RRM
2013 Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
With the exception of histone mRNAs, 3’end formation of all mRNAs in higher eukaryotes is thought to occur via cleavage and polyadenylation. In studies of miRNA action on target mRNAs in S2 cells, a number of mRNAs were analyzed by using a polyA tail length measurement method. While several mRNAs displayed the expected heterogeneous tail, two mRNAs, those encoding Hid and Reaper appeared to have short (less than 10 nucleotides) polyA tails. This phenomenon was observed when the 3’UTR of Hid and Reaper were appended to luciferase reporter constructs. To distinguish between lack of polyadenylation and deadenylation, we knocked down the deadenylase components CCR4 and POP2. Remarkably, in cells depleted of CCR4 and POP2, both Hid and Reaper mRNAs were found to have normal polyA tails. This result clearly indicated that the major form of Hid and Reaper mRNAs at steady state in S2 cells is deadenylated. Further analysis was done using the Reaper 3’UTR. Systematic deletion and substitution analyses demonstrated that several elements, including two cis-acting sequences (20-24 nucleotides) located 178 bases and 118 bases upstream of an AAUAAA hexanucleotide, the hexanucleotide itself, and bases 73-115 downstream of the hexanucleotide, were necessary and sufficient to cause the deadenylated phenotype. The position of the upstream elements relative to the hexanucleotide is also important. Interestingly, we have also found that the deadenylated Reaper mRNAs are engaged with actively translating ribosomes. Importantly, the deadenylated Reaper mRNAs can resume active translation after exposure to conditions that inhibit initiation of protein synthesis, such as hypertonic stress. We are currently attempting to identify the trans-acting factors interacting with the elements in the Reaper 3’UTR, which are required for maintenance of translating deadenylated mRNAs.
Keywords: deadenylation, translation
Abstract:
The functional repertoire of RNA helicases is immense, facilitating the RNA biology of eukaryotic cells and the viruses that infect them. Proteomic approaches have identified host RNA helicases that interact with the cis-acting replication sequences of several retroviruses including HIV-1. Functional assays determined DHX9/RNA helicase A (RHA) specifically and selectively recognizes cis-acting replication elements in the 5’ terminus. Downregulation of RHA impairs the efficient translation of viral mRNAs. Domain analysis demonstrated N-terminal residues of RHA are necessary for RHA’s specific recognition of the cognate viral RNA. Polyribosome analysis determined N-term tethers RHA’s ATPase-dependent helicase domain to catalyze efficient translation. Beyond the catalytic activity, RHA also provides chaperone activity that promotes the infectivity of progeny virions. RHA is incorporated into HIV-1 virions and its stoichiometry of ~2 RHA molecules per particle is in proportion to the viral genomic RNA (n=2). RHA-deficient virions are produced by siRNA downregulation or by deletion of the RHA-responsive elements in the 5’ RNA terminus, indicating incorporation of RHA is viral RNA-dependent. Unexpectedly, the ATPase-deficient RHA allele rescues viral infectivity, demonstrating that a nonenzymatic activity of RHA is sufficient to propagate infectious virions. RHA domains necessary for incorporation into HIV-1 particles have been tested for their ability to rescue infectivity. Results suggest the ability of RHA to bind cognate viral RNA and to form a homodimer is necessary to bolster infectivity. Our results demonstrate two non-redundant functions of RHA are directed by viral RNA elements: ATP-dependent helicase and RNA chaperone activity promote the quality of the viral RNA-protein complex to bolster viral replication.
Keywords: RNA helicase A
Abstract:
Despite having high-resolution structures for eukaryotic large ribosomal subunits, it remained unclear how these ribonucleoprotein complexes are constructed in living cells. Nevertheless, knowing where ribosomal proteins interact with rRNA provides a strategic platform to investigate the connection between spatial and temporal aspects of 60S subunit biogenesis. We previously found that the function of individual yeast large subunit ribosomal proteins (RPLs) in pre-rRNA processing correlates with their location in the structure of mature 60S subunits. This observation suggested that there is an order by which 60S subunits are formed. To directly test this model, we used proteomic approaches to characterize RPLs functioning in early, middle, and late steps of pre-60S assembly by proteomic approaches, assaying the levels of assembly factors and other ribosomal proteins in pre-ribosomes after depleting each RPL. Our results demonstrate that structural domains of eukaryotic 60S complexes are formed in a hierarchical fashion. Assembly begins at the convex solvent-side, followed by the polypeptide exit tunnel, the inter-subunit side, and finally the central protuberance. This model provides an initial paradigm for the sequential assembly of eukaryotic large ribosomal subunits. Our results also reveal striking differences and similarities between assembly of bacterial and eukaryotic large ribosomal subunits, providing insights into the evolution of these RNA-protein particles.
Keywords: ribosome assembly, ribosomal proteins, pre-ribosomes
Abstract:
Eukaryotic ribosome biogenesis requires rapid hybridization between the U3 snoRNA and the pre-rRNA to direct cleavages at the A0, A1, and A2 sites in pre-rRNA that liberate the small subunit precursor. The bases involved in hybridization of one of the three duplexes that U3 makes with pre-rRNA, designated the U3-18S duplex, are buried in conserved structures: box A/A′ stem–loop in U3 snoRNA and helix 1 (H1) in the 18S region of the pre-rRNA. These conserved structures must be unfolded to permit the necessary hybridization. Previously, we reported that Imp3 and Imp4 promote U3-18S hybridization in vitro, but the mechanism by which these proteins facilitate U3-18S duplex formation remained unclear. Here, we directly addressed this question by probing base accessibility with chemical modification and backbone accessibility with ribonuclease activity of U3 and pre-rRNA fragments that mimic the secondary structure observed in vivo. Our results demonstrate that U3-18S hybridization requires only Imp3. Binding to each RNA by Imp3 provides sufficient energy to unfold both the 18S H1 and the U3 box A/A′ stem structures. The Imp3 unfolding activity also increases accessibility at the U3-dependent A0 and A1 sites, perhaps signaling cleavage at these sites to generate the 5′ mature end of 18S. Imp4 destabilizes the U3-18S duplex to aid U3 release, thus differentiating the roles of these proteins. Protein-dependent unfolding of these structures may serve as a switch to block U3-pre-rRNA interactions until recruitment of Imp3, thereby preventing premature and inaccurate U3-dependent pre-rRNA cleavage and folding events in eukaryotic ribosome biogenesis.
Keywords: ribosome synthesis , RNA conformational switch, ribonucleoprotein
Abstract not available online - please check the printed booklet.
Abstract:
The MDM2 oncogene is a regulator of the tumor suppressor protein p53. Under stress and in cancer, MDM2 is alternatively spliced into numerous isoforms. MDM2-ALT1 is comprised of exons 3 and 12, and we have shown that MDM2-ALT1 is expressed in 85% of rhabdomyosarcomas (RMS) and is also predictive of high-grade metastatic disease. There is therefore a critical need to understand MDM2 alternative splicing and to modulate its splicing in cancer.
In order to study the alternative splicing of MDM2 we have developed a damage-inducible minigene system. The MDM2 3-11-12 minigene recapitulates the splicing of the endogenous gene by excluding exon 11 under genotoxic stress. In order to identify cis-elements present on the pre-mRNA we performed chimeric swaps with elements from a non-damage-responsive minigene and showed that exon 11 of MDM2 is sufficient to confer damage-inducible alternative splicing in a heterologous context. Using ESEfinder 3.0 we identified conserved consensus sequences for splicing regulator SF2/ASF in exon 11 of MDM2. Therefore, we hypothesize that SF2/ASF is responsible for damage-induced alternative splicing of MDM2 under genotoxic stress.
We examined the effects of SF2/ASF on MDM2 alternative splicing in vitro through binding and splicing assays using the wild-type and mutant minigenes. Additionally we performed SF2/ASF knockdown and overexpression studies to elucidate its role in stress-induced splicing changes. We report that SF2/ASF promotes the exclusion of exon 11 under damage. Furthermore, antisense oligonucleotides (ASOs) targeting SF2/ASF binding sites push endogenous MDM2 splicing toward the full-length isoform. Importantly, we also observe elevated SF2/ASF expression in RMS samples concomitant with MDM2-ALT1.
Our results provide valuable insight into the regulation of damage-induced MDM2 alternative splicing by SF2/ASF; modulation of its alternative splicing through ASOs therefore presents a potential target for anticancer therapy.
Keywords: MDM2, Alternative Splicing, SF2ASF
Abstract:
We recently identified the molecular basis of a devastating human developmental disorder called microcephalic osteodysplastic primordial dwarfism type 1 (MOPD I) which is caused by mutations in the minor spliceosomal snRNA U4atac. The MOPD I mutations disrupt U12-dependent, minor spliceosomal function and cause aberrant pre-mRNA splicing of downstream target genes. MOPD I is a severely debilitating disorder and the affected infants suffer from severe growth retardation, multiple skeletal abnormalities, abnormal brain development and typically early death. We found that splicing of most genes containing minor class introns is significantly disrupted in patient fibroblast cells. MOPD I is very rare and clinical material is difficult to obtain. Therefore, the aberrantly spliced target genes as well as the individual downstream effects that contribute to the various MOPD I phenotypes are unknown. In order to address these issues and to study the phenotypic consequences of MOPD I mutations of U4atac snRNA in vivo, we have generated MOPD I mouse models. Two human mutations, G51A and G55A were knocked in to the mouse U4atac snRNA gene. Homozygous G51A/G51A mice were more severely affected than human patients with most mice showing embryonic lethality. By contrast, MOPD I homozygous G55A/G55A mice were nearly wild type. Importantly, the phenotype of the compound heterozygous G51A/G55A mutant mice was similar to human MOPD I patients. They were small and displayed craniofacial and long bone malformations. An unexpected phenotype of these mutants was that they developed diabetes due to poor pancreatic beta cell development or survival. Further studies are under way to define the developmental defects behind these phenotypes and to link these to the underlying splicing aberrations.
Keywords: Minor spliceosome , MOPD I , RNA disease
Abstract:
Alternative splicing is a robust mechanism for regulating the functional output of many important genes. In contrast to the well-studied regulation of tissue- and developmental stage-specific alternative splicing, the regulated alternative splicing changes in cells responding to environmental cues is poorly understood.
Using cardiomyocytes isolated from neonatal mice, we found that elevated intracellular calcium levels lead to significantly increased skipping of Nf1 exon 23a. Detailed dose-curve and time-course analyses indicated that the alternative splicing change is dynamic, as the splicing pattern is recovered after calcium levels are restored. These data have provided the first evidence for the robust regulation of alternative splicing by calcium signaling in cardiomyocytes.
Prolonged increase of cytoplasmic calcium levels has been shown to lead to histone hyperacetylation in cardiomyocytes. Indeed, western blots analysis indicated 2-2.5 fold higher total acetylation of histones H4 and H3, as well as H3K9 acetylation than that of control cells. Immuno-staining analysis showed that KCl-induced histone hyperacetylation is correlated with the translocation of class II HDACs from the cell nucleus to the cytoplasm. ChIP performed with pan-acetylated histone H3 antibodies indicated that histone hyperacetylation occurs throughout the Nf1 gene body. We hypothesize that the increase in histone acetylation results in increased rate of transcription leading to increased skipping of Nf1 exon 23a. Consistent with this hypothesis, the KCl-induced skipping of Nf1 exon 23a can be rescued by DRB treatment at low doses.
Importantly, the calcium mediated dynamic splicing pattern change is not limited to Nf1 exon 23a. To date, we have tested 8 alternative exons known to undergo fetal-to-adult splicing pattern switch and found increased skipping for 4 exons in the KCl-treated cardiomyocytes. Interestingly, the splicing patterns of these exons revert to the fetal patterns when the calcium level is abnormally high. Our studies have revealed a hidden network of alternative splicing that is regulated by calcium signaling in cardiomyocytes. We are currently studying the molecular basis of this novel mechanism and its biological relevance in normal and pathological processes.
Keywords: Alternative Splicing
Abstract:
mRNAs targeted by endonuclease decay generally disappear without detectable decay intermediates. The exception to this is nonsense-containing human β-globin mRNA, where the destabilization of full-length mRNA is accompanied by the cytoplasmic accumulation of 5’-truncated transcripts in erythroid cells of transgenic mice and in transfected erythroid cell lines. The relationship of the shortened RNAs to the decay process was characterized using an inducible erythroid cell system and an assay for quantifying full-length mRNA and a truncated RNA with known 5’ end sequence. In cells knocked down for Upf1 a reciprocal increase in full-length and decrease in shortened RNA confirmed the role of NMD in this process. Kinetic analysis demonstrated that the 5’-truncated RNAs are metastable intermediates generated during the decay process. SMG6 previously was identified as an endonuclease involved in NMD. Consistent with involvement of SMG6 in the decay process, full-length nonsense-containing β-globin mRNA was increased and the Δ169 decay intermediate was decreased in cells knocked down for SMG6. This effect was reversed by complementation with siRNA-resistant SMG6, but not by SMG6 with inactivating PIN domain mutations. Importantly, none of these conditions altered the phosphorylation state of Upf1. These data provide the first proof for accumulation of stable NMD products by SMG6 endonuclease cleavage.
References:
Mascarenhas, R*, Dougherty, JA*, and Schoenberg, DR. (2013) PLoS ONE. Accepted 7 Aug 2013.
Bremer KA, Stevens A, Schoenberg DR. (2003) RNA. 9(9):1157-1167.
Lim SK, Maquat LE. (1992) EMBO J. 11(9):3271-3278.
Huntzinger E, Kashima I, Fauser M, Saulière J, Izaurralde E. (2008) RNA. 14(12):2609-2617.
Keywords: nonsense-mediated mRNA decay, SMG6, globin
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The poly(A) binding protein C1 (PABPC1) is a highly conserved and ubiquitously expressed regulatory factor that plays a key role in mRNA polyadenylation, translation and stability. Here we report that PABPC1 protein expression in mice is tightly regulated during postnatal heart development. Western blot analyses showed a surprising 800-fold reduction in PABPC1 protein expression in heart within the first four weeks after birth. However, PABPC1 mRNA levels showed no more than five-fold decrease clearly indicating that the developmental decrease is largely post-transcriptional. We found that both during physiological and pathological cardiac hypertrophy conditions; there is a strong increase in expression of PABPC1 protein.
To investigate a direct role for PABPC1 in cardiac hypertrophy, we generated tetracycline-inducible and cardiac-specific PABPC1 transgenic mice. Histological and echocardiography studies from these mice demonstrate that re-expression of PABPC1 in adult heart was sufficient to induce cardiac hypertrophy without producing any functional or pathological defects. Importantly, PABPC1 depleted neonatal cardiomyocytes failed to exhibit isoproterenol induced hypertrophic growth. Taken together, these results strongly support an important role for PABPC1 in controlling gene regulation programs during normal heart development and in cardiac hypertrophy.
Keywords: PABPC1, Cardiac Hypertrophy, Devlopment
Abstract:
In all retroviruses, reverse transcription is initiated from the 3´ end of a cellular tRNA primer. In the case of HIV-1, the 3´ 18 nucleotides of human tRNALys3 are perfectly complementary to the primer binding site (PBS) within the 5´UTR. After annealing, the 3´ end of tRNALys3 is extended by reverse transcriptase (RT). Only a single copy of tRNALys3 is required to initiate reverse transcription, but all three human tRNALys isoacceptors (~20-25 molecules) are selectively packaged into virions, along with stoichiometric amounts of human lysyl-tRNA synthetase (LysRS). LysRS is specifically packaged into virions via its interaction with the viral Gag protein, thus leading to the enrichment of tRNALys in virions. Recently, we have shown that LysRS binds with high affinity to the viral RNA, in part, due to the presence of a tRNA-like element (TLE) in the U-rich stem loop proximal to the PBS, which mimics the anticodon of tRNALys3 (Jones et al, RNA 2013). However, whether the 5´UTR possesses more extensive tRNA structural mimicry is unknown. In addition, it remains unclear precisely how tRNALys3 annealing affects the conformation of the PBS domain of the HIV-1 genome. Using small-angle X-ray scattering and molecular dynamics simulations, we report the solution structure of the first 223 nucleotides of the 5′UTR comprising the transactivation response (TAR) element, polyadenylation (PolyA) stem loop, and PBS region. These data show that the TAR and PolyA domains adopt long co-axially stacked helices. Analysis of the apo and 18-nt DNA (anti-PBS) primer-annealed PBS domain reveal a tRNA-like tertiary fold. The conformations of the individual domains do not change significantly in the context of the full 223-nt RNA, suggesting the absence of long-range tertiary contacts. SAXS studies of the Psi packaging signal and of complexes between the genomic RNA, anti-PBS, and HIV-1 RT will also be presented.
Keywords: SAXS, HIV, 5UTR
Abstract:
The U1A/U2B´´/SNF family of snRNP proteins has been highly conserved, as indicated by the 70% homology between their first (and RNA binding) RRMs. However, despite their high degree of sequence similarity, each exhibits unique RNA binding properties. Previously, in an effort to understand the evolutionary pressures that have lead to this functional divergence, we reconstructed the protein phylogeny and discovered that the last common ancestor between humans (U1A & U2B´´) and Drosophila (SNF) contained a single ancestral protein (URB). We have gone on to further examine the reconstructed phylogenetic tree to investigate the changes that occurred as the proteins evolved toward the modern vertebrate specimens. Specifically, the predecessors of modern U1A and U2B´´resulted from a gene duplication in ancestors of jawed vertebrates. We have constructed proteins along the lineage from URB to U1A/U2B´´ and investigated their RNA binding affinity and specificity. Using modern U1 snRNA Stemloop II and U2 snRNA Stemloop IV, and selected mutations, we are mapping the path of protein changes that lead to specific RRM recognition. Supported by NIH R01 GM096444 to KBH, F31 GM089576 to SGW.
References:
Williams and Hall (2013) JMB E-pub ahead of print.
Keywords: snRNP, RRM, evolution
Abstract:
RNA functions are intimately linked to their ability to fold into complex secondary and tertiary structures. Thus, understanding how these molecules fold is essential to determining how they function. Current methods for investigating RNA structure often use small molecules, enzymes, or ions that cleave or modify the RNA in a solvent accessible manner. While these methods have been invaluable to understanding RNA structure, they can be fairly labor intensive and often focus on short regions of single RNAs. Here we present a new method and data analysis pipeline, called Mod-seq, for assaying the structure of RNAs by high-throughput sequencing. This technique can be utilized both in vivo and in vitro, with any small molecule that modifies RNA and impedes reverse transcriptase. As proof-of-principle, we used dimethyl sulfate (DMS) to probe the in vivo structure of cellular RNAs in Saccharomyces cerevisiae. Mod-seq analysis simultaneously revealed secondary structural information for all four ribosomal RNAs and 32 additional non-coding RNAs. We further show that Mod-seq can be used to detect structural changes in 5.8S and 25S rRNAs in the absence of ribosomal protein L26, correctly identifying its binding site on the ribosome. While this method is applicable to RNAs of any length, its high-throughput nature makes Mod-seq ideal for studying long RNAs and complex RNA mixtures.
Keywords: RNA structure probing, High-throughput sequencing, RNA folding
Abstract:
The hairpin ribozyme is a catalytic RNA that performs self-cleavage. It consists of two loops, denoted A and B. The two loops undergo major structural rearrangements to form an intricate RNA-RNA tertiary interface. In nature the two loops are connected by a four-way junction. As a complement to spectroscopic studies, we are examining the docking properties of a junctionless (trans-docking) system in which the two loops reside on separate molecules. By utilizing temperature-dependent surface plasmon resonance we have been able to characterize the thermodynamics and kinetics of docking for the juctionless hairpin ribozyme. Using mutations at the acid-base catalytic residue A38, we have also investigated the effect of the 2’-O-methyl modification at the active site that is commonly used to prevent cleavage in biophysical and structural studies. In agreement with recent single-molecule kinetic fingerprinting studies, we find a significant increase in binding affinity in the presence of the native 2’-OH rather than the 2’-O-methyl modification at the active site. Interestingly, docking of the 2’-OH species is more enthalpically favorable but less entropically favorable than the 2’-O-methyl species. Activation energies were determined for docking and undocking in the 2’-OH species but were undeterminable in the 2’-O-methyl species due to nonlinear Arrhenius curves, suggesting a significant disruption of the energy landscape among the two species. Comparison with results in other systems emphasizes the diversity of thermodynamic contributions for RNA-RNA tertiary structure formation.
Keywords: hairpin ribozyme, thermodynamics, RNA-RNA tertiary structure
Abstract not available online - please check the printed booklet.
Abstract:
Eukaryotic initiation factor-4E (eIF-4E) is a cap binding protein and is a component of the eIF-4F complex that plays an important role in translation initiation. eIF-4E is overexpressed in multiple cancer types including prostrate, breast, stomach, colon and lungs.1 Knock down of eIF-4E suppresses tumor growth, enhances apoptosis and increases sensitivity to chemotherapeutics in human breast cancer cells2 which makes it a potent therapeutic target. The presence of a G-quadruplex (GQ) in the 5′-untranslated region (UTR) of an mRNA mostly represses translation.3 Intermolecular GQ structures can be induced in an mRNA to repress the translation. The selective induction of GQ in an mRNA sequence can be achieved by an attached guiding sequence. The above strategy opens up an entirely new domain of cancer therapeutics. We show here that hybrid (DNA:RNA) intermolecular G-quadruplex structure can be induced by targeting a section of the 5prime-UTR of the eIF-4E mRNA with a DNA. The formation of the intermolecular G-quadruplex was established by circular dichroism (CD), gel shift, chemical and enzymatic footprinting. Thermodynamic parameters calculated from CD melting shows deltaG to be -19.22±2.47 kJ which indicates a stable structure under physiological conditions. The specificity of the targeting was established by downregulation of the expression of the reporter gene in a dual luciferase reporter. We also show the inhibition of eIF-4E dependent translation compared to an IRES dependent translation in a bicistronic reporter plasmid. Treatment with the GQ inducing sequence showed significant decrease in cell viability in cancer cells. Our data indicate that expression of specific genes can modulated by inducing G-quadruplex structures in cancer cells.
References:
1. Hsieh, A. C. & Ruggero, D. Targeting Eukaryotic Translation Initiation Factor 4E (eIF4E) in Cancer. Clin. Cancer Res.16, 4914-4920 (2010).
2. Dong, K. et al. Tumor-specific RNAi targeting eIF4E suppresses tumor growth, induces apoptosis and enhances cisplatin cytotoxicity in human breast carcinoma cells. Breast Cancer Res. Treat.113, 443-456 (2009).
3. Bugaut, A. & Balasubramanian, S. 5prime-UTR RNA G-quadruplexes: translation regulation and targeting. Nucleic Acids Res40, 4727-4741 (2012).
Keywords: Translation repression, eIF-4E, G-quadruplex strucutres, Cancer,
Abstract:
Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. We have constructed versatile RNA nanoparticles with solid shapes for targeting cancers specifically by establishing the technology and developing “toolkits”. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.
References:
1. Shu Y, Shu D, Haque F, Guo P. Fabrication of pRNA nanoparticles to deliver therapeutic RNAs and bioactive compounds into tumor cells. Nat Protoc. 2013 Sep;8(9):1635-59.
2. Shu Y, Haque F, Shu D, Li W, Zhu Z, Kotb M, Lyubchenko Y, Guo P. Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs. RNA. 2013 Jun;19(6):767-77.
Keywords: RNA nanotechnology, RNA nanoparticles, Tumor targeting
Abstract:
Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches L-serine or L-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNAAla, as observed in other bacteria, and also transferred alanine, serine, and glycine to tRNAPhe. S. pneumoniae tRNAPhe has an unusual U4:C69 mismatch in its acceptor stem that prevents editing by phenylalanyl-tRNA synthetase (PheRS), leading to the accumulation of misaminoacylated tRNAs that could serve as substrates for translation or for MurM. Although the peptidoglycan layer of S. pneumoniae tolerates a combination of both branched and linear muropeptides, deletion of MurM results in a reversion to penicillin sensitivity in strains that were previously resistant. However, because MurM is not required for cell viability, the reason for its functional conservation across all strains of S. pneumoniae has remained elusive. We now show that MurM can directly function in translation quality control by acting as a broad specificity lipid-independent trans editing factor that deacylates tRNA. This activity of MurM does not require the presence of its second substrate, Lipid II, and can functionally substitute for the activity of widely conserved editing domain homologues of AlaRS, termed AlaXPs proteins, which are themselves absent from S. pneumoniae.
References:
Shepherd and Ibba (2013) Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM, Journal of Biological Chemistry, Vol 288 PMID: 23867453
Keywords: Peptidoglycan, Translation, Aminoacyl-tRNA synthetase
Abstract:
Many microorganisms, including Helicobacter pylori, rely on an indirect pathway for the synthesis of Gln-tRNAGln and/or Asn-tRNAAsn. In these cases, Asn-tRNAAsn production proceeds via misacylation of tRNAAsn (catalyzed by a non-discriminating aspartyl-tRNA synthetase, ND-AspRS) to generate Asp-tRNAAsn, which is subsequently converted to Asn-tRNAAsn by an amidotransferase (AdT). Gln-tRNAGln is produced via an analogous process, relying on a misacylating glutamyl-tRNA synthetase (either ND-GluRS or GluRS2) and AdT. However, efficient delivery of both misacylated tRNAs from the two misacylating enzymes to AdT is required to ensure the stability of the aminoacyl ester bond and to minimize translational errors. Some bacteria, like T. thermophilus, utilize a tRNA-dependent complex called the transamidosome, which contains AdT, tRNAAsn, and ND-AspRS. This Asn-transamidosome traps misacylated Asp-tRNAAsn until it is converted to Asn-tRNAAsn. Recent studies have shown that the H. pylori Asn-transamidosome is more dynamic, suggesting a requirement for an alternative mechanism to facilitate assembly of a stable complex. We have identified a novel ATPase called Hp0100 that is a component of a stable, tRNA-independent H. pylori transamidosome. Hp0100 contains two distinct ATPase active sites, which are activated by misacylated tRNAs. Hp0100 dramatically enhances the capacity of AdT to convert Asp-tRNAAsn into Asn-tRNAAsn but has minimal effects on ND-AspRS function. Our results highlight the importance of the novel ATPase Hp0100, for the stable assembly and function of H. pylori transamidosome.
References:
1. Silva, G. N., Fatma, S., Floyd, A. M., Fischer, F., Chuawong, P., Cruz, A. N., Simari, R. M., Joshi, N., Kern, D., and Hendrickson, T. L. (2013) A tRNA-independent mechanism for transamidosome assembly promotes aminoacyl-tRNA transamidation. J. biol. chem. 288, 3816-3822
2. Fischer F., Huot J. L., Lorber B., Diss G., Hendrickson T. L., Becker H. D., Lapointe J., Kern D. (2012) The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code. Nucleic Acids Res. 40, 4965–4976
Keywords: Transamidosome, Non-discriminating Aspartyl-tRNA Synthetase, Hp0100
Abstract:
The microprocessor, comprised of DGCR8 and Drosha, cleaves primary microRNA (miRNA) hairpins to generate pre-miRNAs, which are subsequently processed to a mature miRNAs. In addition to pri-miRNA hairpins, other regions within coding genes can form RNA hairpins. Increasing evidence indicates that the microprocessor may bind indiscriminately to RNA hairpins, resulting in widespread cleavage of RNA. Prevalent cleavage of RNA by the microprocessor has the potential to impact gene expression and pre-mRNA alternative splicing. Here, we identify the small, alternative exon 5 in the eIF4H gene as a microprocessor substrate. Using a cell-free cleavage assay, we find that this exon is processed into a pre- and mature miRNA by the microprocessor and Dicer, respectively. We also detect the small RNAs generated by the microprocessor in cells. Although cleavage of the exon precludes its inclusion in mRNA, we found that overexpression of Drosha, both wild-type and a mutant form that can bind but not cleave RNA, promotes the inclusion of this alternative exon in a manner that is dependent on RNA structure. This Drosha-mediated increase in exon inclusion is accompanied by non-canonical cleavage within the exon. Additionally, Drosha may be in competition with the SRSF2 and 3 proteins, which are negative regulators of both exon 5 inclusion in the mRNA and the non-canonical cleavage event. These results indicate a previously unknown role for the microprocessor in promoting splicing that may occur through competition with canonical splicing factors and the role in splicing may be in conflict with its role in miRNA biogenesis.
Keywords: Alternative splicing, microRNA, Microprocessor
Abstract not available online - please check the printed booklet.
Abstract:
Ribonucleoprotein enzymes, such as the ribosome, spliceosome, RNase P, RISC Complex, and snRNPs, are essential to life and must interact with a large number of alternative substrates to function. Ribonuclease P , RNase P recognizes all precursor tRNAs (pre-tRNAs) that differ in sequence and structure to perform its role in 5prime end maturation. Work from our lab and others reveals that both the protein and catalytic RNA subunits of RNase P possess sequence specificity in contacts to the 5prime leader of pre-tRNA. We are investigating how variation in the 5prime leader of pre-tRNAs affects binding by the holoenzyme and ribozyme and how these contacts influence one another. We developed a new method allowing us to quantify individual rate constants for a reaction containing a large number of alternative substrates. The power of this technique is the ability to analyze all possible sequences in areas of protein and RNA contact, providing information about thermodynamic coupling. By randomizing regions of the 5prime leader contacting the protein (nucleotides -3 to -8) or the protein and RNA (nucleotides -1 to -6), we can investigate each population for processing by the RNase P ribozyme and holoenzyme. Next-generation sequencing of the residual substrate population along the reactions allows us to calculate the rate constants of each substrate sequence. Comparison of ribozyme and holoenzyme reactions with the same substrate population shows changes in preferential sequence in regions of RNA and protein contact. This combined with the changing shape of the affinity distribution indicates that this crosstalk is occurring. This information will provide key insight into the molecular recognition of RNase P by investigating thermodynamic and kinetic coupling in the RNA-RNA and protein-RNA contacts present in RNase P substrate recognition.
References:
U. Guenther, L.E. Yandek, C.N. Niland, F.E. Campbell, D. Anderson, V.E. Anderson, M.E. Harris, and E. Jankowsky. (2013) Hidden Specificity In An Appartently Non-Specific RNA-Binding Protein. Nature (in press).
L.E. Yandek, H.C. Lin, and M.E. Harris. (2013) Alternative Substrate Kinetics of Eschericia coli Ribonuclease P: Determination of Relative Rate Constants By Internal Competition. J. Biol. Chem. Mar 2013;288(12):8342-54
L. Sun, F.E. Campbell, L.E. Yandek, and M.E. Harris. (2010) Binding of C5 Protein to P RNA Enhances The Rate Constant For Catalysis For P RNA Processing of pre-tRNAs Lacking A Consensus G(+1)/C(+72) Pair. J. Mol. Biol. Feb 2010;395(5):1019-37
N.H. Zahler, E.L. Christian, and M.E. Harris. (2003) Recognition of the 5prime leader of pre-tRNA Substrates By The Active Site of Ribonuclease P. RNA. Jun 2003;9(6):734-45
Keywords: Ribonuclease P, Alternative Substrate Kinetics, RNA Processing
Abstract not available online - please check the printed booklet.
Abstract:
The long-non-coding Steroid Receptor Activator RNA (SRA lncRNA) is part of a SRC-1 enzyme complex that acetylates histones.1 This complex permits the transition of chromatin regions from silenced heterochromatin to expressed euchromatin.2,3 The SRC-1/SRA complex enhances the expression of genes controlled by steroid nuclear receptors.4,5,6 SRA1 RNA also interacts with many proteins, DDX17,6 SHARP,7 and others.8 Nuclear receptors are known to stimulate certain cancers; interestingly SRA RNA is dramatically overexpressed in, and stimulatory to, tumor cells.9 The SRA lncRNA facilitates chromatin remodeling as a non-coding RNA rather than as an mRNA. However, SRA is alternatively spliced into an mRNA encoding the SRA1p protein.10 Thus the gene for SRA is considered both an mRNA and lncRNA coding gene. SRA1p, along with SLIRP and SHARP, are repressors of SRA action and thus important checks on differentiation11 and tumorigenesis. There are no structures of these proteins bound to RNA and the evidence for direct interactions is based on immunoprecipitation. Our NMR structure of the c-terminal domain of hSRA1p, previously thought to be an RRM domain, demonstrates that it is not an RRM nor does it have apparent SRA RNA binding activity. In contrast, RRM domains from SHARP and SLIRP are clearly identifiable and our progress towards the NMR structures of these RNP complexes will be presented.
References:
1. Lanz RB, et al. Tsai SY, Tsai MJ, O Malley BW (1999) Cell 97: 17-27.
2. Onate SA, et al. (1995) Science 270: 1354–7.
3. Onate SA, et al. Edwards DP, O Malley BW (1998) JBC 273: 12101–8.
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6. Watanabe M, et al. (2001) EMBO J. 20: 1341–52.
7. Shi Y, et al. (2001) Genes Dev. 15: 1140–51.
8. Colley SM, Leedman PJ (2011) Biochimie 93:1966-72.
9. Lanz RB, et al. (2003) MCB 23: 7163-76.
10. Chooniedass-Kothari S, et al. (2004) FEBS Lett. 566: 43–7.
11. Hubé F, et al. (2011) NAR 39: 513-25.
Keywords: NMR, lncRNA, epigenetics
