Identification of RNA helicases with unwinding activity on angiogenin-processed tRNAs.

Stress-induced tRNA fragmentation upon environmental insult is a conserved cellular process catalysed by endonucleolytic activities targeting mature tRNAs. The resulting tRNA-derived small RNAs (tsRNAs) have been implicated in various biological processes that impact cell-to-cell signalling, cell survival as well as gene expression regulation during embryonic development. However, how endonuclease-targeted tRNAs give rise to individual and potentially biologically active tsRNAs remains poorly understood. Here, we report on the in vivo identification of proteins associated with stress-induced tsRNAs-containing protein complexes, which, together with a 'tracer tRNA' assay, were used to uncover enzymatic activities that can bind and process specific endonuclease-targeted tRNAs in vitro. Among those, we identified conserved ATP-dependent RNA helicases which can robustly separate tRNAs with endonuclease-mediated 'nicks' in their anticodon loops. These findings shed light on the existence of cellular pathways dedicated to producing individual tsRNAs after stress-induced tRNA hydrolysis, which adds to our understanding as to how tRNA fragmentation and the resulting tsRNAs might exert physiological impact.

Experimental paradigms revisited: oxidative stress-induced tRNA fragmentation does not correlate with stress granule formation but is associated with delayed cell death.

tRNA fragmentation is an evolutionarily conserved molecular phenomenon. tRNA-derived small RNAs (tsRNAs) have been associated with many cellular processes, including improved survival during stress conditions. Here, we have revisited accepted experimental paradigms for modeling oxidative stress resulting in tRNA fragmentation. Various cell culture models were exposed to oxidative stressors followed by determining cell viability, the production of specific tsRNAs and stress granule formation. These experiments revealed that exposure to stress parameters commonly used to induce tRNA fragmentation negatively affected cell viability after stress removal. Quantification of specific tsRNA species in cells responding to experimental stress and in cells that were transfected with synthetic tsRNAs indicated that neither physiological nor non-physiological copy numbers of tsRNAs induced the formation of stress granules. Furthermore, the increased presence of tsRNA species in culture medium collected from stressed cells indicated that cells suffering from experimental stress exposure gave rise to stable extracellular tsRNAs. These findings suggest a need to modify current experimental stress paradigms in order to allow separating the function of tRNA fragmentation during the acute stress response from tRNA fragmentation as a consequence of ongoing cell death, which will have major implications for the current perception of the biological function of stress-induced tsRNAs.

The Regulation of RNA Modification Systems: The Next Frontier in Epitranscriptomics?

RNA modifications, long considered to be molecular curiosities embellishing just abundant and non-coding RNAs, have now moved into the focus of both academic and applied research. Dedicated research efforts (epitranscriptomics) aim at deciphering the underlying principles by determining RNA modification landscapes and investigating the molecular mechanisms that establish, interpret and modulate the information potential of RNA beyond the combination of four canonical nucleotides. This has resulted in mapping various epitranscriptomes at high resolution and in cataloguing the effects caused by aberrant RNA modification circuitry. While the scope of the obtained insights has been complex and exciting, most of current epitranscriptomics appears to be stuck in the process of producing data, with very few efforts to disentangle cause from consequence when studying a specific RNA modification system. This article discusses various knowledge gaps in this field with the aim to raise one specific question: how are the enzymes regulated that dynamically install and modify RNA modifications? Furthermore, various technologies will be highlighted whose development and use might allow identifying specific and context-dependent regulators of epitranscriptomic mechanisms. Given the complexity of individual epitranscriptomes, determining their regulatory principles will become crucially important, especially when aiming at modifying specific aspects of an epitranscriptome both for experimental and, potentially, therapeutic purposes.

Translational engagement of lysophosphatidic acid receptor 1 in skin fibrosis: from dermal fibroblasts of patients with scleroderma to tight skin 1 mouse.

BACKGROUND AND PURPOSE: Genetic deletion and pharmacological studies suggest a role for lysophosphatidic acid (LPA1 ) receptor in fibrosis. We investigated the therapeutic potential in systemic sclerosis (SSc) of a new orally active selective LPA1 receptor antagonist using dermal fibroblasts from patients and an animal model of skin fibrosis. EXPERIMENTAL APPROACH: Dermal fibroblast and skin biopsies from systemic sclerosis patients were used. Myofibroblast differentiation, gene expression and cytokine secretion were measured following LPA and/or SAR100842 treatment. Pharmacolgical effect of SAR100842 was assessed in the tight skin 1 (Tsk1) mouse model. KEY RESULTS: SAR100842 is equipotent against various LPA isoforms. Dermal fibroblasts and skin biopsies from patients with systemic sclerosis expressed high levels of LPA1 receptor. The LPA functional response (Ca2+ ) in systemic sclerosis dermal fibroblasts was fully antagonized with SAR100842. LPA induced myofibroblast differentiation in systemic sclerosis dermal and idiopathic pulmonary fibrosis lung fibroblasts and the secretion of inflammatory markers and activated Wnt markers. Results from systemic sclerosis dermal fibroblasts mirror those obtained in a mouse Tsk1 model of skin fibrosis. Using a therapeutic protocol, SAR100842 consistently reversed dermal thickening, inhibited myofibroblast differentiation and reduced skin collagen content. Inflammatory and Wnt pathway markers were also inhibited by SAR100842 in the skin of Tsk1 mice. CONCLUSION AND IMPLICATIONS: The effects of SAR100842 on LPA-induced inflammation and on mechanisms linked to fibrosis like myofibroblast differentiation and Wnt pathway activation indicate that LPA1 receptor activation plays a key role in skin fibrosis. Our results support the therapeutic potential of LPA1 receptor antagonists in systemic sclerosis.

Production and purification of endogenously modified tRNA-derived small RNAs.

During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions.

tRNA 2'-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila.

2'-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

tRNA-Derived Small RNAs: Biogenesis, Modification, Function and Potential Impact on Human Disease Development.

Transfer RNAs (tRNAs) are abundant small non-coding RNAs that are crucially important for decoding genetic information. Besides fulfilling canonical roles as adaptor molecules during protein synthesis, tRNAs are also the source of a heterogeneous class of small RNAs, tRNA-derived small RNAs (tsRNAs). Occurrence and the relatively high abundance of tsRNAs has been noted in many high-throughput sequencing data sets, leading to largely correlative assumptions about their potential as biologically active entities. tRNAs are also the most modified RNAs in any cell type. Mutations in tRNA biogenesis factors including tRNA modification enzymes correlate with a variety of human disease syndromes. However, whether it is the lack of tRNAs or the activity of functionally relevant tsRNAs that are causative for human disease development remains to be elucidated. Here, we review the current knowledge in regard to tsRNAs biogenesis, including the impact of RNA modifications on tRNA stability and discuss the existing experimental evidence in support for the seemingly large functional spectrum being proposed for tsRNAs. We also argue that improved methodology allowing exact quantification and specific manipulation of tsRNAs will be necessary before developing these small RNAs into diagnostic biomarkers and when aiming to harness them for therapeutic purposes.

RNAs, Phase Separation, and Membrane-Less Organelles: Are Post-Transcriptional Modifications Modulating Organelle Dynamics?

Membranous organelles allow sub-compartmentalization of biological processes. However, additional subcellular structures create dynamic reaction spaces without the need for membranes. Such membrane-less organelles (MLOs) are physiologically relevant and impact development, gene expression regulation, and cellular stress responses. The phenomenon resulting in the formation of MLOs is called liquid-liquid phase separation (LLPS), and is primarily governed by the interactions of multi-domain proteins or proteins harboring intrinsically disordered regions as well as RNA-binding domains. Although the presence of RNAs affects the formation and dissolution of MLOs, it remains unclear how the properties of RNAs exactly contribute to these effects. Here, the authors review this emerging field, and explore how particular RNA properties can affect LLPS and the behavior of MLOs. It is suggested that post-transcriptional RNA modification systems could be contributors for dynamically modulating the assembly and dissolution of MLOs.

Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats.

The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5) RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.

Understanding RNA modifications: the promises and technological bottlenecks of the 'epitranscriptome'.

The discovery of mechanisms that alter genetic information via RNA editing or introducing covalent RNA modifications points towards a complexity in gene expression that challenges long-standing concepts. Understanding the biology of RNA modifications represents one of the next frontiers in molecular biology. To this date, over 130 different RNA modifications have been identified, and improved mass spectrometry approaches are still adding to this list. However, only recently has it been possible to map selected RNA modifications at single-nucleotide resolution, which has created a number of exciting hypotheses about the biological function of RNA modifications, culminating in the proposition of the 'epitranscriptome'. Here, we review some of the technological advances in this rapidly developing field, identify the conceptual challenges and discuss approaches that are needed to rigorously test the biological function of specific RNA modifications.

Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation.

A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.

RNA 5-Methylcytosine Analysis by Bisulfite Sequencing.

Cells have developed molecular machineries, which can chemically modify DNA and RNA nucleosides. One particular and chemically simple modification, (cytosine-5) methylation (m(5)C), has been detected both in RNA and DNA suggesting universal use of m(5)C for the function of these nucleotide polymers. m(5)C can be reproducibly mapped to abundant noncoding RNAs (transfer RNA, tRNA and ribosomal RNA, rRNA), and recently, also nonabundant RNAs (including mRNAs) have been reported to carry this modification. Quantification of m(5)C content in total RNA preparations indicates that a limited number of RNAs carry this modification and suggests specific functions for (cytosine-5) RNA methylation. What exactly is the biological function of m(5)C in RNA? Before attempting to address this question, m(5)C needs to be mapped specifically and reproducibly, preferably on a transcriptome-wide scale. To facilitate the detection of m(5)C in its sequence context, RNA bisulfite sequencing (RNA-BisSeq) has been developed. This method relies on the efficient chemical deamination of nonmethylated cytosine, which can be read out as single nucleotide polymorphism (nonmethylated cytosine as thymine vs. methylated cytosine as cytosine), when differentially comparing cDNA libraries to reference sequences after DNA sequencing. Here, the basic protocol of RNA-BisSeq, its current applications and limitations are described.

Eukaryotic rRNA Modification by Yeast 5-Methylcytosine-Methyltransferases and Human Proliferation-Associated Antigen p120.

Modified nucleotide 5-methylcytosine (m5C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA:m5C-methyltranferases (MTases) Nop2 from S. cerevisiae and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m5C- MTase activity, which restores m5C formation at position 2870 in domain V of 25S rRNA in a nop2Δ yeast strain. We also confirm that yeast proteins Nop2p and Rcm1p catalyze the formation of m5C in domains V and IV, respectively. In addition, we do not find any evidence of m5C residues in yeast 18S rRNA. We also performed functional complementation of Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for yeast growth and m5C-MTase activity. Chimeric protein formed by Nop2 and p120 fragments revealed the importance of Nop2 N-terminal domain for correct protein localization and its cellular function. We also validated that the presence of Nop2, rather than the m5C modification in rRNA itself, is required for pre-rRNA processing. Our results corroborate that Nop2 belongs to the large family of pre-ribosomal proteins and possesses two related functions in pre-rRNA processing: as an essential factor for cleavages and m5C:RNA:modification. These results support the notion of quality control during ribosome synthesis by such modification enzymes.

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability.

In all three domains of life ribosomal RNAs are extensively modified at functionally important sites of the ribosome. These modifications are believed to fine-tune the ribosome structure for optimal translation. However, the precise mechanistic effect of modifications on ribosome function remains largely unknown. Here we show that a cluster of methylated nucleotides in domain IV of 25S rRNA is critical for integrity of the large ribosomal subunit. We identified the elusive cytosine-5 methyltransferase for C2278 in yeast as Rcm1 and found that a combined loss of cytosine-5 methylation at C2278 and ribose methylation at G2288 caused dramatic ribosome instability, resulting in loss of 60S ribosomal subunits. Structural and biochemical analyses revealed that this instability was caused by changes in the structure of 25S rRNA and a consequent loss of multiple ribosomal proteins from the large ribosomal subunit. Our data demonstrate that individual RNA modifications can strongly affect structure of large ribonucleoprotein complexes.

Dnmt2 methyltransferases and immunity: an ancient overlooked connection between nucleotide modification and host defense?

Many species maintain cytosine DNA methyltransferase (MTase) genes belonging to the Dnmt2 family. Prokaryotic modification-restriction systems utilize DNA methylation to distinguish between self and foreign DNA, and cytosine methylation in eukaryotic DNA contributes to epigenetic mechanisms that control gene expression. However, Dnmt2 proteins display only low or no DNA MTase activity, making this protein family the odd and enigmatic family member. Recent evidence showed that Dnmt2 proteins are not DNA but RNA MTases with functions in biological processes as diverse as stress responses and RNA-mediated inheritance. These observations not only raise profound questions regarding the perceived substrate specificities of cytosine MTase, but also suggest links between DNA and RNA modification systems. Here, we speculate that Dnmt2 proteins might be part of an ancient cytosine modification toolbox that is used to successfully respond to environmental challenges, including constantly evolving RNA and DNA substrates.

The RNA methyltransferase Dnmt2 is required for efficient Dicer-2-dependent siRNA pathway activity in Drosophila.

Transfer RNA (tRNA) fragmentation in response to stress conditions has been described in many organisms. tRNA fragments have been found in association with small interfering RNA (siRNA) components, but the biological role of these interactions remains unclear. We report here that the tRNA methyltransferase Dnmt2 is essential for efficient Dicer-2 (Dcr-2) function in Drosophila. Using small RNA (sRNA) sequencing, we confirmed that Dnmt2 limits the extent of tRNA fragmentation during the heat-shock response. tRNAs as well as tRNA fragments serve as Dcr-2 substrates, and Dcr-2 degrades tRNA-derived sequences, especially under heat-shock conditions. tRNA-derived RNAs are able to inhibit Dcr-2 activity on long double-stranded RNAs (dsRNAs). Consequently, heat-shocked Dnmt2 mutant animals accumulate dsRNAs, produce fewer siRNAs, and show misregulation of siRNA pathway-dependent genes. These results reveal the impact of tRNA fragmentation on siRNA pathways and implicate tRNA modifications in the regulation of sRNA homeostasis during the heat-shock response.

Dnmt2-dependent methylomes lack defined DNA methylation patterns.

Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms.

Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2.

Drosophila use small-interfering RNA mechanisms to limit the amplification of viral genomes. However, it is unclear how small RNA interference components recognize and separate viral from cellular RNA. Dnmt2 enzymes are highly conserved RNA methyltransferases with substrate specificity towards cellular tRNAs. We report here that Dnmt2 is required for efficient innate immune responses in Drosophila. Dnmt2 mutant flies accumulate increasing levels of Drosophila C virus and show activated innate immune responses. Binding of Dnmt2 to DCV RNA suggests that Dnmt2 contributes to virus control directly, possibly by RNA methylation. These observations demonstrate a role for Dnmt2 in antiviral defence.

tRNA modifications: necessary for correct tRNA-derived fragments during the recovery from stress?

Endonuclease-mediated tRNA fragmentation has been observed in many species suggesting functional importance for tRNA fragments. The size distribution of tRNA-derived fragments indicates the existence of mechanisms that protect tRNAs and their fragments from total degradation by exonucleases. Could post-transcriptional modifications be important for the controlled processing of tRNAs?