Mario Mörl

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.

Probing the SELEX Process with Next-Generation Sequencing

Background SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. Methodology We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. Conclusions High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.

The final cut: The importance of tRNA 3'- processing

To generate functional tRNA molecules, precursor RNAs must undergo several processing steps. While the enzyme that generates the mature tRNA 5′‐end, RNase P, has been thoroughly investigated, the 3′‐processing activity is, despite its importance, less understood. While nothing is known about tRNA 3′‐processing in archaea, the phenomenon has been analysed in detail in bacteria and is known to be a multistep process involving several enzymes, including both exo‐ and endonucleases. tRNA 3′‐end processing in the eukaryotic nucleus seems to be either exonucleolytic or endonucleolytic, depending on the organism analysed, whereas in organelles, 3′‐end maturation occurs via a single endonucleolytic cut. An interesting feature of organellar tRNA 3′‐processing is the occurrence of overlapping tRNA genes in metazoan mitochondria, which presents a unique challenge for the mitochondrial tRNA maturation enzymes, since it requires not only the removal but also the addition of nucleotides by an editing reaction.

Unbiased RNA–protein interaction screen by quantitative proteomics

Mass spectrometry (MS)-based quantitative interaction proteomics has successfully elucidated specific protein–protein, DNA–protein, and small molecule–protein interactions. Here, we developed a gel-free, sensitive, and scalable technology that addresses the important area of RNA–protein interactions. Using aptamer-tagged RNA as bait, we captured RNA-interacting proteins from stable isotope labeling by amino acids in cell culture (SILAC)-labeled mammalian cell extracts and analyzed them by high-resolution, quantitative MS. Binders specific to the RNA sequence were distinguished from background by their isotope ratios between bait and control. We demonstrated the approach by retrieving known and novel interaction partners for the HuR interaction motif, H4 stem loop, “zipcode” sequence, tRNA, and a bioinformatically-predicted RNA fold in DGCR-8/Pasha mRNA. In all experiments we unambiguously identified known interaction partners by a single affinity purification step. The 5′ region of the mRNA of DGCR-8/Pasha, a component of the microprocessor complex, specifically interacts with components of the translational machinery, suggesting that it contains an internal ribosome entry site.

Integration of group II intron bl1 into a foreign RNA by reversal of the self-splicing reaction in vitro

Group II intron bI1, the first intron of the COB gene in the mitochondria of S. cerevisiae, is able to self-splice in vitro with the basic pathway similar to nuclear pre-mRNA splicing. We show that incubation of the intron lariat with ligated exons bE1 and bE2 leads to a complete reversal of the splicing reaction. The integration of the intron into the ligated exons is correct; the reconstituted preRNA of the reverse reaction can undergo a self-splicing reaction anew. When incubated with a foreign RNA species bearing a sequence motif that is complementary to exon binding site 1, the lariat can integrate into this RNA with the position of insertion immediately downstream of this sequence. This result implies that transposition of group II introns on the RNA level by reversal of the splicing reaction is, in principle, conceivable.

This is the end: Processing, editing and repair at the tRNA 3 `-terminus

Abstract The generation of a mature tRNA 3end is an important step in the processing pathways leading to functional tRNA molecules. While 5end processing by RNase P is similar in all organisms, generation of the mature 3terminus seems to be more variable and complex. The first step in this reaction is the removal of 3trailer sequences. In bacteria, this is a multistep process performed by endo and exonucleases. In contrast, the majority of eukaryotes generate the mature tRNA 3end in a single step reaction, which consists of an endonucleolytic cut at the tRNA terminus. After removal of the 3trailer, a terminal CCA triplet has to be added to allow charging of the tRNA with its cognate amino acid. The enzyme catalyzing this reaction is tRNA nucleotidyltransferase, homologs of which have been found in representatives of all three kingdoms. Furthermore, in metazoan mitochondria, some genes encode 3terminally truncated tRNAs, which are restored in an editing reaction in order to yield functional tRNAs. Interestingly, this reaction is not restricted to distinct tRNAs, but seems to act on a variety of tRNA molecules and represents therefore a more general tRNA repair mechanism than a specialized editing reaction. In this review, the current knowledge about these crucial reactions is summarized.

RNA editing changes the identity of a mitochondrial tRNA in marsupials

In the mitochondrial genome of marsupials, the tRNA gene located at the position where in other mammals an aspartyl‐tRNA is encoded carries the glycine anticodon GCC. Post‐transcriptionally, an RNA editing mechanism affects the second position of the anticodon such that the aspartate anticodon GUC is created in approximately 50% of the mature tRNA pool. We show that the unedited version of this tRNA‘Asp’ (GCC) can be specifically aminoacylated with glycine in vitro, while the edited version becomes aminoacylated with aspartic acid. Furthermore, we show that both forms are aminoacylated to a substantial extent in vivo. By replacing an amino group with a keto group, RNA editing thus changes the identity of this tRNA allowing a single gene to encode two tRNAs.

RNA editing of a group II intron in Oenothera as a prerequisite for splicing.

The trans-splicing group II intron c/d in the Oenothera mitochondrial nad1 gene is modified by RNA editing in domain 6. This C-to-U conversion generates the typical domain 6 structure, which prompted us to speculate that this RNA editing event might be essential for splicing. To test this hypothesis, we investigated the influence of unedited and edited sequences of the Oenothera intron on splicing in vitro. The stem of domain 6 of intron nad1-c/d was transplanted into the autocatalytic yeast intron aI5c, yielding chimeras with the genomic C and the edited U, respectively, 5′ of the branchpoint A. When incubated under self-splicing conditions, only the edited chimera was released as a lariat, while the precursor with the genomically coded C remained inactive. Our results support the hypothesis that Oenothera group II intron nadl-c/d cannot be spliced from the primary transcript without previous editing in domain 6.

New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates

Here we describe three novel reactions of the self-splicing group II intron bI1 (the first intron of the COB gene of yeast mitochondria) demonstrating its catalytic versatility: reversal of the first step of the self-splicing reaction catalyzed by a linear form of the intron utilizing the energy of a phosphoanhydride bond for transesterification, ligation of a single-stranded DNA to an RNA, and cleavage of a single-stranded DNA substrate. These results have the following evolutionary implications: use of the alpha-beta bond of a terminal triphosphate for transesterification suggests that an RNA RNA replicase could use mononucleotide triphosphates as precursors, and cleavage of single-stranded DNA and DNA-RNA ligation suggests that excised group II introns might integrate directly into DNA without prior reverse transcription.

Processing and Editing of Overlapping tRNAs in Human Mitochondria

Overlapping tRNA genes in mitochondria of many metazoans introduce a problem for the processing of such polycistronic primary transcripts. Using runoff transcripts and an S100 extract from HeLa cell mitochondria, the processing of the human mitochondrial tRNATyr/tRNACys precursor (carrying an overlap of one base) was investigated: tRNACys is released in its complete form carrying the overlapping residue at the first position, whereas tRNATyr lacks that nucleotide at the discriminator position. Partial deletion of tRNACys or complete replacement by a non-tRNA-like sequence does not alter the processing reaction and indicates that the upstream tRNATyr alone is recognized by a 3′-endonuclease activity. The truncated 3′-end of this tRNATyr is then completed in an editing reaction that incorporates the missing residue. The processing of this tRNA overlap seems to be species-specific, because an overlapping tRNA precursor (tRNASer(AGY)/tRNALeu(CUN)) from opossum mitochondria is not recognized by the human extract. Because processing activities for overlapping and nonoverlapping tRNA precursors could not be separated, it seems that one general activity is responsible for the 3′-end processing of mitochondrial tRNAs and that this activity coevolved with the particular overlap between tRNATyr and tRNACys in human mitochondria, being unable to recognize overlaps between other tRNAs.