Generating heat and maintaining body temperature is the primary function of brown adipose tissue (BAT). Previous studies have implicated microRNAs, includingmiR-193bandmiR-365-1,in BAT differentiation. We used mouse genetics to further understand the specific contributions of these two miRs. BAT function in mice with an inactivatedmiR-193b-365-1locus, as determined by their response to the selective β3 adrenergic receptor agonist CL316.243 and their tolerance to cold exposure, was normal and expression of genes associated with functional BAT, includingPrdm16andUcp1, was unaffected. In addition, genome-wide expression profiles of miRNAs and mRNAs in BAT in the presence and absence ofmiR-193b-365-1were determined. In summary, these data demonstrate, in contrast to earlier work, that the development, differentiation, and function of BAT do not require the presence ofmiR-193bandmiR-365-1.
RNAs have many important functional properties, including that they are independently controllable and highly tunable. As a result of these advantageous properties, their use in a myriad of sophisticated devices has been widely explored. Yet, the exploitation of RNAs for synthetic applications is highly dependent on the ability to characterize the many new molecules that continue to be discovered by large-scale sequencing and high-throughput screening techniques. In this review, we present an exhaustive survey of the most recent synthetic bacterial riboswitches and small RNAs while emphasizing their virtues in gene expression management. We also explore the use of these RNA components as building blocks in the RNA synthetic biology toolbox and discuss examples of synthetic RNA components used to rewire bacterial regulatory circuitry. We anticipate that this field will expand its catalog of smart devices by mimicking and manipulating natural RNA mechanisms and functions.
Long non-coding RNAs (lncRNAs) are transcripts longer than ~200 nucleotides with little or no protein-coding capacity. Growing evidence shows that lncRNAs present important function in development and are associated with many human diseases such as cancers, Alzheimer disease, and heart diseases. Transcribed ultraconserved region (T-UCR) transcripts are a novel class of lncRNAs transcribed from ultraconserved regions (UCRs). UCRs are absolutely conserved (100%) between the orthologous regions of the human, rat, and mouse genomes. The UCRs are frequently located at fragile sites and at genomic regions involved in cancers. Recent data suggest that T-UCRs are altered at the transcriptional level in human tumorigenesis and the aberrant T-UCRs expression profiles can be used to differentiate human cancer types. The profound understanding of T-UCRs can throw new light on the pathogenesis of human cancers.
Several studies have shown that synthesis of new proteins at the synapse is a prerequisite for the storage of long-term memories. Relatively little is known about the availability of distinct mRNA populations for translation at specific synapses, the process that determines mRNA localization, and the temporal designations of localized mRNA translation during memory storage. Techniques such as synaptosome preparation and microdissection of distal neuronal processes of cultured neurons and dendritic layers in brain slices are general approaches used to identify localized RNAs. Exploration of the association of RNA-binding proteins to the axonal transport machinery has led to the development of a strategy to identify RNAs that are transported from the cell body to synapses by molecular motor kinesin. In this article, RNA localization at the synapse, as well as its mechanisms and significance in understanding long-term memory storage, are discussed.
The term riboswitch usually refers to small molecule sensing regulatory modules in the 5′ untranslated regions of a mRNA. They are typically comprised of separate ligand binding and regulatory domains. The T box riboswitch is unique from other identified riboswitches because its effector is an essential macromolecule, tRNA. It senses the aminoacylation state of tRNA to regulate genes involved in a variety of functions relating to amino acid metabolism and tRNA aminoacylation. T box riboswitches performs an intuitively simple process using a complex structured RNA element and, until recently, the underlying mechanisms were poorly understood. Only two sequence-specific contacts had been previously identified: (1) between the specifier sequence (codon) and the tRNA anticodon and (2) between an anti-terminator stem loop and the tRNA acceptor arm CCA tail. tRNA aminoacylation blocks the latter interaction and therefore serves as the switch between termination and anti-termination. Outside of these two contacts, the structure and functions of T box riboswitches have come to light in some recent studies. We recently described the X-ray crystal structure of the highly conserved T box riboswitch distal Stem I region and demonstrated that this region interacts with the tRNA elbow to anchor it to the riboswitch. Independently, Lehmann et al. used sequence homology search to arrive at a similar model for Stem I-tRNA interactions. The model was further supported by two recent structures of the Stem I-tRNA complex, determined independently by our group and by Zhang and Ferré-D’Amaré. This article highlights some of these contributions to synthesize an updated model for tRNA recognition by the T box riboswitch.
Three-dimensional architectural motifs are increasingly recognized as determinants of RNA functionality. We submit that such motifs can encode spatial information. RNAs are targeted to subcellular localities in many eukaryotic cell types, and especially in neuronal and glial cells, RNAs can be transported over long distances to their final destination sites. Such RNAs contain cis-acting long-range targeting elements, and recent evidence suggests that kink-turn motifs within such elements may act as spatial codes to direct transport. Kink-turns are complex RNA motifs that feature double- and single-stranded components and introduce a signature three-dimensional structure into helical stems. We propose that the overall architectural design as well as the individual character—as specified by nucleotide identity and arrangement—of kink-turn motifs can serve as RNA targeting determinants.
We report here an in-depth characterization of the aptamer domain of the transcriptional adenine-sensing riboswitch (pbuE) by NMR and fluorescence spectroscopy. By NMR studies, the structure of two aptamer sequences with different lengths of the helix P1, the central element involved in riboswitch conformational switching, was characterized. Hydrogen-bond interactions could be mapped at nucleotide resolution providing information about secondary and tertiary structure, structure homogeneity and dynamics. Our study reveals that the elongation of helix P1 has pronounced effects not only on the local but on the global structure of the apo aptamer domain. The structural differences induced by stabilizing helix P1 were found to be linked to changes of the ligand binding affinity as revealed from analysis of kinetic and thermodynamic data obtained from stopped-flow fluorescence studies. The results provide new insight into the sequence-dependent fine tuning of the structure and function of purine-sensing riboswitches.
Hfq is a global regulator of gene expression in bacteria undergoing adaptation to changing environmental conditions. Its major function is to promote RNA-RNA interactions between regulatory small RNAs (sRNAs) and their target mRNAs. Previously, we demonstrated that Hfq binds many antisense RNAs (asRNAs) in vitro and hypothesized that Hfq may play a role in regulating gene expression via asRNAs. To investigate theE. coliHfq-binding transcriptome in more detail, we co-immunoprecipitated and deep-sequenced RNAs bound to Hfq in vivo. We detected many new Hfq-binding sRNAs and observed that almost 300 mRNAs bind to Hfq. Among these, several are known to be sRNA targets. We identified 25 novel RNAs, which are transcribed from within protein coding regions and named them intragenic RNAs (intraRNAs). Furthermore, 67 asRNAs were co-immunoprecipitated with Hfq, demonstrating that Hfq binds antisense transcripts in vivo. Northern blot analyses confirmed the deep sequencing results and demonstrated that many of the novel Hfq-binding RNAs identified are regulated by Hfq.
HOTAIRM1 is a long intergenic non-coding RNA encoded in the human HOXA gene cluster, with gene expression highly specific for maturing myeloid cells. Knockdown of HOTAIRM1 in the NB4 acute promyelocytic leukemia cell line retarded all-trans retinoid acid (ATRA)-induced granulocytic differentiation, resulting in a significantly larger population of immature and proliferating cells that maintained cell cycle progression from G1 to S phases. Correspondingly, HOTAIRM1 knockdown resulted in retained expression of many otherwise ATRA-suppressed cell cycle and DNA replication genes, and abated ATRA induction of cell surface leukocyte activation, defense response, and other maturation-related genes. Resistance to ATRA-induced cell cycle arrest at the G1/S phase transition in knockdown cells was accompanied by retained expression ofITGA4(CD49d) and decreased induction ofITGAX(CD11c). The coupling of cell cycle progression with temporal dynamics in the expression patterns of these integrin genes suggests a regulated switch to control the transit from the proliferative phase to granulocytic maturation. Furthermore,ITGAXwas among a small number of genes showing perturbation in transcript levels upon HOTAIRM1 knockdown even without ATRA treatment, suggesting a direct pathway of regulation. These results indicate that HOTAIRM1 provides a regulatory link in myeloid maturation by modulating integrin-controlled cell cycle progression at the gene expression level.
T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein–nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5′ TOPs (5′ terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.
Expandable (CTG)n repeats in the 3′ UTR of theDMPKgene are a cause of myotonic dystrophy type 1 (DM1), which leads to a toxic RNA gain-of-function disease. Mutant RNAs with expanded CUG repeats are retained in the nucleus and aggregate in discrete inclusions. These foci sequester splicing factors of the MBNL family and trigger upregulation of the CUGBP family of proteins resulting in the mis-splicing of their target transcripts. To date, many efforts to develop novel therapeutic strategies have been focused on disrupting the toxic nuclear foci and correcting aberrant alternative splicing via targeting mutant CUG repeats RNA; however, no effective treatment for DM1 is currently available. Herein, we present results of culturing of human DM1 myoblasts and fibroblasts with two small-molecule ATP-binding site-specific kinase inhibitors, C16 and C51, which resulted in the alleviation of the dominant-negative effects of CUG repeat expansion. Reversal of the DM1 molecular phenotype includes a reduction of the size and number of foci containing expanded CUG repeat transcripts, decreased steady-state levels of CUGBP1 protein, and consequent improvement of the aberrant alternative splicing of several pre-mRNAs misregulated in DM1.
The microphthalmia-associated transcription factor (MITF) is a pivotal regulator of melanogenic enzymes for melanogenesis, and its expression is modulated by many transcriptional factors at the transcriptional level or post-transcriptional level through microRNAs (miRNAs). Although several miRNAs modulate melanogenic activities, there is no evidence of their direct action on MITF expression. Out of eight miRNAs targeting the 3′-UTR ofMitfpredicted by bioinformatic programs, our results show miR-218 to be a novel candidate for direct action on MITF expression. Ectopic miR-218 dramatically reduced MITF expression, suppressed tyrosinase activity, and induced depigmentation in murine immortalized melan-a melanocytes. MiR-218 also suppressed melanogenesis in human pigmented skin organotypic culture (OTC) through the repression of MITF. An inverse correlation betweenMITFand miR-218 expression was found in human primary skin melanocytes and melanoma cell lines. Taken together, our findings demonstrate a novel mechanism involving miR-218 in the regulation of the MITF pigmentary process and its potential application for skin whitening therapy.
In trypanosomes, mRNAs are processed bytrans-splicing; in this process, a common exon, the spliced leader, is added to all mRNAs from a small RNA donor, the spliced leader RNA (SL RNA). However, little is known regarding how this process is regulated. In this study, we investigated the function of two serine-arginine-rich proteins, TSR1 and TSR1IP, implicated intrans-splicing inTrypanosoma brucei. Depletion of these factors by RNAi suggested their role in bothcis- andtrans-splicing. Microarray was used to examine the transcriptome of the silenced cells. The level of hundreds of mRNAs was changed, suggesting that these proteins have a role in regulating only a subset ofT. bruceimRNAs. Mass-spectrometry analyses of complexes associated with these proteins suggest that these factors function in mRNA stability, translation, and rRNA processing. We further demonstrate changes in the stability of mRNA as a result of depletion of the two TSR proteins. In addition, rRNA defects were observed under the depletion of U2AF35, TSR1, and TSR1IP, but not SF1, suggesting involvement of SR proteins in rRNA processing.
The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeastSchizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putativecis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress.
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Since the discovery oflin-4, the founding member of the miRNA family, over 360 miRNAs have been identified forCaenorhabditis elegans(C. elegans). Prediction and validation of targets are essential for elucidation of regulatory functions of these miRNAs. ForC. elegans, crosslinking immunoprecipitation (CLIP) has been successfully performed for the identification of target mRNA sequences bound by Argonaute protein ALG-1. In addition, reliable annotation of the 3′ untranslated regions (3′ UTRs) as well as developmental stage-specific expression profiles for both miRNAs and 3′ UTR isoforms are available. By utilizing these data, we developed statistical models and bioinformatics tools for both transcriptome-scale and developmental stage-specific predictions of miRNA binding sites inC. elegans 3′ UTRs. In performance evaluation via cross validation on the ALG-1 CLIP data, the models were found to offer major improvements over established algorithms for predicting both seed sites and seedless sites. In particular, our top-ranked predictions have a substantially higher true positive rate, suggesting a much higher likelihood of positive experimental validation. A gene ontology analysis of stage-specific predictions suggests that miRNAs are involved in dynamic regulation of biological functions duringC. elegansdevelopment. In particular, miRNAs preferentially target genes related to development, cell cycle, trafficking, and cell signaling processes. A database for both transcriptome-scale and stage-specific predictions and software for implementing the prediction models are available through the Sfold web server athttp://sfold.wadsworth.org.
Recently, we used in vitro selection to identify a new class of naturally occurring GTP aptamer called the G motif. Here we report the discovery and characterization of a second class of naturally occurring GTP aptamer, the “CA motif.” The primary sequence of this aptamer is unusual in that it consists entirely of tandem repeats of CA-rich motifs as short as three nucleotides. Several active variants of the CA motif aptamer lack the ability to form consecutive Watson-Crick base pairs in any register, while others consist of repeats containing only cytidine and adenosine residues, indicating that noncanonical interactions play important roles in its structure. The circular dichroism spectrum of the CA motif aptamer is distinct from that of A-form RNA and other major classes of nucleic acid structures. Bioinformatic searches indicate that the CA motif is absent from most archaeal and bacterial genomes, but occurs in at least 70 percent of approximately 400 eukaryotic genomes examined. These searches also uncovered several phylogenetically conserved examples of the CA motif in rodent (mouse and rat) genomes. Together, these results reveal the existence of a second class of naturally occurring GTP aptamer whose sequence requirements, like that of the G motif, are not consistent with those of a canonical secondary structure. They also indicate a new and unexpected potential biochemical activity of certain naturally occurring tandem repeats.
MicroRNAs (miRNAs) are ubiquitous gene regulators that modulate essential cellular processes at the post-transcriptional level. In metazoans and their viruses, most miRNAs are produced from hairpin-containing primary transcripts that are sequentially cleaved by nuclear Drosha and cytoplasmic Dicer. In the last decade, alternative mechanisms that bypass either the Drosha or Dicer cleavage step have emerged, increasing the complexity of the miRNA regulatory network. Here, we highlight non-canonical pathways that generate miRNAs using a variety of molecular machineries that play fundamental roles in the biogenesis and processing of other classes of cellular RNAs.
N6-methyladenosine (m6A) is a prevalent modification of eukaryotic mRNAs. It regulates yeast cell fate and is essential to the development and fertility of metazoans. Although its presence in mRNA has been known since the early 1970s, the function of m6A remained a mystery until the spate of discoveries in the past three years. Here, we focus on the discovery of m6A “readers” (proteins that specifically recognize m6A), and their functions in tuning mRNA stability, as well as the broader significance of such m6A-dependent regulation of gene expression.