Under certain circumstances, any of the three termination codons can be read through by a near-cognate tRNA; i.e., a tRNA whose two out of three anticodon nucleotides base pair with those of the stop codon. Unless programed to synthetize C-terminally extended protein variants with expanded physiological roles, readthrough represents an undesirable translational error. On the other side of a coin, a significant number of human genetic diseases is associated with the introduction of nonsense mutations (premature termination codons [PTCs]) into coding sequences, where stopping is not desirable. Here, the tRNA's ability to induce readthrough opens up the intriguing possibility of mitigating the deleterious effects of PTCs on human health. In yeast, the UGA and UAR stop codons were described to be read through by four readthrough-inducing rti-tRNAs-tRNATrp and tRNACys, and tRNATyr and tRNAGln, respectively. The readthrough-inducing potential of tRNATrp and tRNATyr was also observed in human cell lines. Here, we investigated the readthrough-inducing potential of human tRNACys in the HEK293T cell line. The tRNACys family consists of two isoacceptors, one with ACA and the other with GCA anticodons. We selected nine representative tRNACys isodecoders (differing in primary sequence and expression level) and tested them using dual luciferase reporter assays. We found that at least two tRNACys can significantly elevate UGA readthrough when overexpressed. This indicates a mechanistically conserved nature of rti-tRNAs between yeast and human, supporting the idea that they could be used in the PTC-associated RNA therapies.
- MeSH
- antikodon MeSH
- cystein * genetika metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- nesmyslný kodon genetika MeSH
- proteosyntéza MeSH
- RNA transferová Cys metabolismus MeSH
- RNA transferová Trp metabolismus MeSH
- RNA transferová Tyr MeSH
- RNA transferová genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika MeSH
- terminační kodon genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs.
Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U-containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U-containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.
Trypanosoma cruzi is a unicellular protistan parasitic species that is comprised of strains and isolates exhibiting high levels of genetic and metabolic variability. In the insect vector, it is known to be highly responsive to starvation, a signal for progression to a life stage in which it can infect mammalian cells. Most mRNAs encoded in its mitochondrion require the targeted insertion and deletion of uridines to become translatable transcripts. This study defined differences in uridine-insertion/deletion RNA editing among three strains and established the mechanism whereby abundances of edited (and, thus, translatable) mitochondrial gene products increase during starvation. Our approach utilized our custom T-Aligner toolkit to describe transcriptome-wide editing events and reconstruct editing products from high-throughput sequencing data. We found that the relative abundance of mitochondrial transcripts and the proportion of mRNAs that are edited varies greatly between analyzed strains, a characteristic that could potentially impact metabolic capacity. Starvation typically led to an increase in overall editing activity rather than affecting a specific step in the process. We also determined that transcripts CR3, CR4, and ND3 produce multiple open reading frames that, if translated, would generate different proteins. Finally, we quantitated the inherent flexibility of editing in T. cruzi and found it to be higher relative to that in a related trypanosomatid lineage. Over time, new editing domains or patterns could prove advantageous to the organism and become more widespread within individual transcriptomes or among strains.
- MeSH
- editace RNA MeSH
- messenger RNA genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA mitochondriální genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- RNA metabolismus MeSH
- savci genetika MeSH
- transkriptom MeSH
- Trypanosoma brucei brucei * genetika MeSH
- Trypanosoma cruzi * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The RNA editing core complex (RECC) catalyzes mitochondrial U-insertion/deletion mRNA editing in trypanosomatid flagellates. Some naphthalene-based sulfonated compounds, such as C35 and MrB, competitively inhibit the auto-adenylylation activity of an essential RECC enzyme, kinetoplastid RNA editing ligase 1 (KREL1), required for the final step in editing. Previous studies revealed the ability of these compounds to interfere with the interaction between the editosome and its RNA substrates, consequently affecting all catalytic activities that comprise RNA editing. This observation implicates a critical function for the affected RNA binding proteins in RNA editing. In this study, using the inhibitory compounds, we analyzed the composition and editing activities of functional editosomes and identified the mitochondrial RNA binding proteins 1 and 2 (MRP1/2) as their preferred targets. While the MRP1/2 heterotetramer complex is known to bind guide RNA and promote annealing to its cognate pre-edited mRNA, its role in RNA editing remained enigmatic. We show that the compounds affect the association between the RECC and MRP1/2 heterotetramer. Furthermore, RECC purified post-treatment with these compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon the addition of recombinant MRP1/2 proteins. This work provides experimental evidence that the MRP1/2 heterotetramer is required for in vitro RNA editing activity and substantiates the hypothesized role of these proteins in presenting the RNA duplex to the catalytic complex in the initial steps of RNA editing.
- MeSH
- editace RNA účinky léků genetika MeSH
- guide RNA, Kinetoplastida účinky léků MeSH
- ligasy antagonisté a inhibitory MeSH
- messenger RNA genetika MeSH
- mitochondriální proteiny genetika MeSH
- mitochondrie účinky léků genetika MeSH
- proteiny vázající RNA genetika MeSH
- protozoální proteiny genetika MeSH
- rekombinantní proteiny genetika MeSH
- RNA mitochondriální genetika MeSH
- RNA protozoální genetika MeSH
- Trypanosoma brucei brucei účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viral and cellular double-stranded RNA (dsRNA) is recognized by cytosolic innate immune sensors, including RIG-I-like receptors. Some cytoplasmic dsRNA is commonly present in cells, and one source is mitochondrial dsRNA, which results from bidirectional transcription of mitochondrial DNA (mtDNA). Here we demonstrate that Trp53 mutant mouse embryonic fibroblasts contain immune-stimulating endogenous dsRNA of mitochondrial origin. We show that the immune response induced by this dsRNA is mediated via RIG-I-like receptors and leads to the expression of type I interferon and proinflammatory cytokine genes. The mitochondrial dsRNA is cleaved by RNase L, which cleaves all cellular RNA including mitochondrial mRNAs, increasing activation of RIG-I-like receptors. When mitochondrial transcription is interrupted there is a subsequent decrease in this immune-stimulatory dsRNA. Our results reveal that the role of p53 in innate immunity is even more versatile and complex than previously anticipated. Our study, therefore, sheds new light on the role of endogenous RNA in diseases featuring aberrant immune responses.
- MeSH
- adenosindeaminasa nedostatek genetika imunologie MeSH
- DEAD box protein 58 genetika imunologie MeSH
- dvouvláknová RNA genetika imunologie MeSH
- embryo savčí MeSH
- endoribonukleasy genetika imunologie MeSH
- fibroblasty cytologie imunologie MeSH
- genetická transkripce MeSH
- IFIH1 genetika imunologie MeSH
- interferonový regulační faktor 7 genetika imunologie MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- nádorový supresorový protein p53 nedostatek genetika imunologie MeSH
- přirozená imunita genetika MeSH
- proteiny genetika imunologie MeSH
- RNA mitochondriální genetika imunologie MeSH
- transfekce MeSH
- transportní proteiny genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MRP1/2 is a heteromeric protein complex that functions in the trypanosomatid mitochondrion as part of the RNA editing machinery, which facilitates multiple targeted insertions and deletions of uridines. MRP1/2 was shown to interact with MRB8170, which initiates RNA editing by marking pre-edited mRNAs, while TbRGG2 is required for its efficient progression on pan-edited mRNAs. Both MRP1/2 and TbRGG2 are capable of modulating RNA-RNA interactions in vitro. As determined by using iCLIP and RIP-qPCR, RNAs bound to MRP1/2 are characterized and compared with those associated with MRB8170 and TbRGG2. We provide evidence that MRP1 and MRB8170 have correlated binding and similar RNA crosslinking peak profiles over minimally and never-edited mRNAs. Our results suggest that MRP1 assists MRB8170 in RNA editing on minimally edited mRNAs.
- MeSH
- editace RNA MeSH
- messenger RNA genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- proteiny vázající RNA metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA mitochondriální genetika metabolismus MeSH
- Trypanosoma genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bordetella pertussis is the causative agent of human whooping cough, a highly contagious respiratory disease which despite vaccination programs remains the major cause of infant morbidity and mortality. The requirement of the RNA chaperone Hfq for virulence of B. pertussis suggested that Hfq-dependent small regulatory RNAs are involved in the modulation of gene expression. High-throughput RNA sequencing revealed hundreds of putative noncoding RNAs including the RgtA sRNA. Abundance of RgtA is strongly decreased in the absence of the Hfq protein and its expression is modulated by the activities of the two-component regulatory system BvgAS and another response regulator RisA. Whereas RgtA levels were elevated under modulatory conditions or in the absence of bvg genes, deletion of the risA gene completely abolished RgtA expression. Profiling of the ΔrgtA mutant in the ΔbvgA genetic background identified the BP3831 gene encoding a periplasmic amino acid-binding protein of an ABC transporter as a possible target gene. The results of site-directed mutagenesis and in silico analysis indicate that RgtA base-pairs with the region upstream of the start codon of the BP3831 mRNA and thereby weakens the BP3831 protein production. Furthermore, our data suggest that the function of the BP3831 protein is related to transport of glutamate, an important metabolite in the B. pertussis physiology. We propose that the BvgAS/RisA interplay regulates the expression of RgtA which upon infection, when glutamate might be scarce, attenuates translation of the glutamate transporter and thereby assists in adaptation of the pathogen to other sources of energy.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Bordetella pertussis genetika metabolismus MeSH
- glutamáty metabolismus MeSH
- lidé MeSH
- malá nekódující RNA genetika MeSH
- regulace genové exprese u bakterií MeSH
- signální transdukce * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Splicing in S. cerevisiae has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the AMA1 intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in prp45(1-169) cells. We also measured pre-mRNA accumulation of the meiotic MER2 gene, which depends on the expression of Mer1 factor for splicing. prp45(1-169) cells accumulated approximately threefold higher levels of MER2 pre-mRNA than WT cells only when splicing was induced. To monitor cotranscriptional splicing, we determined the presence of early spliceosome assembly factors and snRNP complexes along the ECM33 and ACT1 genes. We found that prp45(1-169) hampered the cotranscriptional recruitment of U2 and, to a larger extent, U5 and NTC, while the U1 profile was unaffected. The recruitment of Prp45(1-169) was impaired similarly to U5 snRNP and NTC. Our results imply that Prp45 is required for timely formation of complex A, prior to stable physical association of U5/NTC with the emerging pre-mRNA substrate. We suggest that Prp45 facilitates conformational rearrangements and/or contacts that couple U1 snRNP-recognition to downstream assembly events.
- MeSH
- introny MeSH
- malý jaderný ribonukleoprotein U1 metabolismus MeSH
- malý jaderný ribonukleoprotein U2 metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sestřih RNA * MeSH
- spliceozomy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
ADAR RNA editing enzymes (adenosine deaminases acting on RNA) that convert adenosine bases to inosines were first identified biochemically 30 years ago. Since then, studies on ADARs in genetic model organisms, and evolutionary comparisons between them, continue to reveal a surprising range of pleiotropic biological effects of ADARs. This review focuses on Drosophila melanogaster, which has a single Adar gene encoding a homolog of vertebrate ADAR2 that site-specifically edits hundreds of transcripts to change individual codons in ion channel subunits and membrane and cytoskeletal proteins. Drosophila ADAR is involved in the control of neuronal excitability and neurodegeneration and, intriguingly, in the control of neuronal plasticity and sleep. Drosophila ADAR also interacts strongly with RNA interference, a key antiviral defense mechanism in invertebrates. Recent crystal structures of human ADAR2 deaminase domain-RNA complexes help to interpret available information on Drosophila ADAR isoforms and on the evolution of ADARs from tRNA deaminase ADAT proteins. ADAR RNA editing is a paradigm for the now rapidly expanding range of RNA modifications in mRNAs and ncRNAs. Even with recent progress, much remains to be understood about these groundbreaking ADAR RNA modification systems.
- MeSH
- adenosindeaminasa chemie genetika metabolismus MeSH
- Drosophila melanogaster genetika metabolismus MeSH
- editace RNA * MeSH
- exprese genu MeSH
- interakční proteinové domény a motivy MeSH
- izoenzymy MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- molekulární evoluce MeSH
- nervový systém metabolismus MeSH
- obratlovci MeSH
- proteiny Drosophily genetika metabolismus MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- RNA interference MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
