compared with the plant wild-type gene, supporting the notion that internal regulatory elements contribute to full activity.

Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins.

Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns. Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases. The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end. In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities. Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining. Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing.

Novel upstream and intragenic control elements for the RNA polymerase III-dependent transcription of human 7SL RNA genes.

In the human nuclear genome only a few copies coding for full-length 7SL RNA genes exist. The Hs7SL-1 gene has recently been classified as type 4 of RNA polymerase III (pol III)-transcribed genes as it was demonstrated that mutations in an external transcriptional activator (ATF) binding site and in an internal CG dinucleotide at positions +15/+16 reduced 7SL RNA expression in vivo and in vitro. We have extended the elucidation of external and internal promoter elements and have discovered two novel regulatory sequences: a TATA-like element in the upstream region and internal A and B box-like motifs. This study was greatly facilitated by the identification of a second, new functional human 7SL RNA gene which we called Hs7SL-3. Remarkably, Hs7SL-3 RNA is synthesized twice as efficiently as Hs7SL-1 in HeLa nuclear extract. Comparison of the upstream regions revealed the presence of two conserved elements in the two human 7SL RNA genes, an ATF/CRE binding site at -43 to -50 and a TATA-like box centered around position -25. Mutational analyses indicated that both external promoter elements are important for efficient transcription. In addition, two sequence motifs can be identified in Hs7SL-1 and Hs7SL-3 at positions 10-19 and 50-60, respectively, downstream of the transcription start site that resemble putative A and B boxes. Single and multiple nucleotide substitutions in these regions also influenced transcription activity to a great extent. The requirement of intragenic functional A and B boxes in combination with the external ATF/CRE and TATA-like promoter elements for the efficient transcription of human 7SL RNA genes is reminiscent of at least two other classes of pol III-transcribed genes in human cells, such as Epstein-Barr virus-encoded EBER and vault RNA genes.

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli.

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA(Ser)(CGA), an Arabidopsis intron-containing tRNA(Tyr)(GTA) and an Arabidopsis intron-containing tRNA(Met)(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of beta-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA(Ser) gene and the GUS reporter gene resulted in approximately 10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA(Tyr) or tRNA(Met) genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA(Met)(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA(Tyr) to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA(Tyr) and tRNA(Met) were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.

Plant 7SL RNA and tRNA(Tyr) genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells.

RNA polymerase III-driven cassettes for the expression of antisense RNAs and ribozymes have recently attracted much attention because (1) pol III genes are transcribed abundantly in all kinds of tissues and (2) the transcripts are very stable by virtue of their small and compact size. We have designed two types of pol III-based expression vehicles. Antisense RNA sequences targeted against conserved structural elements or domains in the RNAs of potato spindle tuber viroid, hop latent viroid and potato virus S were either embedded in the anticodon region of a Nicotiana tRNA(Tyr) gene or near the 3' end of an Arabidopsis 7SL RNA gene. Both classes of chimeric genes were transcribed in vitro in a homologous plant extract. Our studies clearly revealed that the modified tRNA and 7SL RNA genes, carrying insertions of up to 90 and 120 bp, respectively, were expressed efficiently in the tobacco nuclear extract, resulting in high levels of stable chimeric transcripts. 7SL RNA (also termed SRP RNA) represents the RNA component of the signal recognition particle. This is the first report of demonstrating the employment of 7SL RNA genes as potential cassettes for the expression of antisense RNA and ribozyme sequences and might be helpful in future experiments to control their localization in specific sub-cellular compartments.