RRM
2009 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:
Prolyl-tRNA synthetase (ProRS) is a multi-domain protein that specifically ligates Pro to its cognate tRNAPro in a reaction catalyzed by its aminoacylation active site. Most bacterial ProRSs also possess an editing domain (INS), a discrete active site that functions to hydrolyze noncognate Ala mischarged onto tRNAPro. In addition, Escherichia coli (Ec) ProRS misactivates Cys but is unable to hydrolyze Cys-tRNAPro via its INS domain. Instead, a small free-standing protein homologous to the INS domain (YbaK) has been shown to hydrolyze mischarged Cys-tRNAPro in trans. YbaK behaves as a general Cys-deacylase in vitro, hydrolyzing all Cys-tRNA substrates independent of tRNA identity. This calls into question how YbaK gains specificity for Cys-tRNAPro and whether there are any other mischarged Cys-tRNA substrates for YbaK in vivo. To gain further mechanistic insights into the biological roles of YbaK, in vivo interactions were probed using a tandem affinity purification (TAP-tag) method to identify proteins interacting with YbaK. Interestingly, in addition to ProRS, three additional synthetases, AlaRS, LysRS and AsnRS, were identified as interacting partners of YbaK when a formaldehyde cross-linking step was included. To further validate these interactions, a split-GFP reassembly technique was used to probe these protein-protein interactions in vivo. We also probed the possible functional implications of these interactions by performing deacylation assays by YbaK in the presence and absence of the interacting partners. Taken together, we propose that YbaK may function in vivo to hydrolyze Cys-tRNA species other than Cys-tRNAPro, and that interactions with the corresponding synthetases may play a role in substrate channeling and translational quality control.
Keywords: ProRS, YbaK, TAP-tag, Split GFP technique
Abstract:
The exit (E) site has been implicated in several ribosomal activities, including translocation, decoding, and maintenance of the translational reading frame. Here, we target the 30S subunit E site by introducing a deletion in rpsG that truncates the beta-hairpin of ribosomal protein S7. This mutation (S7DeltaR77-Y84) increases both -1 and +1 frameshifting but does not increase miscoding, providing evidence that the 30S E site plays a specific role in frame maintenance. Mutation S7DeltaR77-Y84 also stimulates +1 programmed frameshifting during prfB'-lacZ translation in many synthetic contexts. However, no effect is seen when the E codon of the frameshift site corresponds to those found in nature, suggesting that E-tRNA release does not normally limit the rate of prfB frameshifting. Ribosomes containing S7DeltaR77-Y84 exhibit an elevated rate of spontaneous reverse translocation and an increased K (1/2) for E-tRNA. These effects are of similar magnitude, suggesting that both result from destabilization of E-tRNA. Finally, this mutation of the 30S E site does not inhibit EF-G-dependent translocation, consistent with a primary role for the 50S E site in the mechanism
Keywords: Ribosome, E site, Frameshifting
Abstract:
Ribosomes are the machinery responsible for catalyzing protein synthesis in all cells. In eukaryotes, the two ribosomal subunits contain four ribosomal RNA (rRNA) molecules and 78 ribosomal proteins [1]. Eukaryotic ribosome assembly is a very complex process, requiring the action of more than 250 transiently associated protein and RNA accessory factors. These factors are required for precise folding and processing of the rRNAs, as well as for correct incorporation of the ribosomal proteins. While the identities of the necessary assembly factors are known, many of their functions have yet been elucidated. The Saccharomyces cerevisiae protein Nob1 is required for cell viability. Depletion of Nob1 results in a defect in 40S, or small subunit processing. Specifically, it leads to an accumulation of 20S rRNA, the direct precursor to mature 18S rRNA, which is the sole rRNA component of the small subunit [2]. Nob1 contains a N-terminal PIN domain, a ~120 amino acid domain characterized by four conserved acidic amino acids that serve to chelate a metal ion necessary for nuclease activity. Additionally, Nob1 contains a zinc ribbon domain. Nob1 has been shown to copurify with cytoplasmic pre-40S ribosomes [3] along with six other trans-acting factors [4]. Since Nob1 is the only factor bound to pre-40S ribosomes that contains a nuclease domain, it is believed to be the endonuclease responsible for the cleavage event that produces mature 18S rRNA. Here, we show that mutation of basic residues within the protein’s zinc ribbon domain, as well as truncation of its C-terminus results in decreased rRNA binding affinity in vitro accompanied with an increased growth rate in vivo.
References:
1. Fromont-Racine M, et al. (2003) Gene 313, 17-42.
2. Fatica A, et al. (2003) Mol Cell Biol 23, 1798-1807.
3. Schäfer T, et al. (2006) Nature 441, 651-655.
4. Schäfer T, et al. (2003) EMBO J 22, 1370-1380.
Keywords: ribosome assembly, endonuclease
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The ribosome is a potential target for several classes of antibiotics because of its accessibility and structural diversity. Different classes of antibiotics bind to different sites within the ribosome to inhibit the ribosomal assembly and translation. Bacteria have become resistant to most of the known antibiotics; therefore, it is important to design and develop new therapeutic agents. The first step towards the development of a drug is to find ideal targets sites that are not only functionally important, but also accessible to various compounds. Most of the available chemical reagents that have been used to probe RNA structure are limited to in vitro studies due to inability to penetrate the cell wall, high toxicity and or challenges in detection of their reactive sites. Cisplatin, cis-[Pt(NH3)2Cl2], reacts preferentially with purine bases in DNA and RNA and forms various adducts, such as 1,2-GpG-intrastrand cross-links. The adducts formed in the ribosome will give information about accessibility of the nucleotides within the complex 3D structure. We have utilized cisplatin to probe accessible guanine residues in RNA model systems, 16S rRNA, and the ribosome. This method has several advantages over other chemical probes: the adducts are easily detected, and the probe can be used in vivo. In addition, to study the effects of electrostatic interactions and compare the binding sites and reactivity with the cisplatin, we have synthesized a variety of amino-acid-linked platinum complexes. The binding sites of these complexes are distinctly different from those of the parental compound cisplatin. We demonstrate that platinum complexes have the ability to provide information about helix or loop accessibility in the ribosome. Investigation of accessible and functionally important target sites in the ribosome will play a significant role in the design of novel drug leads.
Keywords: Ribosome, Cisplatin, Probing
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
DEAD-box RNA helicases, which catalyze rearrangements of RNA in many cellular processes, are known to unwind RNA secondary structure by a mechanism distinct from canonical helicases. Rather than translocating along the substrate, DEAD-box helicases directly load on a duplex region and then pry the strands apart in an ATP-dependent fashion. This unwinding mechanism consists of multiple steps including loading of the enzyme to duplex regions, binding of ATP, strand separation, ATP hydrolysis, and product release. How these steps are coordinated in the overall kinetic framework of DEAD-box helicases is not known, and the lack of this information complicates the evaluation of structure function relationships for this important enzyme family. Here we describe the first kinetic framework for duplex unwinding by a DEAD-box RNA helicase, Ded1p from S. cerevisiae. We use a combination of complimentary kinetic approaches, including EMSA and stopped flow fluorescence spectroscopy. We find that for short duplexes, which are completely separated in a single enzyme binding event, unwinding occurs with rate constants significantly greater than the rate constant for ATP turnover, consistent with previous results showing that ATP hydrolysis is not required for strand separation. We also find that ATP binding and formation of an activated enzyme conformation are rate limiting for strand separation of short duplexes. As duplex length and stability increases, non-enzymatic strand dissociation becomes rate limiting, and multiple binding events are required for complete strand separation. Collectively, our data reveal how the stability of duplexes controls unwinding rate constants for longer and more stable duplexes. However, for short and less stable duplexes, which are thought to constitute the majority of RNA structures targeted by DEAD-box proteins, ATP binding limits the overall reaction.
Keywords: DEAD-box helicase, ATP binding, RNA metabolism
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Transcription and splicing must proceed over genomic distances of hundreds of kilobases in many human genes. However, the rates and mechanisms of these processes are poorly understood. We have used the compound 5,6-Dichlorobenzimidazole 1-b-D-ribofuranoside (DRB) that reversibly blocks gene transcription in vivo combined with quantitative RT-PCR to analyze the transcription and RNA processing of several long human genes. We found that the rate of RNA polymerase II transcription over long genomic distances is about 3.8 kb per minute and is nearly the same whether transcribing long introns or exon rich regions. We also determined that co-transcriptional pre-mRNA splicing of U2-dependent introns occurs within 5–10 minutes of synthesis irrespective of intron length between 1 kb and 240 kb. Similarly, U12-dependent introns were co-transcriptionally spliced within 10 minutes of synthesis confirming that these introns are spliced within the nuclear compartment. These results show that the expression of large genes is surprisingly rapid and efficient. In summary, we have developed a useful technique to follow the transcription elongation by RNAPII over hundreds of kilobases of DNA in large genes in a highly physiological setting. This system is thus well suited for the investigation of the effects of perturbation of the various components on transcription elongation and RNA processing.
Keywords: RNA Polymerase II, Transcription rate, splicing
Abstract:
Rapid progress in recent years has demonstrated that splicing is tightly coupled to transcription and this coupling provides a possibility that elongation rate of RNAPII can influence splice site selection. While modification of chromatin structure has been shown to regulate splice site choice through affecting transcription elongation rate, no splicing regulators had been shown to directly interact with chromosomal proteins to regulate the transcription elongation rate.
Hu proteins are a family (HuA/R, HuB, HuC and HuD) of mammalian RNA-binding proteins. Our previously studies demonstrated that Hu proteins function as important splicing regulators, which inhibits exon 23a of the neurobromatosis type I (NF1) pre-mRNA inclusion by binding to AU-rich sequences surrounding this exon. In this report, we will discuss a potential novel role of Hu proteins in alternative splicing regulation through affecting transcription elongation rate.
Through a yeast two-hybrid screen, we identified histone deacetylase 2 (HDAC2) as proteins that directly interact with HuC protein. These interactions were then verified in mammalian cells. The interactions between HuC and chromatin-associated proteins prompted us to postulate a role of this protein in transcription. Co-immunoprecipitation assay indicates that Hu proteins interact with both ser5 and ser2 phosphorylated RNAPII in an RNA-independent fashion. An interaction between a transcription elongation factor CDK9 and Hu proteins was also detected. There results suggest that Hu proteins play a role in coupling transcription and splicing.
The ChIP assay also demonstrates that the acetylation level of both histone H3 and H4 at the exon 23a region is much higher in high Hu expressing CA77 cells than low Hu expressing HeLa cells. These results help us to develop a working model that Hu proteins increase transcription elongation rate through modulating the local chromatin structure surrounding exon 23a via its interactions with histones and/or HDAC2. Our studies uncover a novel regulatory mechanism of alternative splicing by Hu proteins. Additional experiments that will provide more mechanistic details will be discussed.
HU
Keywords: Hu proteins, Alternative splicing
Abstract:
Bioactive compounds have been invaluable for dissecting the mechanisms, regulation and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a rapid-response high throughput cell-based assay that measures pre-mRNA splicing utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 hr) thereby obviating potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine, and show that it is a selective inhibitor of specific Clks that phosphorylate SR-protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing, and uncover differential regulation of constitutively spliced introns.
Keywords: Splicing, High throughput screening, SR proteins
Abstract not available online - please check the printed booklet.
Abstract:
Nuclear speckles provide important spatial organization and dynamic regulation for pre-mRNA processing factors in mammalian cells. While the nuclear speckle proteome is complex, little is known at the molecular level about how these factors are organized into nuclear speckles or how alterations in the organization of these factors impacts gene expression. We have discovered a new function for a large (2564 amino acid) nuclear speckle protein called Son in maintaining the organization of pre-mRNA processing factors in nuclear speckles. Depletion of Son by RNAi causes snRNP and serine-arginine rich (SR protein) splicing factors to undergo dramatic disorganization into doughnut-shaped nuclear speckles. Rescue of the disorganized nuclear speckle phenotype requires a region of Son with multiple tandem repeat motifs that are unique to Son. This demonstrates that the tandem repeats of Son are necessary for appropriate localization of pre-mRNA processing factors, and it suggests a potential role for Son as a nuclear speckle scaffold. Son depletion does not alter protein levels of other splicing factors, and it does not reduce global transcription or constitutive splicing. However, Son depletion can affect alternative splice site selection. Surprisingly, in addition to its nuclear functions, Son depletion also results in decreased cell proliferation due to growth arrest in mitosis. Son is critical for promoting the transition from metaphase to anaphase. Son is therefore essential for nuclear organization and function, as well as for normal cell cycle progression.
Keywords: Nuclear Speckles, SR Proteins, Cell Cycle
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Long Non-coding RNAs (lncRNAs) have recently been the focus of much attention due to their apparently ubiquitous regulatory role in eukaryotic cells. In order to gain insight into the cellular function of this novel class of RNAs, we conducted a bioinformatic analysis mainly focused on elucidating any significant differences that might help distinguish between lncRNAs and their protein-coding counterparts and offer clues to their function. Genome-wide analyses of predicted non-coding transcripts have offerred exciting clues to the general characteristics of these transcripts, however, it is not known whether all the predicted lncRNAs indeed serve a cellular function, and it remains formally possible that many of them might code for small polypeptides. To circumvent these issues, we focused our effort on analyzing the well-established lncRNAs for which a cellular function has been identified. A detailed bioinformatic analysis was performed on this group of lncRNAs and a group of randomly selected protein-coding RNAs in parallel.
Keywords: non-coding, bioinformatic, repeat element
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
A major challenge in the fight against cancer, including breast cancer, is early and sensitive detection of neoplasms. Some miRNAs with diagnostic value found in cancer cells have also been detected in body fluids. Thus, circulating miRNAs may afford diagnosis with low invasiveness. However, it remains to be determined, if the released miRNA population is the same as the miRNA population retained by the cell. We find that individual miRNA species are released from cells at different rates. This rate is constant for most miRNAs. Importantly, miRNAs with diagnostic value in breast cancer, such as miR-451, are primarily released into the environment, whereas miR-16, a common cellular miRNA, is mostly retained in the cell. These results suggest that miRNAs are released selectively. In part, this selection may be explained by reports that miRNAs are released from cells still tethered to their mRNA target. However, we find that for some miRNAs, including miR-16 and miR-21, both mature and immature miRNAs are released by cells, whereas only mature miR-451 is released by cells, suggesting that alternative pathways exist. Furthermore, about half of the analyzed mature miRNAs that are released differ from the cell-retained population in size, suggesting that additional processing mechanisms occur during the release of these miRNAs. Some of these miRNA properties can also be detected in human milk and ductal lavages, which contain products of mammary epithelia. This suggests physiological relevance of the observed differences in the released miRNA population. Finally, we find that miRNAs are released from cells in several different nano-vesicles, including exosomes, apoptotic bodies and smaller components. These findings indicate the presence of one or more selection and processing mechanisms in the release of miRNAs. In addition, these data imply that the diagnostic profiles of released miRNAs are expected to be different than the cellular miRNA profile.
Keywords: circulating microRNAs, transport, cancer diagnostics
Abstract:
The pRNA (packaging RNA) of bacteriophage phi29 DNA packaging motor has been reported to have novel applications in nanotechnology and nanomedicine. The unique ability of pRNA to form dimers, trimers, hexamers and patterned superstructures via the interaction of two reengineered interlocking loops makes it a promising polyvalent vehicle to load siRNA and other therapeutic molecules and be applied as a therapeutic nanoparticle in tumor therapy. In this study, several tumor cell lines were used to evaluate the previously reported pRNA nanotechnology for specific siRNA delivery and for the silencing of targeted genes. It was found that MCF-7 and HeLa cells, out of twenty-five tested tumor cell lines, expressed high levels of folate receptors and exhibited specific binding of the FITC-folate-pRNA nanoparticles, while the others expressed low levels and thus, for these, delivery was not feasible using folate as a targeting agent. Folate receptor positive tumor cells were then incubated with the chimeric pRNA dimer harboring both the folate-pRNA and the chimeric pRNA/siRNA (survivin). Knock down effects of survivin expression in these tumor cells were detected at the mRNA level by real time-PCR and at the protein level by western blot. Apoptosis was detected by flow cytometry analysis with dual staining of annexinV-FITC and PI. The data suggest that the chimeric pRNA nanoparticles containing folate-pRNA and pRNA/siRNA (survivin) could be specifically taken up by tumor cells through folate receptor-mediated endocytosis, resulting in significant inhibition of both transcription and expression of survivin in tumor cells and triggering cell apoptosis. Using such protein-free nanoparticles as therapeutic reagents would not only allow specific gene delivery and extend the in vivo retaining time but also allow long-term administration of therapeutic particles, therefor
Keywords: Bacteriophage phi29, folate, pRNA
Abstract:
The Huisgen cycloaddition (Click Chemistry) involves a copper(I)-catalyzed triazole formation from an azide and an alkyne. The azide and alkyne moieties can be used interchangeably, one to tag the molecule of interest and the other in the molecule used for the detection, e.g. DNA and a fluorescent dye respectively. This reaction is not viable in biological systems due to the instability of the Cu(I) catalyst under oxygen rich aqueous solutions. Several stabilizing ligands have been reported that enable this reaction to be used in biological systems containing DNA. The use of systems containing RNA is specially challenging as the Cu(I) disproportionation reaction degrades RNA. Here we report the application of Click chemistry to RNA and a 5’end labeling of RNA with a fluorescent dye. This obviates the need to work with radioactively labeled RNA to study ribozyme kinetics and provides a sensitive and more stable probe that is readily accessible via Click Chemistry. In addition, We have synthesized a 3’-alkyne terminated RNA via solid phase synthesis and used 5’-deoxy 5’-azido guanosine according to literature to furnish a 5’-deoxy-5’azido transcript. Clicking these two fragments yields a ‘Click-Ligated’ RNA.
Keywords: Click Chemistry, RNA, Labeling
Abstract:
Living systems contain a wide variety of nanomachines and highly-ordered structures of macromolecules that could serve as modules, tool boxes, or building blocks in nanotechnology. The ingenious design of the bacteriophage phi29 DNA packaging motor with an elegant and elaborate channel has inspired its application for single molecule detection and sensing. The central component of the phi29 motor is the connector composed of twelve copies of the protein gp10, which form a dodecamer channel. The connector after incorporation into a lipid bilayer can serve as a detector for extremely sensitive, reliable, and precise sensing and fingerprinting of ions and macromolecules at the single molecule level (Nature Nanotechnology, in press). Double stranded and single stranded RNA and DNA can be electrophoretically driven through the channel in a concentration and voltage dependent manner. Information about their structure folding, length and conformational dynamics can then be deduced by their characteristic signatures and dwell times during translocation as well as by their relative percentage in current blockades. This protein nanopore system with explicit engineering capability has potential technological applications such as rapid DNA sequencing, gene therapy and controlled drug delivery.
Keywords: Bacteriophage phi29, Nano-Channel, RNA translocation
Abstract:
Transcription elongation factors NusG and RfaH evolved from a common ancestor and utilize the same binding site on RNA polymerase to modulate transcription. Both proteins increase the rate of RNA synthesis in vitro. However, ChIP-chip analysis indicates that while NusG associates with RNA polymerase transcribing most E. coli genes, RfaH action is restricted to several operons that are devoid of NusG and contain an ops element, a DNA sequence that mediates RfaH recruitment (1). In vitro assays reveal that RfaH and NusG compete for their effects on transcript elongation and termination. Our data argue that RfaH recognizes its specific DNA target even in the presence of NusG. Once recruited, RfaH remains stably associated with RNA polymerase, thereby precluding NusG binding. We propose that E. coli RfaH and NusG play opposite regulatory roles. NusG acts in concert with Rho to suppress expression of foreign DNA (2). By contrast, RfaH inhibits Rho action and activates expression of poorly translated, frequently laterally transferred genes. We envision a pathway by which a specialized regulator has evolved in the background of its ubiquitous paralog.
References:
1. Belogurov, G. A., Mooney, R. A., Svetlov, V., Landick, R. & Artsimovitch, I. (2009). Functional specialization of transcription elongation factors. EMBO J 28, 112-22.
2. Cardinale, C. J., Washburn, R. S., Tadigotla, V. R., Brown, L. M., Gottesman, M. E. & Nudler, E. (2008). Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli. Science 320, 935-8.
Keywords: transcription, termination, regulation
Abstract not available online - please check the printed booklet.
