Led-Seq: ligation-enhanced double-end sequence-based structure analysis of RNA.

Structural analysis of RNA is an important and versatile tool to investigate the function of this type of molecules in the cell as well as in vitro. Several robust and reliable procedures are available, relying on chemical modification inducing RT stops or nucleotide misincorporations during reverse transcription. Others are based on cleavage reactions and RT stop signals. However, these methods address only one side of the RT stop or misincorporation position. Here, we describe Led-Seq, a new approach based on lead-induced cleavage of unpaired RNA positions, where both resulting cleavage products are investigated. The RNA fragments carrying 2', 3'-cyclic phosphate or 5'-OH ends are selectively ligated to oligonucleotide adapters by specific RNA ligases. In a deep sequencing analysis, the cleavage sites are identified as ligation positions, avoiding possible false positive signals based on premature RT stops. With a benchmark set of transcripts in Escherichia coli, we show that Led-Seq is an improved and reliable approach based on metal ion-induced phosphodiester hydrolysis to investigate RNA structures in vivo.

Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes.

Removal of the 5'-leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P-mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5'-leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes-two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

Substrate Affinity Versus Catalytic Efficiency: Ancestral Sequence Reconstruction of tRNA Nucleotidyltransferases Solves an Enzyme Puzzle.

In tRNA maturation, CCA-addition by tRNA nucleotidyltransferase is a unique and highly accurate reaction. While the mechanism of nucleotide selection and polymerization is well understood, it remains a mystery why bacterial and eukaryotic enzymes exhibit an unexpected and surprisingly low tRNA substrate affinity while they efficiently catalyze the CCA-addition. To get insights into the evolution of this high-fidelity RNA synthesis, the reconstruction and characterization of ancestral enzymes is a versatile tool. Here, we investigate a reconstructed candidate of a 2 billion years old CCA-adding enzyme from Gammaproteobacteria and compare it to the corresponding modern enzyme of Escherichia coli. We show that the ancestral candidate catalyzes an error-free CCA-addition, but has a much higher tRNA affinity compared with the extant enzyme. The consequence of this increased substrate binding is an enhanced reverse reaction, where the enzyme removes the CCA end from the mature tRNA. As a result, the ancestral candidate exhibits a lower catalytic efficiency in vitro as well as in vivo. Furthermore, the efficient tRNA interaction leads to a processive polymerization, while the extant enzyme catalyzes nucleotide addition in a distributive way. Thus, the modern enzymes increased their polymerization efficiency by lowering the binding affinity to tRNA, so that CCA synthesis is efficiently promoted due to a reduced reverse reaction. Hence, the puzzling and at a first glance contradicting and detrimental weak substrate interaction represents a distinct activity enhancement in the evolution of CCA-adding enzymes.

Mitochondrial Genomes in Perkinsus Decode Conserved Frameshifts in All Genes.

Mitochondrial genomes of apicomplexans, dinoflagellates, and chrompodellids that collectively make up the Myzozoa, encode only three proteins (Cytochrome b [COB], Cytochrome c oxidase subunit 1 [COX1], Cytochrome c oxidase subunit 3 [COX3]), contain fragmented ribosomal RNAs, and display extensive recombination, RNA trans-splicing, and RNA-editing. The early-diverging Perkinsozoa is the final major myzozoan lineage whose mitochondrial genomes remained poorly characterized. Previous reports of Perkinsus genes indicated independent acquisition of non-canonical features, namely the occurrence of multiple frameshifts. To determine both ancestral myzozoan and novel perkinsozoan mitochondrial genome features, we sequenced and assembled mitochondrial genomes of four Perkinsus species. These data show a simple ancestral genome with the common reduced coding capacity but disposition for rearrangement. We identified 75 frameshifts across the four species that occur as distinct types and that are highly conserved in gene location. A decoding mechanism apparently employs unused codons at the frameshift sites that advance translation either +1 or +2 frames to the next used codon. The locations of frameshifts are seemingly positioned to regulate protein folding of the nascent protein as it emerges from the ribosome. The cox3 gene is distinct in containing only one frameshift and showing strong selection against residues that are otherwise frequently encoded at the frameshift positions in cox1 and cob. All genes lack cysteine codons implying a reduction to 19 amino acids in these genomes. Furthermore, mitochondrion-encoded rRNA fragment complements are incomplete in Perkinsus spp. but some are found in the nuclear DNA suggesting import into the organelle. Perkinsus demonstrates further remarkable trajectories of organelle genome evolution including pervasive integration of frameshift translation into genome expression.

Ligand-dependent tRNA processing by a rationally designed RNase P riboswitch.

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5'-processing by either sequestering or exposing the single-stranded 5'-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5'-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.

CCA-Addition Gone Wild: Unusual Occurrence and Phylogeny of Four Different tRNA Nucleotidyltransferases in Acanthamoeba castellanii.

tRNAs are important players in the protein synthesis machinery, where they act as adapter molecules for translating the mRNA codons into the corresponding amino acid sequence. In a series of highly conserved maturation steps, the primary transcripts are converted into mature tRNAs. In the amoebozoan Acanthamoeba castellanii, a highly unusual evolution of some of these processing steps was identified that are based on unconventional RNA polymerase activities. In this context, we investigated the synthesis of the 3'-terminal CCA-end that is added posttranscriptionally by a specialized polymerase, the tRNA nucleotidyltransferase (CCA-adding enzyme). The majority of eukaryotic organisms carry only a single gene for a CCA-adding enzyme that acts on both the cytosolic and the mitochondrial tRNA pool. In a bioinformatic analysis of the genome of this organism, we identified a surprising multitude of genes for enzymes that contain the active site signature of eukaryotic/eubacterial tRNA nucleotidyltransferases. In vitro activity analyses of these enzymes revealed that two proteins represent bona fide CCA-adding enzymes, one of them carrying an N-terminal sequence corresponding to a putative mitochondrial target signal. The other enzymes have restricted activities and represent CC- and A-adding enzymes, respectively. The A-adding enzyme is of particular interest, as its sequence is closely related to corresponding enzymes from Proteobacteria, indicating a horizontal gene transfer. Interestingly, this unusual diversity of nucleotidyltransferase genes is not restricted to Acanthamoeba castellanii but is also present in other members of the Acanthamoeba genus, indicating an ancient evolutionary trait.

Beyond Plug and Pray: Context Sensitivity and in silico Design of Artificial Neomycin Riboswitches.

Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5' untranslated region of mRNA. Rearrangements of the RNA structure in response, e.g., to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modelling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behaviour in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyse the influence of 5' hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free licence on GitHub (https://github.com/xileF1337/riboswitch_design).

Dual expression of CCA-adding enzyme and RNase T in Escherichia coli generates a distinct cca growth phenotype with diverse applications.

Correct synthesis and maintenance of functional tRNA 3'-CCA-ends is a crucial prerequisite for aminoacylation and must be achieved by the phylogenetically diverse group of tRNA nucleotidyltransferases. While numerous reports on the in vitro characterization exist, robust analysis under in vivo conditions is lacking. Here, we utilize Escherichia coli RNase T, a tRNA-processing enzyme responsible for the tRNA-CCA-end turnover, to generate an in vivo system for the evaluation of A-adding activity. Expression of RNase T results in a prominent growth phenotype that renders the presence of a CCA- or A-adding enzyme essential for cell survival in an E. coli Δcca background. The distinct growth fitness allows for both complementation and selection of enzyme variants in a natural environment. We demonstrate the potential of our system via detection of altered catalytic efficiency and temperature sensitivity. Furthermore, we select functional enzyme variants out of a sequence pool carrying a randomized codon for a highly conserved position essential for catalysis. The presented E. coli-based approach opens up a wide field of future studies including the investigation of tRNA nucleotidyltransferases from all domains of life and the biological relevance of in vitro data concerning their functionality and mode of operation.

Examining tRNA 3'-ends in Escherichia coli: teamwork between CCA-adding enzyme, RNase T, and RNase R.

tRNA maturation and quality control are crucial for proper functioning of these transcripts in translation. In several organisms, defective tRNAs were shown to be tagged by poly(A) or CCACCA tails and subsequently degraded by 3'-exonucleases. In a deep-sequencing analysis of tRNA 3'-ends, we detected the CCACCA tag also in Escherichia coli However, this tag closely resembles several 3'-trailers of tRNA precursors targeted for maturation and not for degradation. Here, we investigate the ability of two important exonucleases, RNase R and RNase T, to distinguish tRNA precursors with a native 3'-trailer from tRNAs with a CCACCA tag. Our results show that the degrading enzyme RNase R breaks down both tRNAs primed for degradation as well as precursor transcripts, indicating that it is a rather nonspecific RNase. RNase T, a main processing exonuclease involved in trimming of 3'-trailers, is very inefficient in converting the CCACCA-tagged tRNA into a mature transcript. Hence, while both RNases compete for trailer-containing tRNA precursors, the inability of RNase T to process CCACCA tails ensures that defective tRNAs cannot reenter the functional tRNA pool, representing a safeguard to avoid detrimental effects of tRNAs with erroneous integrity on protein synthesis. Furthermore, these data indicate that the RNase T-mediated end turnover of the CCA sequence represents a means to deliver a tRNA to a repeated quality control performed by the CCA-adding enzyme. Hence, originally described as a futile side reaction, the tRNA end turnover seems to fulfill an important function in the maintenance of the tRNA pool in the cell.

LOTTE-seq (Long hairpin oligonucleotide based tRNA high-throughput sequencing): specific selection of tRNAs with 3'-CCA end for high-throughput sequencing.

Transfer RNAs belong to the most abundant type of ribonucleic acid in the cell, and detailed investigations revealed correlations between alterations in the tRNA pool composition and certain diseases like breast cancer. However, currently available methods do not sample the entire tRNA pool or lack specificity for tRNAs. A specific disadvantage of such methods is that only full-length tRNAs are analysed, while tRNA fragments or incomplete cDNAs due to RT stops at modified nucleosides are lost. Another drawback in certain approaches is that the tRNA fraction has to be isolated and separated from high molecular weight RNA, resulting in considerable labour costs and loss of material. Based on a hairpin-shaped adapter oligonucleotide selective for tRNA transcripts, we developed a highly specific protocol for efficient and comprehensive high-throughput analysis of tRNAs that combines the benefits of existing methods and eliminates their disadvantages. Due to a 3'-TGG overhang, the adapter is specifically ligated to the tRNA 3'-CCA end. Reverse transcription prior to the ligation of a second adapter allows to include prematurely terminated cDNA products, increasing the number of tRNA reads. This strategy renders this approach a powerful and universal tool to analyse the tRNA pool of cells and organisms under different conditions in health and disease.

Evolving methods for rational de novo design of functional RNA molecules.

Artificial RNA molecules with novel functionality have many applications in synthetic biology, pharmacy and white biotechnology. The de novo design of such devices using computational methods and prediction tools is a resource-efficient alternative to experimental screening and selection pipelines. In this review, we describe methods common to many such computational approaches, thoroughly dissect these methods and highlight open questions for the individual steps. Initially, it is essential to investigate the biological target system, the regulatory mechanism that will be exploited, as well as the desired components in order to define design objectives. Subsequent computational design is needed to combine the selected components and to obtain novel functionality. This process can usually be split into constrained sequence sampling, the formulation of an optimization problem and an in silico analysis to narrow down the number of candidates with respect to secondary goals. Finally, experimental analysis is important to check whether the defined design objectives are indeed met in the target environment and detailed characterization experiments should be performed to improve the mechanistic models and detect missing design requirements.

Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells.

The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.

Small but large enough: structural properties of armless mitochondrial tRNAs from the nematode Romanomermis culicivorax.

As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes.

Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.

Unusual Occurrence of Two Bona-Fide CCA-Adding Enzymes in Dictyostelium discoideum.

Dictyostelium discoideum, the model organism for the evolutionary supergroup of Amoebozoa, is a social amoeba that, upon starvation, undergoes transition from a unicellular to a multicellular organism. In its genome, we identified two genes encoding for tRNA nucleotidyltransferases. Such pairs of tRNA nucleotidyltransferases usually represent collaborating partial activities catalyzing CC- and A-addition to the tRNA 3'-end, respectively. In D. discoideum, however, both enzymes exhibit identical activities, representing bona-fide CCA-adding enzymes. Detailed characterization of the corresponding activities revealed that both enzymes seem to be essential and are regulated inversely during different developmental stages of D. discoideum. Intriguingly, this is the first description of two functionally equivalent CCA-adding enzymes using the same set of tRNAs and showing a similar distribution within the cell. This situation seems to be a common feature in Dictyostelia, as other members of this phylum carry similar pairs of tRNA nucleotidyltransferase genes in their genome.

Adaptation of the Romanomermis culicivorax CCA-Adding Enzyme to Miniaturized Armless tRNA Substrates.

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that-when inserted into the human enzyme-confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3'-end of the tRNA primer in the catalytic core, dramatically increases the enzyme's substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

Post-Transcriptional Regulation of tRNA Pools To Govern the Central Dogma: A Perspective.

Since their initial discovery, tRNAs have risen from sole adapter molecules during protein synthesis to pivotal modulators of gene expression. Through their many interactions with tRNA-associated protein factors, they play a central role in maintaining cell homeostasis, especially regarding the fine-tuning in response to a rapidly changing cellular environment. Here, we provide a perspective on current tRNA topics with a spotlight on the regulation of post-transcriptional shaping of tRNA molecules. First, we give an update on aberrant structural features that a yet functional fraction of mitochondrial tRNAs can exhibit. Then, we outline several aspects of the regulatory contribution of ribonucleases with a focus on tRNA processing versus tRNA elimination. We close with a comment on the possible consequences for the intracellular examination of nascent tRNA precursors regarding respective processing factors that have been shown to associate with the tRNA transcription machinery in alternative moonlighting functions.

Accurate mapping of tRNA reads.

Motivation: Many repetitive DNA elements are transcribed at appreciable expression levels. Mapping the corresponding RNA sequencing reads back to a reference genome is notoriously difficult and error-prone task, however. This is in particular true if chemical modifications introduce systematic mismatches, while at the same time the genomic loci are only approximately identical, as in the case of tRNAs. Results: We therefore developed a dedicated mapping strategy to handle RNA-seq reads that map to tRNAs relying on a modified target genome in which known tRNA loci are masked and instead intronless tRNA precursor sequences are appended as artificial 'chromosomes'. In a first pass, reads that overlap the boundaries of mature tRNAs are extracted. In the second pass, the remaining reads are mapped to a tRNA-masked target that is augmented by representative mature tRNA sequences. Using both simulated and real life data we show that our best-practice workflow removes most of the mapping artefacts introduced by simpler mapping schemes and makes it possible to reliably identify many of chemical tRNA modifications in generic small RNA-seq data. Using simulated data the FDR is only 2%. We find compelling evidence for tissue specific differences of tRNA modification patterns. Availability and implementation: The workflow is available both as a bash script and as a Galaxy workflow from https://github.com/AnneHoffmann/tRNA-read-mapping. Contact: fabian@tbi.univie.ac.at. Supplementary information: Supplementary data are available at Bioinformatics online.

Applicability of a computational design approach for synthetic riboswitches.

Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand-dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.

A Temporal Order in 5'- and 3'- Processing of Eukaryotic tRNAHis.

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3'-terminal CCA sequence that includes the site of aminoacylation, the additional 5'-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3'-CCA and 5'-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3'-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.