Two thirds of the human genome is composed of repetitive sequences. Despite their prevalence, DNA repeats are largely ignored. The vast majority of our genome is transcribed to produce non protein-coding RNAs. Among these, long non protein-coding RNAs represent the most prevalent and functionally diverse class. The relevance of the non protein-coding genome to human disease has mainly been studied regarding the altered microRNA expression and function in human cancer. On the contrary, the elucidation of the involvement of long non-coding RNAs in disease is only in its infancy. We have recently found that a chromatin associated, long non protein-coding RNA regulates a Polycomb/Trithorax epigenetic switch at the basis of the repeat associated facioscapulohumeral muscular dystrophy, a common muscle disorder. Based on this, we propose that long non-coding RNAs produced by repetitive sequences contribute in shaping the epigenetic landscape in normal human physiology and in disease.
repetitive element; non protein-coding RNA; polycomb; trithorax; chromatin; muscular dystrophy
microRNAs (miRNAs) are 20–24 nucleotide RNAs that regulate a variety of developmental and metabolic processes. The accumulation of miRNAs in vivo can be controlled at multiple levels. In addition to miRNA biogenesis, mechanisms that lead to RNA degradation, such as 3′ uridylation and 3′ truncation, also affect the steady-state levels of miRNAs. On the other hand, 2’-O-methylation in plant miRNAs protects their 3′ ends from truncation and uridylation. The recent identification of HESO1 as the key enzyme responsible for miRNA uridylation in Arabidopsis was a first step toward a full understanding of the mechanisms underlying miRNA turnover. Analyses of the heso1 mutant predicted the existence of another uridylation activity and a previously unknown nuclease that act on miRNAs. The future identification of these enzymes will enrich our understanding of miRNA turnover.
argonaute; HEN1; HESO1; methylation; microRNA; miRNA stability; miRNA turnover; uridylation
In the last years post-transcriptional regulation (PTR) of gene expression has been increasingly recognized to be a powerful and general determinant of the quantitative changes in proteomes, and therefore a driving force for cell phenotypes. By means of networks of trans-factors on one hand, and cis-elements found primarily in untranslated regions (UTRs) of mRNA on the other hand, mRNA availability to translation and translation rates are tightly controlled and can be rapidly tuned according to the changing state of the cell. A number of dedicated resources and tools, including databases and predictive algorithms, have been proposed as bioinformatics aids for the study of this fundamental layer of gene expression regulation. Their use, however, is rendered difficult by heterogeneity and fragmentation.
This review aims to locate these resources in their proper space, classifying them according to their goals, limitations and integration capabilities and, in the end, to provide the user with an initial toolbox for the bioinformatic analysis of post-transcriptional regulation of gene expression. The accompanying website, available at www.ptrguide.org, lists all resources, provides summary and features for each one and will be regularly updated in the future.
post-transcriptional regulation; translation; UTR; database; tool; RBP; ncRNA; miRNA; cis-element; trans-factor
Changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways. Recently, genome-wide inhibitor-free technologies for determining RNA stabilities in mammalian cells have been developed. In these methods, endogenous RNAs are pulse labeled by uridine analogs [e.g., 4-thiouridine (4sU), 5-etyniluridine (EU) and 5′-bromo-uridine (BrU)], followed by purification of labeled de novo RNAs. These technologies have revealed that the specific half-life of each mRNA is closely related to its physiological function. Genes with short-lived mRNAs are significantly enriched among regulatory genes, while genes with long-lived mRNAs are enriched among housekeeping genes. This review describes the recent progress of experimental procedures for measuring RNA stability.
genome-wide technology; RNA degradation; RNA decay; RNA transcription; non-coding RNA; 4sU; BrU
It is a prevalent concept that, in line with the Wobble Hypothesis, those tRNAs having an adenosine in the first position of the anticodon become modified to an inosine at this position. Sequencing the cDNA derived from the gene coding for cytoplasmic tRNAArgACG from several higher plants as well as mass spectrometric analysis of the isoacceptor has revealed that for this kingdom an unmodified A in the wobble position of the anticodon is the rule rather than the exception. In vitro translation shows that in the plant system the absence of inosine in the wobble position of tRNAArg does not prevent decoding. This isoacceptor belongs to the class of tRNA that is imported from the cytoplasm into the mitochondria of higher plants. Previous studies on the mitochondrial tRNA pool have demonstrated the existence of tRNAArgICG in this organelle. In moss the mitochondrial encoded distinct tRNAArgACG isoacceptor possesses the I34 modification. The implication is that for mitochondrial protein biosynthesis A-to-I editing is necessary and occurs by a mitochondrion-specific deaminase after import of the unmodified nuclear encoded tRNAArgACG.
wobble hypothesis; inosine; tRNAArg; deaminase; decoding; plant; mitochondrial tRNA import
Alterations in microRNA (miRNA) expression have been observed in cells subjected to exogenous stresses, implying that miRNAs play an important role in cellular stress response; however, the underlying mechanism is still largely unknown. In the present study, we found that miR-3928 was implicated in cellular response to ionizing radiation. After exposed to X-rays, miR-3928 expression increased in 1.5 h and then decreased, meanwhile Dicer, a key component in the miRNA processing machinery, increased gradually. An oscillation was observed in the expression of both mature miR-3928 and Dicer mRNA from 2 h to 3.5 h in irradiated cells. Then, we verified that miR-3928 directly bound to the 3'-untranslated region of Dicer mRNA. Consequently, Dicer expression was suppressed and the maturation of other miRNAs including miR-185, miR-300, and miR-663, was inhibited. Overexpression of miR-3928 induced DNA damage, activated ATR, and phosphorylated Chk1 accompanied by G1 arrest. Taken together, these findings replenished ATR/Chk1 pathway by revealing a novel miRNA regulatory network in response to exogenous stress, in which miR-3928 plays an important role in regulating the expression of Dicer.
DNA damage; Dicer; G1 arrest; ionizing radiation; microRNA
Polyadenylation is a 3′ mRNA processing event that contributes to gene expression by affecting stability, export and translation of mRNA. Human polyadenylation signals (PAS) have core and auxiliary elements that bind polyadenylation factors upstream and downstream of the cleavage site. The majority of mRNAs do not have optimal upstream and downstream core elements and therefore auxiliary elements can aid in polyadenylation efficiency.
Auxiliary elements have previously been identified and studied in a small number of mRNAs. We previously used a global approach to examine auxiliary elements to identify overrepresented motifs by a bioinformatic survey. This predicted information was used to direct our in vivo validation studies, all of which were accomplished using both a tandem in vivo polyadenylation assay and using reporter protein assays measured as luciferase activity. Novel auxiliary elements were placed in a test polyadenylation signal. An in vivo polyadenylation assay was used to determine the strength of the polyadenylation signal. All but one of the novel auxiliary elements enhanced the test polyadenylation signal. Effects of these novel auxiliary elements were also measured by a luciferase assay when placed in the 3′ UTR of a firefly luciferase reporter. Two novel downstream auxiliary elements and all of the novel upstream auxiliary elements showed an increase in reporter protein levels.
Many well known auxiliary polyadenylation elements have been found to occur in multiple sets. However, in our study, multiple copies of novel auxiliary elements brought reporter protein levels as well as polyadenylation choice back to wild type levels. Structural features of these novel auxiliary elements may also affect the role of auxiliary elements. A MS2 structure placed upstream of the polyadenylation signal can affect polyadenylation in both the positive and negative direction. A large change in RNA structure by using novel complementary auxiliary element also decreased polyadenylation choice and reporter protein levels. Therefore, we conclude that RNA structure has an important role in polyadenylation efficiency.
3′ end formation; RNA structure; auxiliary elements; polyadenylation; regulation
MicroRNAs (miRNAs) are important players of post-transcriptional gene regulation. Individual miRNAs can target multiple mRNAs and a single mRNA can be targeted by many miRNAs. We hypothesized that miRNAs select and regulate their targets based on their own expression levels, those of their target mRNAs and triggered feedback loops. We studied the effects of varying concentrations of let-7a-7f and the miR-17-92 cluster plasmids on the reporter genes carrying either DICER- or cMYC -3′UTR in Huh-7 cells. We showed that let-7 significantly downregulated expression of DICER 3′UTR reporter at lower concentrations, but selectively downregulated expression of a cMYC 3′UTR reporter at higher dose. This miRNA dose-dependent target selection was also confirmed in other target genes, including CCND1, CDKN1 and E2F1. After overexpressing let-7a-7f or the miR-17-92 clusters at wide-ranging doses, the target genes displayed a nonlinear correlation to the transfected miRNA. Further, by comparing the expression levels of let-7a and miR-17-5p, along with their selected target genes in 3 different cell lines, we found that the knockdown dose of each miRNA was directly related to their baseline expression level, that of the target gene and feedback loops. These findings were supported by gene modulation studies using endogenous levels of miR-29, -1 and -206 and a luciferase reporter system in multiple cell lines. Finally, we determined that the miR-17-92 cluster affected cell viability in a dose-dependent manner. In conclusion, we have shown that miRNAs potentially select their targets in a dose-dependent and nonlinear fashion that affects biological function; and this represents a novel mechanism by which miRNAs orchestrate the finely tuned balance of cell function.
microRNAs; let-7a-7f; miR-17-92; miRNA target selection; dose-dependent; oncogene; tumor suppressor gene
Sam68, the Src-associated substrate during mitosis of 68 kDa, belongs to the large class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) domain family of RNA-binding proteins. Sam68 contains a single KH domain harboring conserved N- and C-terminal sequences required for RNA binding and homodimerization. The KH domain is one of the most prevalent RNA binding domains that directly contacts single-stranded RNA. Sam68 has been implicated in numerous aspects of RNA metabolism including alternative splicing and polysomal recruitment of mRNAs. Studies in mice have revealed physiological roles linking Sam68 to osteoporosis, obesity, cancer, infertility and ataxia. Recent publications have greatly enhanced our understanding of Sam68 mechanism of action in addition to its cellular role. Herein, we will discuss the latest advances in the literature pertaining to obesity and neuronal development.
RNA binding proteins; Sam68; adipogenesis; alternative splicing; neurogenesis
Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition.1–4 Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e.1, 2 Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology.3, 4 While Cse1 carries out PAM recognition,5 the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer.6 Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop.
CRISPR; DNA; RNA; bacteria; defense; immune; nucleic acids; phage; prokaryotes; ribonucleoprotein complex
The stable linearity of eukaryotic chromosomes depends on special characteristics of their ends, the telomeres. Accurate telomere function in turn requires a sustained presence of repeated DNA elements, which are maintained by the enzyme telomerase. The telomerase holoenzyme is composed of both protein and RNA, and its functions rely on proper expression, maturation, trafficking and assembly of these components. Conflicting models for the recruitment of telomerase at telomeres have been proposed; one suggests a local activation of telomerase at short telomeres, while the other proposes that telomerase is recruited only at short telomeres. To discriminate between these models and investigate the cell cycle-dependent regulation of telomerase in living cells, a GFP reporter system to visualize the yeast telomerase RNA has been recently developed. This assay shed new light on the mechanism of recruitment of telomerase to telomeres, and it uncovered a hitherto unrecognized mechanism for restricting telomerase access to telomeres.
TLC1 RNA; MS2-GFP; Rif1; Rif2; live cell imaging; telomerase; telomeres; yeast
Bacteriophage vectors for achieving single-copy gene expression linked to a colorigenic reporter assay have been used successfully for genetic screening applications. However, the limited number of cloning sites in these vectors, combined with the requirement for lac- strains and the time- and/or media-dependence of the chemical-based colorimetric reaction, have limited the range of applications for these vectors. An alternative approach using a fluorescent reporter gene such as green fluorescent protein (GFP) or GFP derivatives could overcome some of these technical issues and facilitate real-time monitoring of promoter and/or protein activity. Here, we report the development of a novel translational bacteriophage fusion vector encoding enhanced GFP (eGFP) that can be incorporated into the chromosome as a single-copy gene. We identified a Bacillus promoter (BP) that is stably expressed in Escherichia coli and drives ~6-fold more expression of eGFP than the T7 promoter in the absence of inducer. Incorporating this BP and RNase III target signals into a single system enabled clear detection of the absence or downregulation of RNase III activity in vivo, thereby establishing a system for screening and identifying novel RNase III targets in a matter of days. An RNase III target signal identified in this manner was confirmed by post-transcriptional analysis. We anticipate that this novel translational fusion vector will be used extensively to study activity of both interesting RNases and related complex or to identify or validate targets of RNases that are otherwise difficult to study due to their sensitivity to environmental stresses and/or autoregulatory processes.
fluorescence; bacteriophage vector; translational fusion reporter system; ribonuclease III (RNase III); cleavage target
Mirtrons are short intronic microRNA (miRNA) precursors representing an alternative, Drosha/DGCR8-independent miRNA biogenesis pathway. In this study we characterized three predicted human mirtrons. Their expression was proven to be context-independent, since functional mirtrons could be derived either from their endogenous or from a heterologous coding environment. Systematic testing revealed that both 5′- and 3′-arms of mir-877 are capable of producing functional miRNA simultaneously in the various cell types examined. On the other hand, experimental validations revealed that the predicted mir-1233 is not a bona fide mirtron. For functional mirtrons, we were able to detect mature mirtron-derived miRNAs for the first time by qRT-PCR or northern blot analysis, when silencing activity was proven by functional assays. Our results emphasize the need for functional testing of both arms of miRNAs and the importance of experimentally validating human mirtrons since, in spite of being localized in a short intron, predicted species could mature via other miRNA processing pathways.
RNA interference; miRNA; miRNA*; mir-1226; mir-1233; mir-877; mirtron
External guide sequences (EGSs) are RNA molecules that can bind to a target mRNA and direct ribonuclease P (RNase P), a tRNA processing enzyme, for specific cleavage of the target mRNA. Using an in vitro selection procedure, we have previously generated EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, we constructed EGSs from a variant to target the overlapping region of the mRNAs coding for human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The EGS variant was about 40-fold more active in directing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of about 98% and 75% in CSP/assemblin gene expression and a reduction of 7000- and 250-fold in viral growth were observed in HCMV-infected cells that expressed the variant and the tRNA-derived EGS, respectively. Our study shows that the EGS variant is more effective in blocking HCMV gene expression and growth than the tRNA-derived EGS. Moreover, these results demonstrate the utility of highly active EGS RNA variants in gene targeting applications including anti-HCMV therapy.
RNase P; external guide sequence; gene therapy; cytomegalovirus; gene targeting
Computational methods for miRNA target prediction vary in the algorithm used; and while one can state opinions about the strengths or weaknesses of each particular algorithm, the fact of the matter is that they fall substantially short of capturing the full detail of physical, temporal and spatial requirements of miRNA::target-mRNA interactions. Here, we introduce a novel miRNA target prediction tool called Targetprofiler that utilizes a probabilistic learning algorithm in the form of a hidden Markov model trained on experimentally verified miRNA targets. Using a large scale protein downregulation data set we validate our method and compare its performance to existing tools. We find that Targetprofiler exhibits greater correlation between computational predictions and protein downregulation and predicts experimentally verified miRNA targets more accurately than three other tools. Concurrently, we use primer extension to identify the mature sequence of a novel miRNA gene recently identified within a cancer associated genomic region and use Targetprofiler to predict its potential targets. Experimental verification of the ability of this small RNA molecule to regulate the expression of CCND2, a gene with documented oncogenic activity, confirms its functional role as a miRNA. These findings highlight the competitive advantage of our tool and its efficacy in extracting biologically significant results.
computational prediction; microRNA gene targets; experimental verification; luciferase assays; CCND2; oncogene
The largest subunit of RNA polymerase (pol) II, Rpb1, contains an unusual carboxyl-terminal domain (CTD) composed of consecutive repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). During transcription, Ser2, Ser5 and Ser7 are subjected to dynamic phosphorylation and dephosphorylation by CTD kinases and phosphatases, creating a characteristic CTD phosphorylation pattern along genes. This CTD “code” allows the coupling of transcription with co-transcriptional RNA processing, through the timely recruitment of the appropriate factors at the right point of the transcription cycle. In mammals, phosphorylation of Ser7 (Ser7P) is detected on all pol II-transcribed genes, but is only essential for expression of a sub-class of genes encoding small nuclear (sn)RNAs. The molecular mechanisms by which Ser7P influences expression of these particular genes are becoming clearer. Here, I discuss our recent findings clarifying how Ser7P facilitates transcription of these genes and 3′end processing of the transcripts, through recruitment of the RPAP2 phosphatase and the snRNA gene-specific Integrator complex.
CTD phosphatase; RNA polymerase II; RNA processing; carboxyl-terminal domain; snRNA genes; transcription
In the nematode Caenorhabditis elegans (C. elegans), gene inactivation by RNA interference can achieve remarkable potency due to the amplification of initial silencing triggers by RNA-dependent RNA polymerases (RdRPs). RdRPs catalyze the biogenesis of an abundant species of secondary small interfering RNAs (siRNAs) using the target mRNA as template. The interaction between primary siRNAs derived from the exogenous double-stranded RNA (dsRNA) trigger and the target mRNA is required for the recruitment of RdRPs. Other genetic requirements for RdRP activities have not been characterized. Recent studies have identified the RDE-10/RDE-11 complex which interacts with the primary siRNA bound target mRNA and acts upstream of the RdRPs. rde-10 and rde-11 mutants show an RNAi defective phenotype because the biogenesis of secondary siRNAs is completely abolished. In addition, the RDE-10/RDE-11 complex plays a similar role in the endogenous RNAi pathway for the biogenesis of a subset of siRNAs targeting recently acquired, duplicated genes.
C. elegans; rde-10; rde-11; RNAi; small interfering RNAs
Editing by ADAR enzymes is essential for mammalian life. Still, knowledge of the spatio-temporal editing patterns in mammals is limited. By use of 454 amplicon sequencing we examined the editing status of 12 regionally extracted mRNAs from porcine developing brain encompassing a total of 64 putative ADAR editing sites. In total 24 brain tissues, dissected from up to five regions from embryonic gestation day 23, 42, 60, 80, 100 and 115, were examined for editing.
Generally, editing increased during embryonic development concomitantly with an increase in ADAR2 mRNA level. Notably, the Gria2 (GluR-B) Q/R site, reported to be ~100% edited in previous studies, is only 54% edited at embryonic day 23. Transcripts with multiple editing sites in close proximity to each other exhibit coupled editing and an extraordinary incidence of long-range coupling of editing events more than 32 kb apart is observed for the kainate glutamate receptor 2 transcript, Grik2. Our study reveals complex spatio-temporal ADAR editing patterns of coordinated editing events that may play important roles in the development of the mammalian brain.
5-HT2C receptor; ADAR; ADAR2; Blcap; GluR-B; Gria2; Htr2c; Pig; RNA editing
Antisense inhibition of oncogenic or other disease-related miRNAs and miRNA families in vivo may provide novel therapeutic strategies. However, this approach relies on the development of potent miRNA inhibitors and their efficient delivery into cells. Here, we introduce short seed-directed LNA oligonucleotides (12- or 14-mer antiseeds) with a phosphodiester backbone (PO) for efficient miRNA inhibition. We have analyzed such LNA (PO) antiseeds using a let-7a-controlled luciferase reporter assay and identified them as active miRNA inhibitors in vitro. Moreover, LNA (PO) 14-mer antiseeds against ongogenic miR-17–5p and miR-20a derepress endogenous p21 expression more persistently than corresponding miRNA hairpin inhibitors, which are often used to inhibit miRNA function. Further analysis of the antiseed-mediated derepression of p21 in luciferase reporter constructs - containing the 3′-UTR of p21 and harboring two binding sites for miRNAs of the miR-106b family - provided evidence that the LNA antiseeds inhibit miRNA families while hairpin inhibitors act in a miRNA-specific manner. The derepression caused by LNA antiseeds is specific, as demonstrated via seed mutagenesis of the miR-106b target sites. Importantly, we show functional delivery of LNA (PO) 14-mer antiseeds into cells upon complexation with polyethylenimine (PEI F25-LMW), which leads to the formation of polymeric nanoparticles. In contrast, attempts to deliver a functional seed-directed tiny LNA 8-mer with a phosphorothioate backbone (PS) by formulation with PEI F25-LMW remained unsuccessful. In conclusion, LNA (PO) 14-mer antiseeds are attractive miRNA inhibitors, and their PEI-based delivery may represent a promising new strategy for therapeutic applications.
miRNA; miR-17-92; let-7a; antimiR; antiseed; LNA; PEI; p21; cancer
cyclin D3 (CCND3) is one of the three D-type cyclins that regulate the G1/S phase transition of the cell cycle. Expression of CCND3 is observed in nearly all proliferating cells; however, the presence of high levels of CCND3 has been linked to a poor prognosis for several types of cancer. Therefore, further mechanistic studies on the regulation of CCND3 expression are urgently needed to provide therapeutic implications. In this study, we report that a conserved RNA G-quadruplex-forming sequence (hereafter CRQ), located in the 5′ UTR of mammalian CCND3 mRNA, is able to fold into an extremely stable, intramolecular, parallel G-quadruplex in vitro. The CRQ G-quadruplex dramatically reduces the activity of a reporter gene in human cell lines, but it has little impact on its mRNA level, indicating a translational repression. Moreover, the CRQ sequence in its natural context inhibits translation of CCND3. Disruption of the G-quadruplex structure by G/U-mutation or deletion results in an elevated expression of CCND3 and an increased phosphorylation of Rb, a downstream target of CCND3, which promotes progression of cells through the G1 phase. Our results add to the growing understanding of the regulation of CCND3 expression and provide a potential therapeutic target for cancer treatment.
CCND3; RNA; cell cycle; quadruplex; translation
Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome Entry Site (IRES) present in the 5′-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.
HCV IRES; Translation inhibition; Sendai virosome; La autoantigen; Ribosome assembly
Negative-strand (NS) RNA viruses initiate infection with a unique polymerase complex that mediates both mRNA transcription and subsequent genomic RNA replication. For nearly all NS RNA viruses, distinct enzymatic domains catalyzing RNA polymerization and multiple steps of 5′ mRNA cap formation are contained within a single large polymerase protein (L). While NS RNA viruses include a variety of emerging human and agricultural pathogens, the enzymatic machinery driving viral replication and gene expression remains poorly understood. Recent insights with Machupo virus and vesicular stomatitis virus have provided the first structural information of viral L proteins, and revealed how the various enzymatic domains are arranged into a conserved architecture shared by both segmented and nonsegmented NS RNA viruses. In vitro systems reconstituting RNA synthesis from purified components provide new tools to understand the viral replicative machinery, and demonstrate the arenavirus matrix protein regulates RNA synthesis by locking a polymerase–template complex. Inhibition of gene expression by the viral matrix protein is a distinctive feature also shared with influenza A virus and nonsegmented NS RNA viruses, possibly illuminating a conserved mechanism for coordination of viral transcription and polymerase packaging
5′ cap formation; L protein structure; Machupo virus; Negative-strand RNA virus; arenavirus; matrix protein; polymerase; single-particle electron microscopy; template recognition; vesicular stomatitis virus
Correct codon-anticodon pairing promotes translational fidelity, with these interactions greatly facilitated by modified nucleosides found in tRNA. We hypothesized that wobble uridine modifications catalyzed by tRNA methyltransferase 9 (Trm9) are essential for translational fidelity. In support, we have used phenotypic, reporter and protein-based assays to demonstrate increased translational infidelity in trm9Δ Saccharomyces cerevisiae cells. Codon reengineering studies suggest that Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific genes, those rich in arginine and glutamic acid codons from mixed boxes. Using quantitative tRNA modification analysis, we determined that trm9Δ cells are only deficient in 2 of 23 tRNA modifications, with those 2, 5-methoxycarbonylmethyluridine (mcm5U) and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), classified as key determinants of translational fidelity. We also show that in the absence of mcm5U and mcm5s2U, the resulting translational infidelity promotes protein errors and activation of unfolded protein and heat shock responses. These data support a model in which Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific transcripts, with decreased wobble base modification leading to translational infidelity, protein errors and activation of protein stress response pathways.
Trm9; codon; heat shock response; modification; protein stress; tRNA; translation infidelity; unfolded protein response; wobble base
Post-transcriptional gene regulation by microRNAs (miRNAs) and RNA-binding proteins (RBPs) is central to many biological functions. Aberrant gene expression patterns underlie many metabolic diseases that represent major public health concerns and formidable therapeutic challenges. Several studies have established a number of post-transcriptional regulators implicated in metabolic diseases such as diabetes and obesity. In addition, emerging knowledge of metabolically active and insulin-sensitive organs, such as the pancreas, liver, muscle and adipose compartment, is rapidly expanding the panel of potential therapeutic targets for the treatment of metabolic diseases. Here, we review our current understanding of miRNAs and RBPs that affect glucose and lipid homeostasis, and their roles in normal physiology and metabolic disorders, especially type 2 diabetes and obesity.
posttranscriptional gene regulation; microRNAs; RNA-binding proteins; glucose homeostasis; lipid homeostasis; diabetes; metabolic disease
The tumor suppressor p53 is a sequence-specific transcription factor that activates the expression of genes involved in apoptosis, cell cycle arrest and senescence. p53 can also inhibit gene expression and this effect is partly mediated by inducing several microRNAs (miRNAs). MiRNAs have emerged as a new class of regulators of the expression and function of eukaryotic genomes. Tumor suppressive or oncogenic functions have been attributed to some miRNAs. Recent studies have shown that p53 can alter the transcription of several miRNAs, and in some cases, it can also influence miRNA maturation. Conversely, miRNAs can also modulate the abundance and activity of p53 by direct or indirect mechanisms. Moreover, mutant p53 can actively repress the expression of some miRNAs that are activated by wild-type p53. In this review, we discuss recent evidences of this crosstalk between miRNAs and the p53 network and also highlight its implications in cancer.
cancer; miRNA; miRNA dysregulation; mutant p53; p53; wild-type p53