RRM
2011 Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract:
Correct prediction of RNA 3D structure from sequence is a major challenge and a bottle-neck in genomic analysis. Most of the eukaryl genome is transcribed, and most transcripts are non-coding RNA (ncRNA). Moreover, many ncRNAs comprise one or more structured domains. An important sub-goal in the prediction of global 3D structures is the inference of the structures of recurrent, modular motifs corresponding to hairpin and internal "loop" regions in secondary structures. Such recurrent 3D motifs can play architectural roles (e.g., kink-turns), serve to anchor RNA tertiary interactions (e.g., GNRA loops), or provide binding sites for proteins or ligands. Different sequences can form the same 3D motif. We extract all hairpin and internal loops from a non-redundant (NR) set of RNA 3D structures from the PDB/NDB and cluster them in geometrically similar families. For each motif, we construct a probabilistic model for sequence variability based on a hybrid Stochastic Context-Free Grammar/Markov Random Field (SCFG/MRF) method. To parameterize each model, we use all instances of the motif found in the NR dataset and RNA knowledge of nucleotide interactions, especially isosteric basepairs and their substitution patterns. SCFG techniques can account for nested pairs and insertions, while MRF ideas can handle non-nested interactions. Given the sequence of a hairpin or internal loop as input, each SCFG/MRF model calculates the probability that it forms the corresponding 3D motif. If the score is in the same range as sequences known to form the 3D structure, we infer that the new sequence forms the same 3D structure. This approach correctly infers the 3D structures of nearly all structured internal loops when using as input sequences from 3D structures, a first validation step. A web server will be presented for users to input motif sequences and obtain the most likely 3D structures, or a statistical indication that the motif is likely to be new.
Keywords: RNA, Motif, SCFG
Abstract not available online - please check the printed booklet.
Abstract:
The activation parameters, ∆*H and ∆*S, for four dodecamers with consistent composition but varying sequence (5’-CACACAAGCGGC-3’/5’-GCCGCTTGTGTG-3’, 5’-CACAAGCGGCAC-3’/5’-GTGCCGCTTGTG-3’, 5’-CAAGCGGCACAC-3’/5’-GTGTGCCGCTTG-3’, 5’-GCGGCAACACAC-3’/5’-GTGTGTTGCCGC-3’) were measured using the stopped-flow technique from 278 K to 303 K. The kinetic profiles were collected over a period of six to eight half-lives and the percentage change in absorbance showed no recognizable correlation with respect to reaction temperature, which indicated complete formation of the duplex. The change in enthalpy of activation ranged from approximately 18 kcal/mol to 27 kcal/mol depending on the sequence. The change in entropy of activation ranged from approximately 31 cal/mol K to 62 cal/mol K. The sequence in which the GC-rich region started at the fifth base-pair had the highest activation parameters, while the sequence in which this region started at the third base-pair position has the lowest activation parameters. The data are interpreted using a model in which the duplex formation is initiation in the GC-rich region.
Keywords: DNA, Activation parameters, Kinetics
Abstract:
Noncoding RNAs (ncRNAs) play a pivotal role in the function and regulation of essential cell processes. Small RNA repertoire, biogenesis and function are largely unknown in malaria parasite Plasmodium falciparum that kills 3-5 million people worldwide, each year. We are particularly interested in understanding the specific role of ncRNAs in malaria parasite P. falciparum. Towards this end, we have performed a genome wide single stranded and a double stranded RNA high throughput sequencing. This allowed us to catalog all small RNAs that are overrepresented in the genome and to initiate investigation on their function. Our studies indicate antisense small RNA transcription as a major event that can regulate essential genes in malaria parasites. Additionally, we are in the process of in vivo structure probing by hydroxyl radical footprinting, which provides information on tertiary structure and intermolecular interface with a single nucleotide resolution. We have included tight controls to validate some of our genome wide RNA sequencing and structure probing data. All results tie up to enlighten our understanding of the properties and function of small RNAs in malaria parasite P. falciparum at a genome wide scale.
Keywords: ncRNA, hydroxyl radical footprinting, ss and dsRNA-Seq
Abstract:
Vaccinia is a member of the Pox virus family containing a double-stranded DNA genome. Early genes, the first of three transcriptional classes, are transcribed by enzymes packaged within the Vaccinia virion. Nucleoside Triphosphate Phosphohydrolase I (NPH I) is an integral component of Vaccinia early gene transcription termination complex. NPH I releases paused transcripts by hydrolyzing ATP or deoxyATP. NPH I ATPase activity requires ssDNA as a cofactor, which is hypothesized to originate from the non-template strand of the DNA being transcribed. To test this hypothesis we constructed bead-bound transcription DNA templates lacking portions of the non-template strand. Transcription was paused using nucleotide starvation and NPH I mediated transcript release was measured. Lengthening the non-template strand restored NPH I mediated transcript release activity. Hence the non-template strand may be the source of the ssDNA cofactor. Another possibility is that transcription bubble collapse may be restored when the non-template strand is lengthened. Transcription bubble collapse is an important component of terminator hairpin function, rho mediated termination, and MFD mediated transcript release in bacteria. To test whether bubble collapse is an important component of NPH I mediated transcript release, templates were constructed with sequences of non-complementarity within the transcription bubble and assayed for NPH I mediated transcript release and oligonucleotide mediated transcript release. Preliminary results suggest that bubble collapse is an important component of NPH I mediated transcript release. Future directions will include attempting to crosslink NPH I to regions of single stranded DNA within the transcription bubble.
Keywords: Vaccinia, Early Gene Transcription
Abstract:
In the trpEDCFBA operon of Bacillus subtilis, transcription is regulated by the trp RNA-binding attenuation protein (TRAP) using an attenuation mechanism involving two competing RNA secondary structures. These mutually exclusive RNA structures, an antiterminator and terminator, are in the 203-nt 5’ leader region of the transcript upstream of trpE. When tryptophan is present the TRAP protein is activated and binds to a region in the trp leader RNA that contains 11 (G/U)AG repeats. This binding prevents formation of the antiterminator and allows formation of a terminator that induces transcription termination and the genes in the operon are not expressed. When tryptophan is limiting, TRAP does not bind to the RNA and an alternative antiterminator structure forms allowing expression of the trp genes. Mutations in the trp leader that prevent formation of the anti-terminator and allow the terminator structure to form constitutively fail to terminate in the leader region in the absence of TRAP in vitro. In the presence of TRAP, termination occurs suggesting that TRAP may have an additional role in attenuation other than changing the secondary structure of RNA. The characteristics of these mutants were also examined in vivo. Since the trp terminator does not function as an intrinsic terminator in the absence of TRAP, we wanted to determine the importance of the RNA structure/sequence in TRAP-mediated transcription termination. To do so mutations were made in the hairpin stem and the U-stretch of the trp terminator and transcription termination was examined in vitro and in vivo. While TRAP was able to cause termination at these mutant terminators in vivo, it was not able to do so in vitro. Therefore, there may be an additional factor present in vivo that allows TRAP to cause transcription termination at a deficient terminator. A genetic screen using transposon mutagenesis was performed and found a possible additional transcription factor, yceG.
Keywords: TRAP
Abstract:
Massively parallel sequencing has identified a large number of capped small RNAs and 5’ truncated forms of RefSeq transcripts. We previously described a cytoplasmic capping enzyme complex that contains capping enzyme and a kinase that converts 5’-monophosphate ends to a diphosphate capping substrate. Targets for cytoplasmic capping were identified by first overexpressing a tet-inducible dominant-negative form capping enzyme that is restricted to the cytoplasm (DN-cCE). Cytoplasmic RNA from control and induced cells was then treated with Xrn1 to degrade uncapped mRNAs and analyzed on Affymetrix Human Exon 1.0 arrays. Scoring was based on changes in probe intensity as a function of position on each RefSeq gene to derive a factor (alpha) that could be compared between sets. Transcriptome-wide changes were only evident in Xrn1-treated samples, indicating the general impact of DN-cCE is limited. This analysis identified 2,666 mRNAs with a population that is uncapped at any given time, 672 mRNAs that are enriched in the uncapped pool only in cells expressing DN-cCE, and 835 mRNAs common to both populations. Ten re-capping targets and 5 controls were selected for detailed analysis of changes in cap status by their; a) susceptibility to in vitro degradation by Xrn1, b) recovery following ligation of an RNA oligonucleotide onto uncapped 5’ ends and recovery with a biotin-tagged antisense primer, and c) separation on a high affinity cap-binding matrix containing immobilized heterodimer of eIF4E+eIF4G. In all cases this confirmed the cap status of re-capping targets identified on exon arrays. Re-capping substrates are enriched for products involved in general cellular metabolism and for downstream Kozak consensus translation initiation sites. In keeping with a role in translation, expression of DN-cCE reduces the ability of cells to recover from stress and increases the population of non-translating mRNPs.
Supported by PHS grant GM084177 and NSF agreement No. 0931642
Keywords: Uncapped transcriptome, cytoplasmic capping
Abstract:
PMR1 is an mRNA endonuclease that degrades its targets while they are engaged by translating ribosomes. To date all of the work on this enzyme has been done on the Xenopus protein because of difficulties in identifying an obvious human ortholog. Xenopus PMR1 is an alternatively processed form of eosinophil peroxidase gene, but there is no evidence for a similar protein from this or any of the other human peroxidase genes. Results in this study identify the newly discovered gene for peroxidasin-like protein as the source of human PMR1. The two peroxidasin genes are a subgroup of the large peroxidase gene family. Both encode a 164 kDa membrane-bound protein with a heme peroxidase domain. In addition peroxidasin-like protein undergoes alternative splicing, the predicted product of which is a 57 kDa protein that shares critical features of Xenopus PMR1, including active site histidines, c-Src binding site, tyrosine phosphorylation site and SH2 binding site. We show here that this is the major form of peroxidasin-like protein in a number of cell lines, and using criteria developed in the course of characterizing the Xenopus protein demonstrate that this is human PMR1.
Keywords: mRNA decay, PMR1, RNA endonuclease
Abstract:
The accumulation of unfolded proteins in the endoplasmic reticulum triggers transcriptional and translational reprogramming. This unfolded protein response (UPR) protects cells during transient stress and can lead to apoptosis during prolonged stress. Two key mediators of the UPR are PERK, which phosphorylates the translation initiation factor eIF2α, resulting in decreased protein synthesis, and IRE1α, which initiates cytoplasmic splicing of the mRNA encoding the transcription factor XBP1. XBP1 induces transcription of genes involved in protein quality control. This report describes a cross-talk between these two pathways: phosphorylation of eIF2α was required for maximal induction of spliced XBP1 (XBP1s) protein levels via a mechanism that involved stabilization of XBP1s mRNA. Using MEFs deficient in UPR signaling pathways, we demonstrate that stress-induced stabilization of XBP1s mRNA requires cytoplasmic splicing of the mRNA and inhibition of its translation. Because XBP1s protein promotes transcription of its own gene, the UPR induced mRNA stabilization is part of a positive feedback loop that induces XBP1s protein accumulation and transcription of target genes during stress. We propose a model where eIF2α phosphorylation-mediated control of mRNA turnover is a molecular switch that regulates the stress-response transcription program and the ER’s capacity to handle protein folding during stress.
Keywords: ER stress, mRNA stability
Abstract:
Eukaryotic ribosome biogenesis involves regulated processing and folding of precursor rRNAs (pre-rRNAs) and concomitant assembly of ribosomal proteins (r-proteins). Over 200 trans-acting factors are known to be required for pre-rRNA processing, association of proteins, and transit of preribosomes from the nucleolus to the cytoplasm. Recently, it has become more evident that mutations in r-proteins result in a variety of human diseases. In vitro reconstitution studies in bacteria have revealed the hierarchical and cooperative nature of assembly characterized by a sequence of conformational changes in pre-rRNPs and association of r-proteins. However, such studies could not reveal important facets of ribosome synthesis in vivo. We have systematically investigated the roles of thirty-one r-proteins in the synthesis of Saccharomyces cerevisiae 60S subunits by generating yeast strains conditionally expressing each one of these proteins. In vivo depletion of individual r-proteins caused defects in large subunit assembly. Interestingly, we found that these r-proteins fall into broad phenotypic categories: early (processing of 27SA to 27SB pre-rRNA), middle (conversion of 27SB to 7S and 25.5S pre-rRNAs), and late (final maturation into 5.8S and 25S rRNAs ) steps in pre-rRNA processing. Surprisingly, these classes correlate with location of the proteins in mature ribosomes. We examined in further detail two (rpL7 and rpL8) and three r-proteins (rpL17, rpL35, and rpL37) from the early and middle classes, respectively. In the absence of these proteins, preribosomal particles are largely stable. However, when either rpL7 or rpL8 is depleted, assembly factors required for 27SA3 pre-rRNA processing cannot associate into preribosomes. On the other hand, key assembly factors required for cleavage at the C2 site fail to join preribosomes lacking rpL17, rpL35, or rpL37.
Keywords: ribosome assembly, ribosomal proteins, pre-rRNA processing
Abstract:
Truncated mRNAs missing a stop codon cause synthesis of defective proteins and trap ribosomes at the end of their incomplete message. The stalled ribosome causes 2 major problems: first, a non-functional protein, which can be toxic for the cell; second, depletion of the pool of ribosomes available for translation. In bacteria, these problems can be resolved by a rescue process called trans-translation, which employs a noncoding RNA and its cofactor SmpB. Previous studies showed the importance of tmRNA during gene expression and for virulence in many pathogenic bacteria, making it an attractive target for antimicrobial drug discovery. However, key aspects of this mechanism including how the tmRNA-SmpB complex enters into and interacts with stalled ribosomes are not fully understood.
We are developing a Single Molecule Fluorescence Resonance Energy Transfer (smFRET) assay to investigate how this complex identifies and binds the stalled ribosome. We have labeled P-site phe-tRNA with a FRET donor and the 5’ end of the tmRNA open reading frame with a FRET acceptor. Our preliminary data show multiple FRET states indicating that tmRNA binding and accommodation into the ribosome. We are also labeling SmpB to further characterize the tmRNA trans-translation mechanism.
Keywords: tmRNA, SmpB, Single molecule
Abstract:
Recent transcriptome analyses have indicated that a large part of mammalian genomes are transcribed into long non-protein-coding RNAs (lncRNAs). However, only a very small fraction of them have been individually studied and whether the majority of predicted lncRNAs obtained in large-scale studies have a cellular role is debated. To gain insight into the sequence features and genomic architecture of the subset of lncRNAs which have been proven to be functional, we created a database containing studied lncRNAs manually culled from the literature along with a parallel database containing all annotated protein-coding human RNAs. The Functional lncRNA Database, which contains 204 lncRNAs and their splicing variants, is available at valadkhanlab.org/database. Analysis of the lncRNAs and their comparison to protein-coding transcripts revealed sequence features including paucity of introns and low GC content in lncRNAs which could explain several biological characteristics of these transcripts, such as their nuclear localization and low expression level. The predicted ORFs in lncRNAs have poor start codon and ORF contexts which would lead to activation of the nonsense-mediated decay pathways and thus make it unlikely for most lncRNAs to code for even short peptides.Interestingly, our analyses revealed significant similarities between the lncRNAs and the 3' untranslated regions (3'UTRs) in protein-coding RNAs in structural features and sequence composition. The presence of these intriguing parallels between the lncRNAs and 3'UTRs, which constitute the two main components of the RNA-mediated cellular regulatory system, indicate that highly similar evolutionary constraints govern the function of regulatory RNA sequences in the cell.
References:
Birney, E. et al. 2007. Identification and analysis of functional elements in 1% of the human genome by
the ENCODE pilot project. Nature 447: 799-816.
Keywords: lncRNA, long noncoding RNAs, 3'UTR
Abstract:
A major focus of our laboratory is to identify novel mechanisms of regulating gene expression with the ultimate goal of developing new approaches to understand and treat disease. Recent large-scale studies of the human and mouse genomes have revealed that although there are approximately 22,000 protein-coding genes in human and in mouse, significantly larger portions of both genomes are transcribed. Such analyses suggest that protein-coding genes alone are not sufficient to account for the complexity of higher eukaryotic organisms. It is estimated that a significant portion of the transcriptional output of the human genome represents RNA that does not encode protein and therefore either these non-coding RNAs are largely useless transcripts or they are fulfilling a wide range of unexpected functions in eukaryotic biology. We are focusing our efforts on a class of non-coding RNAs that are large (>1 kb) and retained in the nuclei of cells. We suggest that within this class of large non-coding RNAs will be found a diverse group of key regulatory molecules that will provide significant insight into basic cellular functions, developmental regulation, and disease. Such non-coding RNAs may play a much more crucial role in developmental and pathological processes than currently anticipated. I will discuss our recent progress and unexpected findings characterizing two nuclear-retained long ncRNAs.
Keywords: long noncoding RNA, gene expression, nuclear organization
Abstract:
Aminoacyl tRNA-synthetases (aaRSs) are essential for aminacylating tRNAs and thereby providing substrates for ribosomal translation. Several aaRS paralogs of these highly conserved enzymes have been identified across all domains of life and their roles often fall outside tRNA aminoacylation for protein synthesis. Bacterial poxA, which encodes a paralog of type II lysyl tRNA-synthetase (LysRS), is often located near two other genes yjek and efp, encoding a lysine aminomutase and a poorly understood translation factor (EF-P) respectively. These three genes demonstrate strong phenotypic pleiotropy in Salmonella and appear to be needed for efficient translation of a subset of messages during virulence and stress adaptation. Here we show PoxA efficiently modifies EF-P with (R)-beta-lysine, the product of YjeK, by mimicking the activity of LysRS. The modified form of EF-P is able to stimulate peptide bound synthesis on the ribosome suggesting the PoxA/YjeK/EF-P (PYE) pathway regulates bacterial gene expression directly at the level of the ribosomal translation machinery.
Keywords: tRNA synthetase, translation factor EF-P, posttranslational modification
Abstract not available online - please check the printed booklet.
Abstract:
Trypanosomes perform RNA editing, one of the most bizarre biochemical processes, in which the transcripts of mitochondrial genes are made translatable after extensive site-specific insertion and deletion of uridines. Editing in turn also creates uridine-rich mRNAs, whereby, in order to restore open-reading frames, poly-uridines often occur within coding regions. A potential problem of translation accuracy then makes itself apparent following well-documented cases in other eukaryotes, where multiple consecutive uridines in mRNA can lead to -1 ribosomal frameshifting; creating nonfunctional polypeptides. Since the codon UUU codes for phenylalanine, correct reading frame maintenance at repeated phenylalanine codons requires the presence of the hyper-modified nucleotide wybutosine 3’ of the anticodon (position 37) of phenylalanyl tRNA. In its most common form seen in eukaryotes, wybutosine is the product of five highly conserved wybutosine-synthesizing enzymes that use a guanosine (G37) precursor.
We have hypothesized that in systems that undergo massive mRNA editing, such as the trypanosome mitochondria, similar mechanisms must exist to prevent frameshifting and ensure fidelity during translation. However, wybutosine has not been reported in the mitochondrial tRNAs of any organism. In the present work, we show that mitochondrial tRNAPhe undergoes unusual modifications at position 37 and in addition nearly all of the wybutosine-synthesizing machinery has been duplicated in the trypanosome mitochondria, suggesting a potential role in reading frame maintenance and lending credence to our proposed hypothesis.
References:
de Crecy-Lagard, V., et al.
Keywords: tRNA, Wybutosine, Trypanosome
Abstract not available online - please check the printed booklet.
Abstract:
Pre-tRNA splicing is an essential process in eukaryotic cells. In yeast, the tRNA splicing machinery is composed of three essential enzymes: the heterotetrameric tRNA splicing endonuclease (SEN complex), the tRNA ligase, and the 2'-phosphotransferase. While the SEN complex is conserved from yeast to vertebrates, the subcellular location where pre-tRNA splicing occurs is not conserved. In yeast, the SEN complex is located at the cytoplasmic surface of mitochondria and pre-tRNA splicing occurs in the cytoplasm whereas in vertebrates pre-tRNA splicing is nuclear. To understand why yeast pre-tRNA splicing occurs in the cytoplasm, we engineered yeast that express nuclear tRNA splicing machinery and investigated whether it can functionally substitute pre-tRNA splicing at mitochondria. We demonstrate that all three steps of pre-tRNA splicing and nuclear export of spliced tRNAs to the cytoplasm occur efficiently when endonucleolytic cleavage of introns occurs in the nucleus. However, pre-tRNA splicing in the nucleus fails to complement the growth defects of cells with defective mitochondrially-located SEN complex. The data support the hypothesis that the pre-tRNA splicing machinery in yeast surprisingly serves at least two essential functions, one of which is unrelated to pre-tRNA splicing and occurs in the cytoplasm/mitochondrial surface. Initial studies reveal that yeast tRNA splicing endonuclease subunits influence pre-ribosomal RNA processing.
Keywords: tRNA processing, rRNA processing, tRNA ligase
Abstract not available online - please check the printed booklet.
Abstract:
In the fission yeast Schizosaccharomyces pombe, polyadenylation signals utilized for productive expression of meiotic transcripts during sexual differentiation are bypassed in proliferating cells (1). For the early meiotic crs1 (cyclin regulated by splicing) gene, a bipartite 3' regulatory signal triggers destruction of the nascent transcript (2). Here, we describe a distinct regulatory mechanism for the rem1 gene, which encodes a meiosis-specific cyclin with a later expression window. Proper temporal regulation of rem1 requires positive and negative control of both splicing and 3' RNA processing, which are governed by distinct sequences located upstream from the transcription start site. Although 3' maturation and splicing peak at the same time during meiosis, polyadenylation is still restricted to meiosis when the intron is deleted and is not blocked in mitotic cells by inactivating the 5' splice site. Consistent with the presence of multiple forkhead transcription factor recognition motifs in the 5' control regions, inhibition of rem1 mRNA production in mitotic cells is partially reversed by over-expressing the mei4+ gene, encoding a forkhead protein normally produced only during meiosis, or by deleting fkh2+, encoding a constitutive family member. However, both genetic perturbations are necessary to fully overcome negative control and accumulate rem1 mRNA at levels comparable to those in meiotic cells. These findings, in conjunction with ChIP data demonstrating a nearly identical distribution of Fkh2p and Mei4p across the entire rem1 locus in meiotic cells, support a model in which the two forkhead factors collaborate to promote RNA processing during meiosis. The expression patterns for other middle meiotic genes, including one that lacks introns, are similar to that of rem1, setting the stage for genome-wide studies to comprehensively identify members of the forkhead regulon.
References:
1. Cremona, N., Potter, K. and Wise, J. A. (2011) A meiotic gene regulatory cascade driven by alternative fates for newly synthesized transcripts. Molec. Biol. of the Cell 22, 66-77.
2. McPheeters, D.S., Cremona, N., Sunder, S., Averbeck, N., Chen, H.-M., Leatherwood, J. and Wise, J. A. (2009) A Complex Gene Regulatory Mechanism that Operates at the Nexus of Multiple RNA Processing Decisions. Nat. Struct. Molec. Biol. 16, 255-263.
Keywords: Polyadenylation, Splicing, Transcription factor
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
In mammalian genomes, majority of the genes (92%) contain introns. The average length of human introns is about 5.5kb, although human genome has more than 1200 introns larger than 100 kb and there are 9 introns that are larger than 500 kb in length. Large introns face a unique set of problems during splicing. For example, owing to their size, 5’ and 3’ splice sites might be far from each other spatially; the recognition of correct splice sites from the cryptic ones, since there are a number of cryptic exonic sequences present inside the large introns; and initial stability of 100s of kb long pre mRNA until it is spliced out. A number of potential mechanisms have been proposed to explain large intron splicing such as Recursive splicing which has been demonstrated in Drosophila, intra-splicing, intron skipping by RNA polymerase II or normal splicing. In this study, we used a number of different assays including use of transcription and splicing inhibitors, absolute quantitation of intronic sequences in the large introns, and shRNA dependent down-regulation of intron lariat debranching enzyme DBR1 to investigate the large intron splicing in human cells. Here we present evidence that the large introns are spliced in a normal fashion as their smaller counterparts in human cells and not by any other method as previously hypothesized.
Keywords: splicing, large introns
Abstract not available online - please check the printed booklet.
Abstract:
microRNA (miRNA) mediated gene regulation is an evolutionarily conserved cellular RNA interference (RNAi) pathway wherein cellular non-(protein)coding RNAs ~22-nucleotide in length, associated with a cohort of proteins, bind to partially complementary regions on messenger RNA (mRNA) targets to repress protein expression. While standard ensemble assays, including intracellular fluorescence microscopy, have revealed a wealth of information on miRNA function and intracellular localization, the mechanism of miRNA mediated mRNA repression is still under intense debate. We have developed a novel method to investigate miRNA activity inside living (and fixed) human cells using single particle tracking and single molecule fluorescence microscopy with 30 nm spatial accuracy and 100 ms time resolution. Upon microinjection into the cytoplasm of HeLa cells, fluorophore labeled repression competent mature miRNAs were visualized as distinct diffraction limited particles freely diffusing with multifarious diffusive behaviors. More specifically, two major populations particles bearing diffusion coefficients (0.26 µm2/s and 0.034 µm2/s) closely resembling those of messenger RNPs and processing bodies were obtained. The number of miRNA molecules per fluorescent particle was then quantified using the stochastic photobleaching property of fluorescent probes in formaldehyde fixed cells. A majority of miRNA particles contained single miRNA molecules, however, a significant proportion of spots had more than one molecule (up to seven). Time dependent changes in miRNA diffusion and distribution were also observed in live and fixed cells respectively, suggesting a miRNA multiple turnover model. These results demonstrate the ability of our method to unravel the dynamic nature of an important gene regulatory pathway, and the potential for it to be extended to many other small, non-coding RNAs.
Keywords: microRNA, Single particle tracking microscopy, Single molecule fluorescence microscopy
Abstract:
MDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. Recently, MDM4 mRNA was also observed to be down-regulated in response to DNA damage, contributing to p53 activation. Here we report that MDM4 mRNA is a target of hsa-mir-34a. MDM4 mRNA contains a lengthy 3’ untranslated region; however, we find that it is a miR-34a site in the ORF of the last exon that is responsible for the repression. Overexpression of miR34a, but not a mutant miR34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR34a target genes. Likewise, MDM4 protein levels are decreased by miR34a overexpression, independent of HDM2 activity. A luciferase reporter gene fused to a portion of exon 11 of MDM4 containing a miR34a site is sufficient to decrease reporter expression, and is inhibited by additional expression of exogenous mir34a. These data establish a mechanism for the recently observed negative regulation of MDM4 and provide a means to manipulate MDM4 expression without introducing DNA damage.
Keywords: MDM4, microRNA, p53
Abstract not available online - please check the printed booklet.
