Recently, we have determined the secondary structure of the 5′-terminal region of p53 mRNA that starts from the P1 transcription initiation site and includes two major translation initiation codons responsible for the synthesis of p53 and ΔNp53 isoform. Here, we showed that when this region was extended into 5′ direction to the P0 transcription start site, the two characteristic hairpin motifs found in this region were preserved. Moreover, the presence of alternatively spliced intron 2 did not interfere with the formation of the larger hairpin in which the initiation codon for p53 was embedded. The impact of the different variants of p53 5′-terminal region, which start at P0 or P1 site and end with the initiation codon for p53 or ΔNp53, on the translation of luciferase reporter protein was compared. Strikingly, the efficiency of translation performed in rabbit reticulocyte lysate differed by two orders of magnitude. The toe-printing analysis was also applied to investigate the formation of the ribosomal complex on the model mRNA constructs. The relative translation efficiencies in HeLa and MCF-7 cells were similar to those observed in the cell lysate, although some differences were noted in comparison with cell-free conditions. The results were discussed in terms of the role of secondary structure folding of the 5′-terminal region of p53 mRNA in translation and possible modes of p53 and ΔNp53 translation initiation.
hnRNP A2 is a cellular protein that is important for nucleocytoplasmic and cytosolic trafficking of the HIV-1 genomic RNA. Both hnRNP A2’s interaction with HIV-1 RNA and its expression levels influence the activities of Rev in mediating nucleocytoplasmic export of the HIV-1 genomic RNA. While the lack of Rev expression during HIV-1 gene expression results in nuclear retention of HIV-1 genomic RNA, we show here by fluorescence in situ hybridization and fractionation studies that the genomic RNA translocates to the cytoplasm when hnRNP A2/B1 are depleted from cells. Polyribosome analyses revealed that the genomic RNA was shunted into a cytoplasmic, dense polyribosomal fraction. This fraction contained several RNA-binding proteins involved in viral gene expression and RNA trafficking but did not contain the translation initiation factor, eIF4G1. Amino acid incorporation into nascent polypeptides in this fraction was also greatly reduced, demonstrating that this fraction contains mRNAs that are poorly translated. These results demonstrate that hnRNP A2/B1 expression plays roles in the nuclear retention of the HIV-1 genomic RNA in the absence of Rev and in the release of the genomic RNA from translationally inactive, cytoplasmic RNP complexes.
Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNAtrans-splicing of SV40 early transcripts in transformed rodent cells. Enhancedtrans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5′ donor splice site (ss). This mutation impairedcis-splicing in favor of an alternativetrans-splice reaction via a cryptic 5′ss within a secondcis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5′ss we identified additionaltrans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNAtrans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes.
Mitochondrial ribosomes ofTrypanosoma bruceiare composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing.
Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.
Huntington’s disease (HD) is an adult-onset neurodegenerative disorder caused by a mutated CAG repeat in the huntingtin gene that is translated into an expanded polyglutamine tract. The clinical manifestation of HD is a progressive physical, cognitive, and psychiatric deterioration that is eventually fatal.
RNA transcripts are generally classified into polyA-plus and polyA-minus subgroups due to the presence or absence of a polyA tail at the 3′ end. Even though a number of physiologically and pathologically important polyA-minus RNAs have been recently identified, a systematic analysis of the expression and function of these transcripts in adipogenesis is still elusive. To study the potential function of the polyA-minus RNAs in adipogenesis, a dynamic expressional profiling was performed in the induced differentiation of 3T3-L1 cells. In addition to identifying thousands of novel intergenic transcripts, differentiation-synchronized expression was characterized for many of them. Among these, several large intergenic transcripts were found to be upregulated by more than 19-fold during differentiation. Further study demonstrated a fat tissue-specific expression pattern for these regions and identified an adipogenesis-associated long non-coding RNA. Collectively, these lines of evidence contribute to the characterization of a super-long intergenic transcript functioning in adipogenesis.
RNA molecules have highly versatile structures that can fold into myriad conformations, providing many potential pockets for binding small molecules. The increasing number of available RNA structures, in complex with proteins, small ligands and in free form, enables the design of new therapeutically useful RNA-binding ligands. Here we studied RNA ligand complexes from 10 RNA groups extracted from the protein data bank (PDB), including adaptive and non-adaptive complexes. We analyzed the chemical, physical, structural and conformational properties of binding pockets around the ligand. Comparing the properties of ligand-binding pockets to the properties of computed pockets extracted from all available RNA structures and RNA-protein interfaces, revealed that ligand-binding pockets, mainly the adaptive pockets, are characterized by unique properties, specifically enriched in rare conformations of the nucleobase and the sugar pucker. Further, we demonstrate that nucleotides possessing the rare conformations are preferentially involved in direct interactions with the ligand. Overall, based on our comprehensive analysis of RNA-ligand complexes, we suggest that the unique conformations adopted by RNA nucleotides play an important role in RNA recognition by small ligands. We term the recognition of a binding site by a ligand via the unique RNA conformations “RNA conformational readout.” We propose that “conformational readout” is a general way by which RNA binding pockets are recognized and selected from an ensemble of different RNA states.
Fungi of the CTG clade translate the Leu CUG codon as Ser. This genetic code alteration is the only eukaryotic sense-to-sense codon reassignment known to date, is mediated by an ambiguous serine tRNA (tRNACAGSer), exposes unanticipated flexibility of the genetic code and raises major questions about its selection and fixation in this fungal lineage. In particular, the origin of the tRNACAGSerand the evolutionary mechanism of CUG reassignment from Leu to Ser remain poorly understood. In this study, we have traced the origin of the tDNACAGSergene and studied critical mutations in the tRNACAGSeranticodon-loop that modulated CUG reassignment. Our data show that the tRNACAGSeremerged from insertion of an adenosine in the middle position of the 5′-CGA-3′anticodon of a tRNACGASerancestor, producing the 5′-CAG-3′ anticodon of the tRNACAGSer, without altering its aminoacylation properties. This mutation initiated CUG reassignment while two additional mutations in the anticodon-loop resolved a structural conflict produced by incorporation of the Leu 5′-CAG-3′anticodon in the anticodon-arm of a tRNASer. Expression of the mutant tRNACAGSerin yeast showed that it cannot be expressed at physiological levels and we postulate that such downregulation was essential to maintain Ser misincorporation at sub-lethal levels during the initial stages of CUG reassignment. We demonstrate here that such low level CUG ambiguity is advantageous in specific ecological niches and we propose that misreading tRNAs are targeted for degradation by an unidentified tRNA quality control pathway.
We established new methods for cloning cDNA ends that start with reverse transcription (RT) and soon proceed with the synthesis of the second cDNA strand, avoiding manipulations of fragile RNA. Our 3′-end cloning method does not involve poly-dT primers and polymerase chain reactions (PCR), is low in efficiency but high in fidelity and can clone those RNAs without a poly-A tail. We also established a cDNA protection assay to supersede RNA protection assay. The protected cDNA can be amplified, cloned and sequenced, enhancing sensitivity and fidelity. We report that RT product using gene-specific primer (GSP) cannot be gene- or strand-specific because RNA sample contains endogenous random primers (ERP). The gene-specificity may be improved by adding a linker sequence at the 5′-end of the GSP to prime RT and using the linker as a primer in the ensuing PCR. The strand-specificity may be improved by using strand-specific DNA oligos in our protection assay. The CDK4 mRNA and TSPAN31 mRNA are transcribed from the opposite DNA strands and overlap at their 3′ ends. Using this relationship as a model, we found that the overlapped sequence might serve as a primer with its antisense as the template to create a wrong-template extension in RT or PCR. We infer that two unrelated RNAs or cDNAs overlapping at the 5′- or 3′-end might create a spurious chimera in this way, and many chimeras with a homologous sequence may be such artifacts. The ERP and overlapping antisense together set complex pitfalls, which one should be aware of.
RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of protein-protein and RNA-protein interactions within a cell to achieve efficient replication and spreading. Deciphering these interactions is essential to reach a comprehensive understanding of the viral infection process. To study RNA-protein complexes directly in infected cells, we developed a new approach based on recombinant viruses expressing tagged viral proteins that were purified together with their specific RNA partners. High-throughput sequencing was then used to identify these RNA molecules. As a proof of principle, this method was applied to measles virus nucleoprotein (MV-N). It revealed that in addition to full-length genomes, MV-N specifically interacted with a unique population of 5′ copy-back defective interfering RNA genomes that we characterized. Such RNA molecules were able to induce strong activation of interferon-stimulated response element promoter preferentially via the cytoplasmic pattern recognition receptor RIG-I protein, demonstrating their biological functionality. Thus, this method provides a new platform to explore biologically active RNA-protein networks that viruses establish within infected cells.
In the last decade, an ever-growing number of connections between microRNAs (miRNAs) and RNA-binding proteins (RBPs) have uncovered a new level of complexity of gene expression regulation in cancer. In this review, we examine several aspects of the functional interactions between miRNAs and RBPs in cancer models. We will provide examples of reciprocal regulation: miRNAs regulating the expression of RBPs, or the converse, where an RNA-binding protein specifically regulates the expression of a specific miRNA, or when an RBP can exert a widespread effect on miRNAs via the modulation of a key protein for miRNA production or function. Moreover, we will focus on the ever-growing number of functional interactions that have been discovered in the last few years: RBPs that were shown to cooperate with microRNAs in the downregulation of shared target mRNAs or, on the contrary, that inhibit microRNA action, thus resulting in a protection of the specific target mRNAs. We surely need to obtain a deeper comprehension of such intricate networks to have a chance of understanding and, thus, fighting cancer.
Long non-coding RNAs (lncRNAs) have been found to perform various functions in a wide variety of important biological processes. To make easier interpretation of lncRNA functionality and conduct deep mining on these transcribed sequences, it is convenient to classify lncRNAs into different groups. Here, we summarize classification methods of lncRNAs according to their four major features, namely, genomic location and context, effect exerted on DNA sequences, mechanism of functioning and their targeting mechanism. In combination with the presently available function annotations, we explore potential relationships between different classification categories, and generalize and compare biological features of different lncRNAs within each category. Finally, we present our view on potential further studies. We believe that the classifications of lncRNAs as indicated above are of fundamental importance for lncRNA studies, helpful for further investigation of specific lncRNAs, for formulation of new hypothesis based on different features of lncRNA and for exploration of the underlying lncRNA functional mechanisms.
In the Gac/Rsm signal transduction pathway ofPseudomonas fluorescensCHA0, the dimeric RNA-binding proteins RsmA and RsmE, which belong to the vast bacterial RsmA/CsrA family, effectively repress translation of target mRNAs containing a typical recognition sequence near the translation start site. Three small RNAs (RsmX, RsmY, RsmZ) with clustered recognition sequences can sequester RsmA and RsmE and thereby relieve translational repression. According to a previously established structural model, the RsmE protein makes optimal contacts with an RNA sequence 5′-A/UCANGGANGU/A-3′, in which the central ribonucleotides form a hexaloop. Here, we questioned the relevance of the hexaloop structure in target RNAs. We found that two predicted pentaloop structures, AGGGA (inpltAmRNA encoding a pyoluteorin biosynthetic enzyme) and AAGGA (in mutatedpltAmRNA), allowed effective interaction with the RsmE protein in vivo. By contrast, ACGGA and AUGGA were poor targets. Isothermal titration calorimetry measurements confirmed the strong binding of RsmE to the AGGGA pentaloop structure in an RNA oligomer. Modeling studies highlighted the crucial role of the second ribonucleotide in the loop structure. In conclusion, a refined structural model of RsmE-RNA interaction accommodates certain pentaloop RNAs among the preferred hexaloop RNAs.
MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the “RNA-induced silencing complex” (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity.
Synthetic biology approaches often combine natural building blocks to generate new cellular activities. Here, we make use of two RNA elements to design a regulatory device with novel functionality. The system is based on a hammerhead ribozyme (HHR) that cleaves itself to generate a liberated ribosome-binding site and, thus, permits expression of a downstream gene. We connected a temperature-responsive RNA hairpin to the HHR and, thus, generated a temperature-controlled ribozyme that we call thermozyme. Specifically, aSalmonellaRNA thermometer (RNAT) known to modulate small heat shock gene expression by temperature-controlled base-pairing and melting was fused to the ribozyme. Following an in vivo screening approach, we isolated two functional thermozymes. In vivo expression studies and in vitro structure probing experiments support a mechanism in which rising temperatures melt the thermometer structure impairing the self-cleavage reaction of the ribozyme. Since RNA cleavage is necessary to liberate the RBS, these engineered thermozymes shut off gene expression in response to a temperature increase and, thus, act in a reverse manner as the natural RNAT. Our results clearly emphasize the highly modular nature and biotechnological potential of ribozyme-based RNA thermometers.
Post-synthetic modifications of nucleic acids have long been known to affect their functional and structural properties. For instance, numerous different chemical modifications modulate the structural organization, stability or translation efficiency of tRNAs and rRNAs. In contrast, little is known about modifications of poly(A)RNAs. Here, we demonstrate for the first time that the two well-studied regulatory long non-coding RNAs HOTAIR and XIST are targets of site-specific cytosine methylation. In both XIST and HOTAIR, we found methylated cytosines located within or near functionally important regions that are known to mediate interaction with chromatin-associated protein complexes. We show that cytosine methylation in the XIST A structure strongly affects binding to the chromatin-modifying complex PRC2 in vitro. These results suggest that cytosine methylation may serve as a general strategy to regulate the function of long non-coding RNAs.
Development, growth and adult survival are coordinated with available metabolic resources, ascertaining that the organism responds appropriately to environmental conditions. MicroRNAs are short (21–23 nt) regulatory RNAs that confer specificity on the RNA-induced silencing complex (RISC) to inhibit a given set of mRNA targets. We profiled changes in miRNA expression during adult life inDrosophila melanogasterand determined that miR-277 is downregulated during adult life. Molecular analysis revealed that this miRNA controls branched-chain amino acid (BCAA) catabolism and as a result it can modulate the activity of the TOR kinase, a central growth regulator, in cultured cells. Metabolite analysis in cultured cells as well as flies suggests that the mechanistic basis may be an accumulation of branched-chain α-keto-acids (BCKA), rather than BCAAs, thus avoiding potentially detrimental consequences of increased branched chain amino acid levels on e.g., translational fidelity. Constitutive miR-277 expression shortens lifespan and is synthetically lethal with reduced insulin signaling, indicating that metabolic control underlies this phenotype. Transgenic inhibition with a miRNA sponge construct also shortens lifespan, in particular on protein-rich food. Thus, optimal metabolic adaptation appears to require tuning of cellular BCAA catabolism by miR-277.