Cellular RNA Metabolism Research Articles

Structural insight into precursor ribosomal RNA processing by ribonuclease MRP.

Ribonuclease (RNase) MRP is a conserved eukaryotic ribonucleoprotein complex that plays essential roles in precursor ribosomal RNA (pre-rRNA) processing and cell cycle regulation. In contrast to RNase P, which selectively cleaves transfer RNA-like substrates, it has remained a mystery how RNase MRP recognizes its diverse substrates. To address this question, we determined cryo-electron microscopy structures of Saccharomyces cerevisiae RNase MRP alone and in complex with a fragment of pre-rRNA. These structures and the results of biochemical studies reveal that coevolution of both protein and RNA subunits has transformed RNase MRP into a distinct ribonuclease that processes single-stranded RNAs by recognizing a short, loosely defined consensus sequence. This broad substrate specificity suggests that RNase MRP may have myriad yet unrecognized substrates that could play important roles in various cellular contexts.

RNase AM, a 5' to 3' exonuclease, matures the 5' end of all three ribosomal RNAs in E. coli.

Bacterial ribosomal RNAs (rRNAs) are transcribed as precursors and require processing by Ribonucleases (RNases) to generate mature and functional rRNAs. Although the initial steps of rRNA processing in Escherichia coli (E. coli) were described several decades ago, the enzymes responsible for the final steps of 5S and 23S rRNA 5′-end maturation have remained unknown. Here, I show that RNase AM, a recently identified 5′ to 3′ exonuclease, performs the last step of 5S rRNA 5′-end maturation. RNase AM was also found to generate the mature 5′ end of 23S rRNA, subsequent to a newly identified prior processing step. Additionally, RNase AM was found to mature the 5′ end of 16S rRNA, a reaction previously attributed to RNase G. These findings indicate a major role for RNase AM in cellular RNA metabolism and establish a biological role for the first 5′ to 3′ RNA exonuclease identified in E. coli.

Arsenite Increases Linc-ROR in Human Bronchial Epithelial Cells that Can Be Inhibited by Antioxidant Factors.

Oxidative stress is the main mechanism of arsenite toxicity. Long intergenic non-coding RNA regulator of reprogramming is a newly found stress-response long non-coding RNA that is activated in various stress conditions. However, whether long intergenic non-coding RNA, regulator of reprogramming (linc-ROR) is involved in arsenite-induced oxidative stress has not been explored. In this study, we found that arsenite dose responsively increased the expression of linc-ROR in human bronchial epithelial (HBE) cells, along with elevated oxidative stress demonstrated by increased intracellular reactive oxygen species (ROS) and DNA damage, as well as decreased antioxidant glutathione and superoxide dismutase. We further found that the pre-treatment with N-acetylcysteine, a widely used ROS scavenger, and the over-expression of antioxidant NRF2 protein, both significantly reduced arsenite-induced oxidative stress in arsenite-treated HBE cells, and the linc-ROR over-expression was also inhibited, suggesting that oxidative stress is a key factor for the increase of linc-ROR in arsenite-treated HBE cells. Moreover, our results of bio-informatic analysis showed that arsenite-induced oxidative stress might modulate linc-ROR expression via 3 genes and the up-regulated linc-ROR in arsenite-induced oxidative stress may get involved in cellular processes such as cellular stress response, RNA metabolism, and DNA repair. Collectively, our study demonstrates that oxidative stress plays the key role in arsenite-induced over-expression of linc-ROR, and linc-ROR may be a new clue for exploring the mechanism of arsenite toxicity.

DEAD-Box Helicases: Sensors, Regulators, and Effectors for Antiviral Defense.

DEAD-box helicases are a large family of conserved RNA-binding proteins that belong to the broader group of cellular DExD/H helicases. Members of the DEAD-box helicase family have roles throughout cellular RNA metabolism from biogenesis to decay. Moreover, there is emerging evidence that cellular RNA helicases, including DEAD-box helicases, play roles in the recognition of foreign nucleic acids and the modulation of viral infection. As intracellular parasites, viruses must evade detection by innate immune sensing mechanisms and degradation by cellular machinery while also manipulating host cell processes to facilitate replication. The ability of DEAD-box helicases to recognize RNA in a sequence-independent manner, as well as the breadth of cellular functions carried out by members of this family, lead them to influence innate recognition and viral infections in multiple ways. Indeed, DEAD-box helicases have been shown to contribute to intracellular immune sensing, act as antiviral effectors, and even to be coopted by viruses to promote their replication. However, our understanding of the mechanisms underlying these interactions, as well as the cellular roles of DEAD-box helicases themselves, is limited in many cases. We will discuss the diverse roles that members of the DEAD-box helicase family play during viral infections.

Stress granules and neurodegeneration.

Recent advances suggest that the response of RNA metabolism to stress has an important role in the pathophysiology of neurodegenerative diseases, particularly amyotrophic lateral sclerosis, frontotemporal dementias and Alzheimer disease. RNA-binding proteins (RBPs) control the utilization of mRNA during stress, in part through the formation of membraneless organelles termed stress granules (SGs). These structures form through a process of liquid-liquid phase separation. Multiple biochemical pathways regulate SG biology. The major signalling pathways regulating SG formation include the mammalian target of rapamycin (mTOR)-eukaryotic translation initiation factor 4F (eIF4F) and eIF2α pathways, whereas the pathways regulating SG dispersion and removal are mediated by valosin-containing protein and the autolysosomal cascade. Post-translational modifications of RBPs also strongly contribute to the regulation of SGs. Evidence indicates that SGs are supposed to be transient structures, but the chronic stresses associated with ageing lead to chronic, persistent SGs that appear to act as a nidus for the aggregation of disease-related proteins. We suggest a model describing how intrinsic vulnerabilities within the cellular RNA metabolism might lead to the pathological aggregation of RBPs when SGs become persistent. This process might accelerate the pathophysiology of many neurodegenerative diseases and myopathies, and it suggests new targets for disease intervention.

N4BP1 restricts HIV-1 and its inactivation by MALT1 promotes viral reactivation.

RNA-modulating factors not only regulate multiple steps of cellular RNA metabolism, but also emerge as key effectors of the immune response against invading viral pathogens including human immunodeficiency virus type-1 (HIV-1). However, the cellular RNA-binding proteins involved in the establishment and maintenance of latent HIV-1 reservoirs have not been extensively studied. Here, we screened a panel of 62 cellular RNA-binding proteins and identified NEDD4-binding protein 1 (N4BP1) as a potent interferon-inducible inhibitor of HIV-1 in primary T cells and macrophages. N4BP1 harbours a prototypical PilT N terminus-like RNase domain and inhibits HIV-1 replication by interacting with and degrading viral mRNA species. Following activation of CD4+ T cells, however, N4BP1 undergoes rapid cleavage at Arg 509 by the paracaspase named mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1). Mutational analyses and knockout studies revealed that MALT1-mediated inactivation of N4BP1 facilitates the reactivation of latent HIV-1 proviruses. Taken together, our findings demonstrate that the RNase N4BP1 is an efficient restriction factor of HIV-1 and suggest that inactivation of N4BP1 by induction of MALT1 activation might facilitate elimination of latent HIV-1 reservoirs.

The Host DHX9 DExH-Box Helicase Is Recruited to Chikungunya Virus Replication Complexes for Optimal Genomic RNA Translation.

Beyond their role in cellular RNA metabolism, DExD/H-box RNA helicases are hijacked by various RNA viruses in order to assist replication of the viral genome. Here, we identify the DExH-box RNA helicase 9 (DHX9) as a binding partner of chikungunya virus (CHIKV) nsP3 mainly interacting with the C-terminal hypervariable domain. We show that during early CHIKV infection, DHX9 is recruited to the plasma membrane, where it associates with replication complexes. At a later stage of infection, DHX9 is, however, degraded through a proteasome-dependent mechanism. Using silencing experiments, we demonstrate that while DHX9 negatively controls viral RNA synthesis, it is also required for optimal mature nonstructural protein translation. Altogether, this study identifies DHX9 as a novel cofactor for CHIKV replication in human cells that differently regulates the various steps of CHIKV life cycle and may therefore mediate a switch in RNA usage from translation to replication during the earliest steps of CHIKV replication.IMPORTANCE The reemergence of chikungunya virus (CHIKV), an alphavirus that is transmitted to humans by Aedes mosquitoes, is a serious global health threat. In the absence of effective antiviral drugs, CHIKV infection has a significant impact on human health, with chronic arthritis being one of the most serious complications. The molecular understanding of host-virus interactions is a prerequisite to the development of targeted therapeutics capable to interrupt viral replication and transmission. Here, we identify the host cell DHX9 DExH-Box helicase as an essential cofactor for early CHIKV genome translation. We demonstrate that CHIKV nsP3 protein acts as a key factor for DHX9 recruitment to replication complexes. Finally, we establish that DHX9 behaves as a switch that regulates the progression of the viral cycle from translation to genome replication. This study might therefore have a significant impact on the development of antiviral strategies.

Comparative proteomics illustrates the molecular mechanism of potato (Solanum tuberosum L.) tuberization inhibited by exogenous gibberellins in vitro.

Among the multiple environmental signals and hormonal factors regulating potato tuberization, gibberellins (GAs) are important components of the signaling pathways in these processes. To understand the GAs-signaling response mechanism of potato tuberization, a comparative proteomics approach was applied to analyze proteome change of potato tuberization in vitro subjected to a range of exogenous GA3 treatments (0, 0.01, 0.1 and 1.0 μM) using two-dimensional gel electrophoresis. Quantitative image analyses showed that a total of 37 protein spots have their abundance significantly altered more than 2-fold. Among these proteins, 13 proteins were up-regulated, 13 proteins were down-regulated, one protein was absent and 10 proteins were induced after treatment by exogenous GA3 . The MALDI-TOF/TOF MS analyses led to the identification of differentially abundant proteins that are mainly involved in bioenergy and metabolism, storage, signaling, cell defense and rescue, transcription, chaperones, transport. Furthermore, the comparative analysis of GA3 -responsive proteome allowed for general elucidation of underlying molecular mechanisms of potato tuberization inhibited by exogenous GA3 . Most of these cellular processes were not conducive to the transition from stolon elongation to tuber formation, including a blockage of starch and storage protein accumulation, the accelerated carbohydrate catabolism, a blockage of JA biosynthesis but an elevated endogenous GAs level, the amplification of GA3 signal transduction by other signaling pathways, and the regulation of cellular RNA metabolism for controlling tuberization. Our results firstly integrated physiology and proteome data to provide new insights into GA3 -signaling response mechanisms of potato tuberization in vitro.

Distinct mechanisms obviate the potentially toxic effects of inverted-repeat Alu elements on cellular RNA metabolism.

RNA-binding proteins are shown in two independent studies to mediate distinct and beneficial effects to cells by binding to the extensive double-stranded RNA structures of inverted-repeat Alu elements. One study reports stress-induced export of ADAR1p110 to the cytoplasm where it binds inverted-repeat Alu elements so as to protect many mRNAs encoding anti-apoptotic proteins from degradation; the other demonstrates binding of the nuclear helicase DHX9 to inverted-repeat Alu elements embedded within RNAs to minimize defects in RNA processing.

Expression of Sam68 Correlates With Cell Proliferation and Survival in Epithelial Ovarian Cancer.

Src associated in mitosis, 68 kDa (Sam68) is a KH domain RNA-binding protein that belongs to the signal transduction and activation of RNA family. It is a multifunctional protein known to regulate cellular signal transduction, transcription, RNA metabolism, proliferation, and apoptosis, thus implicated in tumor growth. Herein, we investigated the clinical significance of Sam68 in human epithelial ovarian cancer (EOC). Western blot and immunohistochemical staining demonstrated that Sam68 expression was upregulated in EOC tissues and cell lines. Statistical analysis showed that high expression of Sam68 correlated with poor prognosis of patients with EOC. In vitro, serum starvation-refeeding experiment was primarily performed to confirm that Sam68 participated in the cell cycle progression of EOC cell lines. Then knocking down Sam68 level with small interfering RNA, cell cycle was arrested at G1 phase and cell proliferation impaired. Furthermore, we demonstrated that the antiproliferative effect of silencing Sam68 in EOC cells was associated with the upregulation of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, along with the downregulation of p-FOXO3a, p-Akt, and p-GSK-3β. Taken together, our findings uncovered that Sam68 played an important role in promoting the proliferation of human ovarian cancer, thereby might be a novel therapeutic target for EOC.

Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs.

Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19.

Reprogramming, Circular Reasoning and Self versus Non-self: One-Stop Shopping with RNA Editing

Transcription of genetic information from archival DNA into RNA molecule working copies is vital for proper cellular function and is highly accurate. In turn, RNAs serve structural, enzymatic, and regulatory roles, as well as being informational templates for the ribosomal translation of proteins. Following RNA synthesis, maturing of RNA molecules occurs through various RNA processing events. One component of the collection of processes involving RNA species, broadly defined as RNA metabolism, is the RNA-editing pathway and is found in all animals. Acting specifically on RNA substrates with double-stranded character, RNA editing has been shown to regulate a plethora of genomic outputs, including gene recoding, RNA splicing, biogenesis and targeting actions of microRNAs and small interfering RNAs, and global gene expression. Recent evidence suggests that RNA modifications mediated via RNA editing influence the biogenesis of circular RNAs and safeguard against aberrant innate immune responses generated to endogenous RNA sources. These novel roles have the potential to contribute new insights into molecular mechanisms underlying pathogenesis mediated by mishandling of double-stranded RNA. Here, we discuss recent advances in the field, which highlight novel roles associated with the RNA-editing process and emphasize their importance during cellular RNA metabolism. In addition, we highlight the relevance of these newly discovered roles in the context of neurological disorders and the more general concept of innate recognition of self versus non-self.

Vinca alkaloid drugs promote stress-induced translational repression and stress granule formation.

Resistance to chemotherapy drugs is a serious therapeutic problem and its underlying molecular mechanisms are complex. Stress granules (SGs), cytoplasmic ribonucleoprotein complexes assembled in cells exposed to stress, are implicated in various aspects of cancer cell metabolism and survival. SGs promote the survival of stressed cells by reprogramming gene expression and inhibiting pro-apoptotic signaling cascades. We show that the vinca alkaloid (VA) class of anti-neoplastic agents potently activates a SG-mediated stress response program. VAs inhibit translation initiation by simultaneous activation of eIF4E-BP1 and phosphorylation of eIF2α, causing polysome disassembly and SG assembly. VA-induced SGs contain canonical SG components but lack specific signaling molecules. Blocking VA-induced SG assembly by inactivating eIF4EBP1 or inhibiting eIF2α phosphorylation decreases cancer cell viability and promotes apoptosis. Our data describe previously unappreciated effects of VAs on cellular RNA metabolism and illuminate the roles of SGs in cancer cell survival.

Regulation of axon regeneration by the RNA repair and splicing pathway.

Mechanisms governing a neuron’s regenerative ability are important but not well understood. We identified Rtca, RNA 3′-terminal phosphate cyclase, as an inhibitor for axon regeneration. Removal of dRtca cell-autonomously enhanced axon regrowth in the Drosophila central nervous system, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair/splicing pathway important for stress induced mRNA splicing, including that of Xbp1, a cellular stress sensor. dRtca and dArchease had opposing effects on Xbp1 splicing, and deficiency of dArchease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma.

Deletion of the Sm1 encoding motif in the lsm gene results in distinct changes in the transcriptome and enhanced swarming activity of Haloferax cells.

Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility.

Techniques for RNA in vivo imaging in plants.

Since the discovery of small RNAs and RNA silencing, RNA biology has taken a centre stage in cell and developmental biology. Small RNAs, but also mRNAs and other types of cellular and viral RNAs are processed at specific subcellular localizations. To fully understand cellular RNA metabolism and the various processes influenced by it, techniques are required that permit the sequence-specific tracking of RNAs in living cells. A variety of methods for RNA visualization have been developed since the 1990s, but plant cells pose particular challenges and not all approaches are applicable to them. On the other hand, plant RNA metabolism is particularly diverse and RNAs are even transported between cells, so RNA imaging can potentially provide many valuable insights into plant function at the cellular and tissue level. This Short Review briefly introduces the currently available techniques for plant RNA in vivo imaging and discusses their suitability for different biological questions.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Flaviviral RNAs: weapons and targets in the war between virus and host.

Flaviviruses are a genus of (+)ssRNA (positive ssRNA) enveloped viruses that replicate in the cytoplasm of cells of diverse species from arthropods to mammals. Many are important human pathogens such as DENV-1-4 (dengue virus types1-4), WNV (West Nile virus), YFV (yellow fever virus), JEV (Japanese encephalitis virus) and TBEV (tick-borne encephalitis). Given their RNA genomes it is not surprising that flaviviral life cycles revolve around critical RNA transactions. It is these we highlight in the present article. First, we summarize the mechanisms governing flaviviral replication and the central role of conserved RNA elements and viral protein-RNA interactions in RNA synthesis, translation and packaging. Secondly, we focus on how host RNA-binding proteins both benefit and inhibit flaviviral replication at different stages of their life cycle in mammalian hosts. Thirdly, we cover recent studies on viral non-coding RNAs produced in flavivirus-infected cells and how these RNAs affect various aspects of cellular RNA metabolism. Together, the article puts into perspective the central role of flaviviral RNAs in modulating both viral and cellular functions.

Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target

hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans.

Post-transcriptional control of HIV-1 Gag expression

Background The HIV-1 genomic RNA (gRNA) has several roles in the viral life cycle. In the nucleus, the gRNA is the pre-mRNA that is alternatively and incompletely spliced into mRNAs encoding Tat, Rev, Nef, Vif, Vpr, Vpu and Env. However, approximately 50% of the gRNA remains unspliced and is exported from the nucleus by the viral protein Rev using the CRM1 nuclear export pathway. In the cytoplasm, the gRNA serves as the mRNA for Gag and Gag-Pol and is also packaged into virions. As the gRNA is an unspliced, intron-containing mRNA with a long, highly structured 5’ UTR and rare codon usage in the open reading frames, how it interacts with cellular RNA metabolism and translation machinery to promote abundant Gag expression and infectious virion production remains unclear.