Folding of the SAM-I riboswitch: A tale with a twist
Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be “captured” upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.
Poly(ADP-ribose) regulates post-transcriptional gene regulation in the cytoplasm
Since its discovery in 1963, poly(ADP-ribose) (pADPr) has been shown to play important functions in the nucleus of multicellular eukaryotes. Each of these functions centers upon DNA metabolism, including DNA-damage repair, chromatin remodeling, transcription and telomere functions. We recently described two novel functions for pADPr in the cytoplasm, both of which involve RNA metabolism – 1) the assembly of cytoplasmic stress granules, cellular macrostructures that aggregate translationally stalled mRNA/protein complexes, and 2) modulation of microRNA activities. Multiple stress granule-localized, post-transcriptional gene regulators, including microRNA-binding argonaute family members, are substrates for pADPr modification and are increasingly modified by pADPr upon stress. Interestingly, the cytoplasmic RNA regulatory functions for PARPs are likely mediated through activities of catalytically inactive PARP-13/ARTD13/ZC3HAV1/ZAP and mono/poly(ADP-ribose)-synthesizing enzymes, including PARP-5a/ARTD5/TNKS1, PARP-12/ARTD12/ZC3HDC1 and PARP-15/ARTD7/BAL3. These data are consistent with other recent work, which suggests that mono(ADP-ribosyl)ated residues can be poly(ADP-ribosyl)ated by different enzymes.
The Bacterial CRISPR/Cas System as Analog of the Mammalian Adaptive Immune System
Bacteria, like mammals, have to constantly defend themselves from viral attack. Like mammals, they use both innate and adaptive defense mechanisms. In this point of view we highlight the commonalities between defense systems of bacteria and mammals. Our focus is on the recently discovered bacterial adaptive immune system, the clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas). We suggest that fundamental aspects of CRISPR/Cas immunity may be viewed in light of the vast accumulated knowledge on the mammalian immune system, and propose that further insights will be revealed by thorough comparison between the systems.
New kid on the ID block: Neural functions of the Nab2/ZC3H14 class of Cys3His tandem zinc-finger polyadenosine RNA binding proteins
Polyadenosine RNA binding proteins (Pabs) play critical roles in regulating the polyadenylation, nuclear export, stability, and translation of cellular RNAs. Although most Pabs are ubiquitously expressed and are thought to play general roles in post-transcriptional regulation, mutations in genes encoding these factors have been linked to tissue-specific diseases including muscular dystrophy and now intellectual disability (ID). Our recent work defined this connection to ID, as we showed that mutations in the gene encoding the ubiquitously expressed Cys3His tandem zinc-finger (ZnF) Pab, ZC3H14 (Zinc finger protein, CCCH-type, number 14) are associated with non-syndromic autosomal recessive intellectual disability (NS-ARID). This study provided a first link between defects in Pab function and a brain disorder, suggesting that ZC3H14 plays a required role in regulating RNAs in nervous system cells. Here we highlight key questions raised by our study of ZC3H14 and its ortholog in the fruit fly Drosophila melanogaster, dNab2, and comment on future approaches that could provide insights into the cellular and molecular roles of this class of zinc finger-containing Pabs. We propose a summary model depicting how ZC3H14-type Pabs might play particularly important roles in neuronal RNA metabolism.
The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells.
The untranslated regions (UTRs) at the 3'end of mRNA transcripts contain important sequences that influence the fate of mRNA and thus proteosynthesis. In this review, we summarize the information known to date about 3'end processing, sequence characteristics including related binding proteins and the role of 3'UTRs in several selected signaling pathways to delineate their importance in the regulatory processes in mammalian cells. In addition to reviewing recent advances in the more well known aspects, such as cleavage and polyadenylation processes that influence mRNA stability and location, we concentrate on some newly emerging concepts of the role of the 3'UTR, including alternative polyadenylation sites in relation to proliferation and differentiation and the recognition of the multi-functional properties of non-coding RNAs, including miRNAs that commonly target the 3'UTR. The emerging picture is of a highly complex set of regulatory systems that include autoregulation, cooperativity and competition to fine tune proteosynthesis in context-dependent manners.
An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity in E. Coli cell lysate
Post-transcriptional modifications of RNA diversify the genetically encoded sequences and play important roles in fundamental cellular processes like mRNA and rRNA maturation. Most RNA methylations are catalyzed by S-adenosylmethionine-dependent RNA methyltransferases. An assay for rapid and easy detection of this enzymatic activity would be highly desirable to identify novel RNA methyltransferases, test S-adenosylmethionine analogs or screen RNA methyltransferase inhibitors. We have developed an enzyme-coupled assay to determine the activity of trimethylguanosine synthases, a class of RNA methyltransferases responsible for RNA cap hypermethylation in eukaryotes. We show that this assay can be used in E. coli cell lysate to measure the activity of recombinantly produced trimethylguanosine synthase. Potential hits are validated for formation of hypermethylated product using HPLC and MALDI-TOF-MS. Furthermore, we demonstrate how this assay can be implemented to screen methyltransferase inhibitors.
Satellite DNA-associated siRNAs as mediators of heat shock response in insects
Conversion of environmental signals into epigenetic information is thought to occur widely but has been poorly studied as yet. It is proposed that changes in the expression of molecules involved in chromatin modifications might play a role in this process. Here we study the expression of abundant satellite DNA TCAST that makes up 35% of genome of the red flour beetle Tribolium castaneum and is located within the constitutive pericentromeric heterochromatin. RNA polymerase II promotes the transcription of TCAST satellite DNA from both strands, and long primary transcripts are rapidly processed into 21–30 nt siRNAs. Expression of TCAST satellite DNA-associated siRNAs is developmentally regulated, the most intense being at specific stages of embryogenesis. Moreover, the expression is strongly induced following heat shock and is accompanied by increase in repressive epigenetic modifications of histones at TCAST regions. Upon recovery from heat stress, the expression of satellite DNA-associated siRNAs as well as histone modifications is quickly restored. Our results indicate that satellite DNA-associated siRNAs, transiently activated after heat shock, affect epigenetic state of constitutive heterochromatin in Tribolium. It can be hypothesized that transient remodeling of heterochromatin is part of a physiological gene expression program activated under stress conditions in insects.
Multiple insert size paired-end sequencing for deconvolution of complex transcriptomes
Deep sequencing of transcriptomes allows quantitative and qualitative analysis of many RNA species in a sample, with parallel comparison of expression levels, splicing variants, natural antisense transcripts, RNA editing and transcriptional start and stop sites the ideal goal. By computational modeling, we show how libraries of multiple insert sizes combined with strand-specific, paired-end (SS-PE) sequencing can increase the information gained on alternative splicing, especially in higher eukaryotes. Despite the benefits of gaining SS-PE data with paired ends of varying distance, the standard Illumina protocol allows only non-strand-specific, paired-end sequencing with a single insert size. Here, we modify the Illumina RNA ligation protocol to allow SS-PE sequencing by using a custom pre-adenylated 3′ adaptor. We generate parallel libraries with differing insert sizes to aid deconvolution of alternative splicing events and to characterize the extent and distribution of natural antisense transcription in C. elegans. Despite stringent requirements for detection of alternative splicing, our data increases the number of intron retention and exon skipping events annotated in the Wormbase genome annotations by 127% and 121%, respectively. We show that parallel libraries with a range of insert sizes increase transcriptomic information gained by sequencing and that by current established benchmarks our protocol gives competitive results with respect to library quality.
PRMT1 is required for RAP55 to localize to processing bodies
In eukaryotic cells, components of messenger ribonucleoproteins (mRNPs) are often detected in cytoplasmic granules, such as processing bodies (P-bodies) and stress granules (SGs) where translationally repressed mRNAs accumulate. RAP55A, which is an RNA binding component of mRNPs, acts as a translational repressor and localizes to P-bodies and SGs. We found here that a homologous protein RAP55B also localized to P-bodies when expressed in human cultured cells. When RAP55A or RAP55B was highly expressed in the cells, they induced the formation of SG-like large cytoplasmic mRNP granules that contained both P-body and SG components, indicating that RAP55 is important for the assembly of cytoplasmic mRNP granules. In addition, we found that RAP55A associated with protein arginine methyltransferases PRMT1 and PRMT5. Multiple arginine residues of RAP55A were indeed asymmetrically dimethylated in the cell and PRMT1 was shown to be a component of large mRNP granules induced by RAP55A overexpression. Although PRMT1 did not accumulate in P-bodies, siRNA-mediated knockdown of PRMT1 impaired the localization of RAP55A to P-bodies, while other components were still retained in these structures. Thus, our data indicate that RAP55 is important for the assembly of cytoplasmic mRNP granules and that PRMT1 is required for RAP55A to localize to P-bodies.
Z’ Factor including siRNA design quality parameter in RNAi screening experiments
RNA interference (RNAi) high-content screening (HCS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high quality HCS assays. The Z’ factor statistic provides a way to evaluate whether or not screening run conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z’ factor, introduced by Zhang et al.,1 is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data. This paper describe a new extension of the Z' factor, which integrates bioinformatics RNAi non-target compounds for screening quality assessment. Currently presented Z’ factor is based on positive and negative control, which may not be sufficient for RNAi experiments including oligonucleotides (oligo) with lack of knock-down. This paper proposes an algorithm which extends existing algorithm by using additional controls generetaed from on-target analysis.
Drosophila Pur-α binds to trinucleotide-repeat containing cellular RNAs and translocates to the early oocyte
Pur-α was identified as a DNA-binding protein with high affinity for the single-stranded PUR-motif (GGN)n. Bound to DNA, Pur-α can both activate and repress transcription. In addition, Pur-α binds to RNA and may participate in nuclear RNA export as well as transport of cytoplasmic neuronal mRNP granules. The heritable trinucleotide-repeat expansion disease Fragile X associated Tremor and Ataxia Syndrome (FXTAS) leads to interaction of Pur-α with mutant, abnormally long r(CGG)n stretches, which appears to titrate the protein away from its physiologic mRNA targets into nuclear RNA-protein aggregates. We examined the function of Drosophila Pur-α and demonstrate that the protein accumulates in the growing oocyte early in oogenesis. Co-purifying proteins reveal that Pur-α is part of transported mRNP complexes, analogous to its reported role in nerve cells. We analyzed the subcellular localization of mutant GFP-Pur-α fusion proteins where either nucleic acid binding or dimerization, or both, were prevented. We propose that association with mRNAs occurs in the nucleus and is required for nuclear export of the complex. Furthermore, efficient translocation into the oocyte also requires RNA binding as well as dimerization. RNA binding assays demonstrate that recombinant Drosophila Pur-α can bind r(CGG)4 with higher affinity than previously thought. Related sequences, such as r(CAG)4 and the consensus sequence of the opa-repeat r(CAG)3CAA, can also associate with Pur-α in vitro and in vivo. The mRNA target spectrum of Pur-α may therefore be larger than previously anticipated.
The role of decapping proteins in the miRNA accumulation in Arabidopsis thaliana
Decapping 1 (DCP1), Decapping 2 (DCP2) and VARICOSE (VCS) are components of the decapping complex that removes the 7-methyl-guanosine 5′-diphosphate from the 5′ end of mRNAs. In animals, the decapping proteins are involved in miRNA-mediated gene silencing, whereas in plants the roles of the decapping proteins in the miRNA pathway are not well understood. Here we demonstrated that the accumulation of miRNAs decreased in dcp1, dcp2 and vcs mutants, indicating that DCP1, DCP2 and VCS are important for the miRNA pathway in Arabidopsis thaliana. The primary miRNAs (pri-miRNAs) did not increase and miRNA biogenesis components did not decrease in these mutants, suggesting that the miRNA decrease in decapping mutants is not due to the defect of pri-miRNA processing. We showed that the accumulation of miRNA targets increased concomitantly with the decrease of miRNA in the decapping mutants. Our results suggested that the seedling lethal phenotypes in the dcp1, dcp2 and vcs mutants are caused not only by the defect in decapping, but also by the disruption of miRNA-mediated gene regulation.
Long-distance movement of Arabidopsis FLOWERING LOCUS T RNA participates in systemic floral regulation
The finding of mRNA acting as a systemic information molecule is one of the most exciting discoveries in recent plant biology. However, evidence demonstrating the functional significance of non-cell autonomous RNA remains limited. Recent analyses of Arabidopsis and rice revealed FLOWERING LOCUS T (FT) protein as a systemic florigenic signal. However, whether the FT RNA also participates in systemic floral regulation remains controversial. By using Arabidopsis cleft-grafting experiments, we showed that the RNA of Arabidopsis FT undergoes long-distance movement from the stock to the scion apex in both FT transformants and non-transformants. In addition, the sequences of FT RNA are sufficient to target a cell-autonomous RNA for long-distance movement. Therefore, FT RNA is a bona fide non-cell autonomous RNA. To examine the systemic action of FT RNA, we uncoupled the movement of FT RNA from protein by fusing FT with RED FLUORESCENT PROTEIN (RFP). When RFP-FT protein was retained in companion cells, the detection of RFP-FT RNA correlates with floral promotion in the scion. Further depletion of the translocated RFP-FT RNA by RNAi or artificial miRNA against FT delayed the floral promotion, indicating that the translocated FT RNA acts as a part of the systemic floral signaling. Our results indicate that both FT RNA and protein move long distance and act redundantly to integrate the photoperiodic signals.
The SID-1 double-stranded RNA transporter is not required for systemic RNAi in the migratory locust
Systemic RNAi, the spreading of RNAi effects to cells and tissues that have not initially encountered a dsRNA trigger, is a common phenomenon in many organisms. However, the underlying mechanisms of systemic RNAi remain largely unknown. Here, we studied the characteristics and possible mechanisms of systemic RNAi in Locusta migratoria. We observed that the locust has pronounced sensitive systemic RNAi in response to dsRNA injection for both broadly-expressed as well as tissue-specific genes. Only a 30 pg (dsRNA / mg tissues) dose could induce significant systemic RNAi effects. Only one SID-1 ortholog (LmSID-1) was identified in the locust genome, which exhibited a progressively increasing expression pattern with development and its expression was enriched in the gonad. To test the role of LmSID-1 in systemic RNAi, we performed in vitro and in vivo experiments. The results from in vivo experiments showed that silencing of LmSID-1 gene dose not influence RNAi effects of other genes. The results from in vitro experiments confirmed that the expression of the LmSID-1 protein in Drosophila S2 cells could not enhance dsRNA uptake. Thus, these findings imply the existence of alternative mechanisms underlying insect systemic RNAi, which may be different from Caenorhabditis elegans or mammals.
Mechanisms for differentiation between cognate and near-cognate ligands by purine riboswitches
Riboswitches are elements in the 5′-untranslated region of mRNAs that regulate gene expression by directly interacting with metabolites related to their own gene products. A remarkable feature of this gene regulation mechanism is the high specificity of riboswitches for their cognate ligands. In this study, we used a combination of static and time-resolved NMR-spectroscopic methods to investigate the mechanisms for ligand specificity in purine riboswitches. We investigate the xpt-aptamer domain from a guanine-responsive riboswitch and the mfl-aptamer domain from a 2’-deoxyguanosine-responsive riboswitch. The xpt-aptamer binds the purine nucleobases guanine/hypoxanthine with high affinity, but, unexpectedly, also the nucleoside 2’-deoxyguanosine. On the other hand, the mfl-aptamer is highly specific for its cognate ligand 2’-deoxyguanosine, and does not bind purine ligands. We addressed the question of aptamer`s ligand specificity by real-time NMR spectroscopy. Our studies of ligand binding and subsequently induced aptamer folding revealed that the xpt-aptamer discriminates against non-cognate ligands by enhanced life-times of the cognate complex compared with non-cognate complexes, whereas the mfl-aptamer rejects non-cognate ligands at the level of ligand association, employing a kinetic proofreading mechanism.
The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element
Selenocysteine insertion into selenoproteins involves the translational recoding of UGA stop codons. In mammals, selenoprotein expression further depends on selenium availability, which has been particularly described for glutathione peroxidase 1 and 4 (Gpx1 and Gpx4). The SECIS element located in the 3′UTR of the selenoprotein mRNAs is a modulator of UGA recoding efficiency in adequate selenium conditions. One of the current models for the UGA recoding mechanism proposes that the SECIS binds SECIS-binding protein 2 (SBP2), which then recruits a selenocysteine-specific elongation factor (EFsec) and tRNASec to the ribosome, where L30 acts as an anchor. The involvement of the SECIS in modulation of UGA recoding activity was investigated, together with SBP2 and EFsec, in Hek293 cells cultured with various selenium levels. Luciferase reporter constructs, in transiently or stably expressing cell lines, were used to analyze the differential expression of Gpx1 and Gpx4. We showed that, upon selenium fluctuation, the modulation of UGA recoding efficiency depends on the nature of the SECIS, with Gpx1 being more sensitive than Gpx4. Attenuation of SBP2 and EFsec levels by shRNAs confirmed that both factors are essential for efficient selenocysteine insertion. Strikingly, in a context of either EFsec or SBP2 attenuation, the decrease in UGA recoding efficiency is dependent on the nature of the SECIS, GPx1 being more sensitive. Finally, the profusion of selenium of the culture medium exacerbates the lack of factors involved in selenocysteine insertion.
Conservation and regulation of alternative splicing by dynamic inter- and intra-intron base pairings in Lepidoptera 14-3-3z pre-mRNAs
Alternative splicing of pre-mRNAs greatly contributes to diversity in products generated from a single gene. However, the underlying regulatory mechanisms are poorly understood. In the present study, we describe evolutionarily conserved intra-intronic and inter-intronic RNA secondary structures. Mutation experiments revealed that intra-intronic RNA secondary structure causes steric hindrance to enforce mutual splicing exclusivity, while inter-intronic RNA pairing largely functions through a looped-out mechanism. Moreover, mutually exclusive splicing may be regulated by RNA pairing competition between intra- and inter-introns. Importantly, the resulting dynamic RNA architecture largely controls mutually exclusive splicing, although cis-acting regulatory sequences may fine-tune this process. Our results emphasize the importance of dynamic RNA architecture in alternative splicing.