Roland K. Hartmann

tRNAdb 2009: compilation of tRNA sequences and tRNA genes

One of the first specialized collections of nucleic acid sequences in life sciences was the ‘compilation of tRNA sequences and sequences of tRNA genes’ (http://www.trna.uni-bayreuth.de). Here, an updated and completely restructured version of this compilation is presented (http://trnadb.bioinf.uni-leipzig.de). The new database, tRNAdb, is hosted and maintained in cooperation between the universities of Leipzig, Marburg, and Strasbourg. Reimplemented as a relational database, tRNAdb will be updated periodically and is searchable in a highly flexible and user-friendly way. Currently, it contains more than 12 000 tRNA genes, classified into families according to amino acid specificity. Furthermore, the implementation of the NCBI taxonomy tree facilitates phylogeny-related queries. The database provides various services including graphical representations of tRNA secondary structures, a customizable output of aligned or un-aligned sequences with a variety of individual and combinable search criteria, as well as the construction of consensus sequences for any selected set of tRNAs.

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G–ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G–ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit ‘pe/E’ hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the ‘missing link’ in terms of tRNA intermediates involved in the universally conserved translocation process.

MicroRNA Replacement Therapy for miR-145 and miR-33a Is Efficacious in a Model of Colon Carcinoma

MicroRNAs (miRNA) aberrantly expressed in tumors may offer novel therapeutic approaches to treatment. miR-145 is downregulated in various cancers including colon carcinoma in which in vitro studies have established proapoptotic and antiproliferative roles. miR-33a was connected recently to cancer through its capacity to downregulate the oncogenic kinase Pim-1. To date, miRNA replacement therapy has been hampered by the lack of robust nonviral delivery methods for in vivo administration. Here we report a method of miRNA delivery by using polyethylenimine (PEI)-mediated delivery of unmodified miRNAs, using miR-145 and miR-33a to preclinically validate the method in a mouse model of colon carcinoma. After systemic or local application of low molecular weight PEI/miRNA complexes, intact miRNA molecules were delivered into mouse xenograft tumors, where they caused profound antitumor effects. miR-145 delivery reduced tumor proliferation and increased apoptosis, with concomitant repression of c-Myc and ERK5 as novel regulatory target of miR-145. Similarly, systemic injection of PEI-complexed miR-33a was validated as a novel therapeutic targeting method for Pim-1, with antitumor effects comparable with PEI/siRNA-mediated direct in vivo knockdown of Pim-1 in the model. Our findings show that chemically unmodified miRNAs complexed with PEI can be used in an efficient and biocompatible strategy of miRNA replacement therapy, as illustrated by efficacious delivery of PEI/miR-145 and PEI/miR-33a complexes in colon carcinoma.

Locked Nucleic Acid Oligonucleotides

Locked nucleic acid (LNA) is the term for oligonucleotides that contain one or more nucleotide building blocks in which an extra methylene bridge fixes the ribose moiety either in the C3′-endo (β-D-LNA) or C2′-endo(α-L-LNA) conformation. The β-D-LNA modification results in significant increases in melting temperature of up to several degrees per LNA residue. The α-L-LNA stereoisomer, which also stabilizes duplexes, lends itself to use in triplex-forming oligonucleotides and transcription factor decoys, which have to maintain a B-type (C2′-endo) DNA conformation. LNA oligonucleotides are synthesized in different formats, such as all-LNA, LNA/DNA mixmers, or LNA/DNA gapmers. Essentially, all aspects of antisense technology have profited from LNAdue to its unprecedented affinity, good or even improved mismatch discrimination, low toxicity, and increased metabolic stability. LNA is particularly attractive for in vivo applications that are inaccessible to RNAinterference technology, such as suppression of aberrant splice sites or inhibition of oncogenic microRNAs. Furthermore, the extreme antisense-target duplex stability (formation of persistent steric blocks) conferred by β-D-LNAalso contributes to the capacity to invade stable secondary structures of RNA targets. The in vivo studies reported so far indeed point to LNA as a promising antisense player at the horizon of clinical applications.

The proto-oncogene Pim-1 is a target of miR-33a

The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR-33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3′-untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.

RP-PHOSPHOROTHIOATE MODIFICATIONS IN RNASE P RNA THAT INTERFERE WITH TRNA BINDING

We have used Rp‐phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp‐phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre‐tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non‐binding fractions by gel retardation. Rp‐phosphorothioate modifications that interfered with tRNA binding were found 5′ of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long‐range base‐pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp‐phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro‐Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA‐RNase P RNA complex.

RNA interference as a gene-specific approach for molecular medicine.

The discovery of RNA interference (RNAi) in eukaryotic cells has been the major recent breakthrough in molecular and cell biology. RNAi machineries exert biological functions in gene regulation, genome defense and chromatin architecture and dynamics. The potential of RNAi to silence any gene of interest in a highly specific and efficient manner via double-stranded RNA (dsRNA) has literally revolutionized modern genetics. RNAi-based functional genomics now permits, for the first time, to evaluate the cellular role of individual gene products on a genome-wide scale in higher organisms like mammals, presenting an alternative to the generation of animal knockouts often doomed to failure because of a lethal phenotype. RNAi has had an enormous impact on the development of novel disease models in animals, and it is likely that small interfering RNAs (siRNAs), which are the trigger molecules for RNA silencing, will become an invaluable tool for the treatment of genetic diseases. First clinical trials, using siRNAs directed against the vascular endothelial growth factor (VEGF) or one of its receptors, have been initiated recently for the treatment of age-related macular degeneration. Improving guidelines for the rational design of siRNAs, based on recent progress in understanding the mechanisms underlying RNAi, as well as the introduction of chemical modifications into siRNAs are expected to improve their pharmacokinetic and pharmacodynamic properties for in vivo applications. Finally, successful therapeutic application of RNAi will depend on the development of improved siRNA delivery strategies that combine high specificity and efficiency with a low immunostimulatory and tumorigenic potential.

Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA.

We have identified by nucleotide analog interference mapping (NAIM) exocyclic NH2 groups of guanosines in RNase P RNA from Escherichia coli that are important for tRNA binding. The majority of affected guanosines represent phylogenetically conserved nucleotides. Several sites of interference could be assigned to direct contacts with the tRNA moiety, whereas others were interpreted as reflecting indirect effects on tRNA binding due to the disruption of tertiary contacts within the catalytic RNA. Our results support the involvement of the 2-NH2 groups of G292/G293 in pairing with C74 and C75 of tRNA CCA-termini, as well as formation of two consecutive base triples involving C75 and A76 of CCA-ends interacting with G292/A258 and G291/G259, respectively. Moreover, we present first biochemical evidence for two tertiary contacts (L18/P8 and L8/P4) within the catalytic RNA, whose formation has been postulated previously on the basis of phylogenetic comparative analyses. The tRNA binding interference data obtained in this and our previous studies are consistent with the formation of a consecutive nucleotide triple and quadruple between the tetraloop L18 and helix P8. Formation of the nucleotide triple (G316 and A94:U104 in wild-type E. coli RNase P RNA) is also supported by mutational analysis. For the mutant RNase P RNA carrying a G94:C104 double mutation, an additional G316-to-A mutation resulted in a restoration of binding affinity for mature and precursor tRNA.

Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its γ-proteobacterial homologs (∼185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the γ-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the γ-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

Catalysis by RNase P RNA: Unique features and unprecedented active site plasticity

Metal ions are essential cofactors for precursor tRNA (ptRNA) processing by bacterial RNase P. The ribose 2′-OH at nucleotide (nt) –1 of ptRNAs is known to contribute to positioning of catalytic Me2+. To investigate the catalytic process, we used ptRNAs with single 2′-deoxy (2′-H), 2′-amino (2′-N), or 2′-fluoro (2′-F) modifications at the cleavage site (nt –1). 2′ modifications had small (2.4–7.7-fold) effects on ptRNA binding to E. coli RNase P RNA in the ground state, decreasing substrate affinity in the order 2′-OH > 2′-F > 2′-N > 2′-H. Effects on the rate of the chemical step (about 10-fold for 2′-F, almost 150-fold for 2′-H and 2′-N) were much stronger, and, except for the 2′-N modification, resembled strikingly those observed in the Tetrahymena ribozyme-catalyzed reaction at corresponding position. Mn2+ rescued cleavage of the 2′-N but also the 2′-H-modified ptRNA, arguing against a direct metal ion coordination at this location. Miscleavage between nt –1 and –2 was observed for the 2′-N-ptRNA at low pH (further influenced by the base identities at nt –1 and +73), suggesting repulsion of a catalytic metal ion due to protonation of the amino group. Effects caused by the 2′-N modification at nt –1 of the substrate allowed us to substantiate a mechanistic difference in phosphodiester hydrolysis catalyzed by Escherichia coli RNase P RNA and the Tetrahymena ribozyme: a metal ion binds next to the 2′ substituent at nt –1 in the reaction catalyzed by RNase P RNA, but not at the corresponding location in the Tetrahymena ribozyme reaction.