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Eleanor Eustis Farrington Chair of Cancer Research
Professor, RNA Therapeutics Institutes (RTI)

Eukaryotic RNA Processing and Metabolism

melissa moore

Melissa Moore’s work encompasses a broad array of topics involved in post-transcriptional gene regulation in eukaryotes via mechanisms involving RNA.

Our research currently focuses on three distinct but interconnected areas involving the basic mechanisms of eukaryotic gene expression: (1) the structure and mechanism of the spliceosome, (2) the effects of nuclear-acquired proteins on cytoplasmic messenger RNA (mRNA) metabolism, and (3) the fate of functionally defective ribosomal RNAs (rRNAs) and mRNAs.
Introns are incoherent strings of nucleotides that interrupt the coding regions of genes. They are removed from nascent RNA transcripts by the process of precursor mRNA (pre- mRNA) splicing.

Since the majority of genes in multicellular organisms contain introns, their timely and precise removal is essential for proper gene expression. Most introns are excised by the major spliceosome, a complex macromolecular machine containing five stable, small nuclear RNAs (snRNAs) and a multitude of proteins. The spliceosome must be at once precise (e.g., a 1-nucleotide shift in a splice site will throw the protein-coding region completely out of frame) and adaptable (in humans it must recognize >10^5 different splice site pairs in diverse sequence contexts). In metazoans, the recognition problem is compounded by poor conservation of the sequences defining splice sites and the presence of multiple introns per pre-mRNA. Also, a remarkably high percentage of metazoan pre-mRNAs are subject to alternative splicing, which greatly expands the repertoire of proteins that can be expressed from relatively small genomes.

A major goal of our research is to elucidate the basic mechanisms by which mammalian spliceosomes accurately identify splice sites in pre-mRNAs and then catalyze intron excision. For some time, our primary focus has been the second step of splicing, wherein the intron is excised and the expressed regions (or exons) are ligated together. Recently we succeeded in purifying, in their native state, spliceosomes poised to perform this reaction. Mass spectrometry revealed more than 100 polypeptides associated with this structure. Using techniques for single-particle image reconstruction from electron micrographs, we obtained an initial three-dimensional structural map to ~30-Å resolution. The structure, with dimensions ~240 x 270 Å, exhibits three major domains connected via a series of bridges and tunnels. Further structural analysis is under way. (This work has been carried out in collaboration with Nikolaus Grigorieff [HHMI, Brandeis University].)

On the mechanistic front, we have recently developed methodologies for following pre- mRNA splicing at the single-molecule level. All previous in vitro mechanistic studies of splicing have utilized ensemble assays that report only the average behavior of a population. Although such bulk assays have provided a wealth of mechanistic insight, they are ultimately limited in their ability to tease out finer mechanistic details. Over the past two decades, single-molecule techniques that complement ensemble measurements have emerged as powerful tools to elucidate the enzyme mechanism. These approaches permit observation of the stochastic behavior of individual binding and catalytic events. They also allow observation of many individual events that would otherwise go undetected.

Using a pre-mRNA attached to a glass surface via end and containing fluorescent labels in the 5' exon and intron, we are now able to observe individual splicing events in Saccharomyces cerevisiae extracts, using a multiwavelength total internal reflection fluorescence (TIRF) microscope system developed by Jeff Gelles (Brandeis University) and his colleagues. Chemical biology tools are being used to fluorescently label core spliceosomal proteins and snRNAs, as well as a number of transiently associating splicing factors. This system allows us to analyze the dynamic characteristics of individual spliceosomes in real time. It should provide a new window into previously unaddressable questions regarding spliceosome assembly and internal structural transitions, as well as the comings and goings of key splicing factors. (All of the spliceosome structure and mechanism work is supported by a grant from the National Institutes of Health.)

Structure and Assembly of the Exon Junction Complex

In addition to removing introns, the process of pre-mRNA splicing has significant consequences for the subsequent metabolism of the product mRNA. That is, mRNAs produced by splicing are subject to different subcellular localization, different

efficiencies of translation into proteins, and different decay rates than otherwise identical mRNAs produced from intronless genes. Splicing affects downstream mRNA metabolism by altering the complement of proteins that associate with the mRNA to form an mRNP (mRNA ribonucleoprotein particle). Several years ago, in collaboration with Lynne Maquat (University of Rochester) and Elisa Izaurralde (European Molecular Biology Laboratory, Heidelberg), we showed that spliceosomes stably deposit a complex of proteins (the EJC) on mRNAs at a conserved position 20–24 nucleotides upstream of exon-exon junctions. Such EJCs accompany spliced mRNAs to the cytoplasm, where they are ultimately displaced by the process of translation.

A major unresolved question regarding the EJC had been how this complex manages to bind so tightly to a specific position on mRNA in what seems to be an entirely RNA structure- and sequence-independent fashion. We solved this mystery by identifying eIF4AIII as the EJC anchor. A member of the DEAD-box family of RNA helicases, eIF4AIII represents a new functional class of such proteins that act as RNA "placeholders" or "clothespins" rather than RNA translocases. Such place-holding DEAD-box proteins could serve as a general means for attaching factors that add functionality to an RNP without requiring any special consensus sequences in the RNA.

Functional Consequences of EJC Deposition

As stated above, spliced mRNAs exhibit metabolic fates different from the metabolic fates of mRNAs not produced by splicing. We have been investigating to what extent and by what mechanism(s) EJC deposition contributes to these differences. One area of investigation is the efficiency by which mRNAs are utilized as templates for making proteins. Quantitative analysis revealed that two to three times as many protein molecules are made per spliced mRNA molecule than per identical mRNA molecules not made by splicing. Polysome analysis revealed that spliced mRNAs interact more efficiently with ribosomes, the macromolecular machines that use mRNAs as the blueprints to synthesize proteins, than do unspliced mRNAs. This effect may facilitate the rapid expression of newly made mRNAs by enabling them to outcompete translationally experienced mRNAs (that no longer carry EJCs) for limiting translation initiation factors.

Recently, in collaboration with Gina Turrigiano (Brandeis University) and Christopher Burge (Massachusetts Institute of Technology), we found that eIF4AIII remains associated with dendritically localized mRNAs in mammalian neurons. eIF4AIII knockdown up-regulates at least two proteins involved in postsynaptic function and markedly increases synaptic strength. Thus eIF4AIII appears to act as a key brake on expression of proteins required for synaptic function. One mechanism for this braking action is via the translation-dependent decay of Arc mRNA, the gene for which contains two conserved introns in its 3'-untranslated region (3'-UTR). This is a highly unusual gene structure in mammals, as EJCs downstream of stop codons trigger nonsense- mediated mRNA decay (NMD). A bioinformatics approach revealed 148 other mammalian genes with this same feature, suggesting that translation-dependent mRNA decay mechanisms such as NMD might be widely employed in mammalian cells as a means to limit the amount of protein produced from certain mRNAs. Curiously, a large number of these genes are expressed in hematopoietic cells, suggesting that some feature of blood cells may particularly favor their evolution there. Future experiments will probe the role of the EJC in modulating expression from some of these new 3'-UTR intron- containing genes.

Clearance of Nonfunctional Ribosomes

The ribosome is the most abundant macromolecular machine in the cell. Its highly complex structure, composed of both ribosomal RNAs (rRNAs) and proteins, necessitates an intricate assembly mechanism in which pre-rRNA processing and nucleotide modification are coupled with chaperone-assisted rRNA folding and protein association.

Although the mechanics of this assembly process are becoming increasingly understood, surprisingly little is known about the mechanisms assuring its overall fidelity. Furthermore, given their inordinately long half-lives in eukaryotic cells, it is to be expected that some ribosomes will become nonfunctional over time as they accumulate oxidative damage due to normal cellular metabolism. We therefore wondered whether eukaryotes might possess any mechanisms for eliminating ribosomes that are fully assembled but functionally defective, akin to their abilities to eliminate mRNAs that are fully processed but defective. To test this, we introduced point mutations into the peptidyltransferase center of 25S rRNA and the decoding center of 18S rRNA in S. cerevisiae. These mutant rRNAs are assembled into ribosomes, but they display markedly decreased steady-state levels compared to wild-type rRNAs.

Preliminary analyses of knockout strains have revealed several candidate genes important for decreased expression of the translationally defective mutant rRNAs. Our results therefore indicate that budding yeast do contain a quality control system capable of recognizing and eliminating translationally deficient ribosomes so as to prevent their interference with normal cellular function. We continue to study the trans-acting factors and molecular mechanisms involved in this process.

Rotation Projects

Our laboratory combines biochemical, biophysical, molecular and cell biological approaches to investigate various aspects of pre-mRNA processing, mRNA metabolism and RNA quality control in eukaryotic cells. Potential rotation projects are available in all areas of the laboratory's interests (see Research section).

One or more keywords matched the following items that are connected to Moore, Melissa

Item TypeName
Academic Article Nuclear RNA turnover.
Academic Article Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.
Academic Article RNA events. No end to nonsense.
Academic Article Capturing splicing complexes to study structure and mechanism.
Academic Article A quantitative analysis of intron effects on mammalian gene expression.
Academic Article How introns influence and enhance eukaryotic gene expression.
Academic Article Splicing enhances translation in mammalian cells: an additional function of the exon junction complex.
Academic Article The ever-increasing complexities of the exon junction complex.
Academic Article Effects of the U1C L13 mutation and temperature regulation of yeast commitment complex formation.
Academic Article Association of ribosomal proteins with nascent transcripts in S. cerevisiae.
Academic Article Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core.
Academic Article Spliceosome assembly and composition.
Academic Article The immediate early gene arc/arg3.1: regulation, mechanisms, and function.
Academic Article Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes.
Academic Article New insights into the spliceosome by single molecule fluorescence microscopy.
Academic Article The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing.
Academic Article The spliceosome: a flexible, reversible macromolecular machine.
Academic Article Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling.
Academic Article Introns in UTRs: why we should stop ignoring them.
Academic Article 5' exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly.
Academic Article An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes.
Academic Article A long noncoding RNA mediates both activation and repression of immune response genes.
Academic Article Pre-mRNA splicing: awash in a sea of proteins.
Academic Article Three-dimensional structure of C complex spliceosomes by electron microscopy.
Academic Article eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay.
Academic Article From birth to death: the complex lives of eukaryotic mRNAs.
Academic Article Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay.
Academic Article The three-dimensional arcitecture of the EJC core.
Academic Article A late-acting quality control process for mature eukaryotic rRNAs.
Academic Article The nuclear nurture and cytoplasmic nature of localized mRNPs.
Academic Article The EJC factor eIF4AIII modulates synaptic strength and neuronal protein expression.
Academic Article Visualizing the splicing of single pre-mRNA molecules in whole cell extract.
Academic Article Interactions between eIF4AI and its accessory factors eIF4B and eIF4H.
Academic Article The biflavonoid isoginkgetin is a general inhibitor of Pre-mRNA splicing.
Academic Article Pre-mRNA processing reaches back to transcription and ahead to translation.
Academic Article A convergence of rRNA and mRNA quality control pathways revealed by mechanistic analysis of nonfunctional rRNA decay.
Academic Article Meayamycin inhibits pre-messenger RNA splicing and exhibits picomolar activity against multidrug-resistant cells.
Academic Article RNA metabolism and the pathogenesis of motor neuron diseases.
Academic Article Ordered and dynamic assembly of single spliceosomes.
Academic Article Genome analysis reveals interplay between 5'UTR introns and nuclear mRNA export for secretory and mitochondrial genes.
Academic Article The cellular EJC interactome reveals higher-order mRNP structure and an EJC-SR protein nexus.
Academic Article Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site.
Academic Article Defining piRNA primary transcripts.
Concept RNA
Concept RNA, Small Nuclear
Concept RNA, Small Interfering
Concept RNA, Ribosomal
Concept Sequence Analysis, RNA
Concept RNA Precursors
Concept RNA Interference
Concept RNA, Nuclear
Concept RNA Editing
Concept RNA, Catalytic
Concept RNA, Guide
Concept RNA-Induced Silencing Complex
Concept RNA, Ribosomal, 18S
Concept RNA, Fungal
Concept RNA, Untranslated
Concept RNA Helicases
Concept RNA Stability
Concept RNA Splice Sites
Concept RNA, Messenger
Concept RNA, Transfer, Gly
Concept RNA Processing, Post-Transcriptional
Concept DEAD-box RNA Helicases
Concept RNA Polymerase II
Concept Codon, Nonsense
Concept Embryo, Nonmammalian
Concept RNA Splicing
Concept RNA-Binding Proteins
Concept RNA Transport
Academic Article Staufen1 senses overall transcript secondary structure to regulate translation.
Academic Article ASPeak: an abundance sensitive peak detection algorithm for RIP-Seq.
Academic Article Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy.
Academic Article RIPiT-Seq: a high-throughput approach for footprinting RNA:protein complexes.
Academic Article Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes.
Academic Article The spliceosome: disorder and dynamics defined.
Academic Article Post-transcriptional regulation of gene expression in innate immunity.
Academic Article An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments.
Academic Article Spliceosomes.
Academic Article Twenty years of technology.
Academic Article Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation.
Academic Article The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion.
Academic Article Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides.
Academic Article Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.
Academic Article Redefining the Translational Status of 80S Monosomes.
Academic Article Single Molecule Approaches in RNA-Protein Interactions.
Academic Article A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation.
Academic Article A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and N1-methyladenosine modification.
Academic Article ASC1 and RPS3: new actors in 18S nonfunctional rRNA decay.
Academic Article Corrigendum: ASC1 and RPS3: new actors in 18S nonfunctional rRNA decay.
Academic Article mRNA structure regulates protein expression through changes in functional half-life.

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