Please note: Melissa Moore is relocating to the University
of Massachusetts Medical School on September 1, 2007.
More information.
Our research is directed toward understanding the molecular
mechanisms of and interconnections between several processes
involved in the intracellular metabolism of RNA. Areas currently
under investigation include (1) mechanistic and structural
studies of the human spliceosome, (2) the effects of pre-mRNA
splicing on mRNP structure and function, and (3) clearance
of nonfunctional ribosomes.
Mechanistic and Structural Studies of Spliceosomes
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 >105 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-Ĺ resolution. The structure, with dimensions ~240
× 270 Ĺ, exhibits three major domains connected via a series
of bridges and tunnels. Further refinement and subunit mapping
with site-specific labels is now under way. (This work,
done in collaboration with Nikolaus Grigorieff [HHMI, Brandeis
University], is supported by a grant from the National Institutes
of Health.)
Structure and Assembly of the Exon Junction Complex
In addition to removing introns, pre-mRNA splicing has
significant consequences for the subsequent metabolism of
the product messenger RNAs. 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 exon junction complex,
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 removed
by the process of translation.
A major unresolved question had been how the EJC manages
to bind so tightly to a specific position on mRNA in what
seems to be an entirely RNA structure- and sequence-independent
fashion. Two years ago, we identified eIF4AIII as the EJC
anchor. A member of the DEAD-box family of RNA helicases,
eIF4AIII represents a new functional class of 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. More recently, we showed that the
minimal stable EJC core consists of eIF4AIII plus three
interaction partners: MLN51, Y14, and Magoh. A three-dimensional
structure of this tetramer obtained by electron microscopy
represents the first structure of a DEAD-box protein in
complex with its binding partners.
Functional Consequences of EJC Deposition
As stated above, spliced mRNAs exhibit different metabolic
fates than 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. By using mRNAs with a first
exon too short or just long enough to accept an EJC, we
showed that this increased translational yield is due to
EJC deposition. The effect of splicing on translational
yield could also be replicated by tethering one of several
EJC proteins as RNA-binding fusion proteins to a reporter
mRNA.
Polysome analysis revealed that both spliced mRNAs and
mRNAs carrying tethered EJC proteins interact more efficiently
with ribosomes—the macromolecular machines that use mRNAs
as the blueprints to synthesize proteins—than do unspliced
mRNAs. Current experiments are focused on elucidating the
molecular mechanisms by which the EJC and its components
mediate this effect. We are also investigating the potential
role of EJC proteins in the localization and regulated translation
of mRNAs in neuronal dendrites.
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 Saccharomyces 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.
Selected Publications
Anderson K, Moore MJ. (1997) Bimolecular exon ligation
by the human spliceosome. Science 276:1712-6.
[abstract
in PubMed]
Luo HR, Moreau GA, Levin N, Moore MJ. (1999) The human
Prp8 protein is a component of both U2- and U12-dependent
spliceosomes. RNA 5:893-908. [abstract
in PubMed]
Moore MJ, Query CC. (2000) Joining of RNAs by splinted
ligation. Methods Enzymol 317:109-23. [abstract
in PubMed]
Anderson K, Moore MJ. (2000) Bimolecular exon ligation
by the human spliceosome bypasses early 3' splice site AG
recognition and requires NTP hydrolysis. RNA 6:16-25.
[abstract
in PubMed]
Chen S, Anderson K, Moore MJ. (2000) Evidence for a linear
search in bimolecular 3' splice site AG selection. Proc
Natl Acad Sci U S A. 2000 Jan 18;97(2):593-8. [abstract
in PubMed]
Le Hir H, Moore MJ, Maquat LE. (2000) Pre-mRNA splicing
alters mRNP composition: evidence for stable association
of proteins at exon-exon junctions. Genes Dev. 2000 May
1;14(9):1098-108. [abstract
in PubMed]
Le Hir H, Izaurralde E, Maquat LE, Moore MJ. (2000) The
spliceosome deposits multiple proteins 20-24 nucleotides
upstream of mRNA exon-exon junctions. EMBO J 19:6860-6869.
[abstract
in PubMed]
Mühlemann O, Mock-Casagrande CS, Wang J, Li S, Custódio
N, Carmo-Fonseca M, Wilkinson MF and Moore MJ. (2001). Precursor
RNAs harboring nonsense codons accumulate near the site
of transcription. Molecular Cell, 8:33-44.
[abstract
in PubMed]
Le Hir H, Gatfield D, Izaurralde E and Moore MJ. (2001).
The exon-exon junction complex provides a binding platform
for factors involved in mRNA export and NMD. EMBO J
20:4987-97 [abstract
in PubMed]
Moore MJ, Rosbash M. (2001). CELL BIOLOGY: TAPping into
mRNA Export. Science 294:1841-2 [abstract
in PubMed] [Summary]
[Full
Text]
Reichert VL, Le Hir H, Jurica MS, Moore MJ. (2002) 5' exon
interactions within the human spliceosome establish a framework
for exon junction complex structure and assembly. Genes
Dev 16:2778-91 [abstract
in PubMed]
Jurica MS, Licklider LJ, Gygi SR, Grigorieff N, Moore MJ.
(2002) Purification and characterization of native spliceosomes
suitable for three- dimensional structural analysis. RNA
8:426-39. [abstract
in PubMed]
Moore MJ.(2002) Nuclear RNA turnover.Cell 108:431-4.
Review. [abstract
in PubMed]
Moore MJ. (2002) RNA events. No end to nonsense. Science
11;298(5592):370-1 [Full
Text]
Jurica MS, Moore MJ. (2002) Capturing splicing complexes
to study structure and mechanism. Methods. 28:336-45.
[abstract]
Le Hir H, Nott A, Moore MJ. (2003) How introns influence
and enhance eukaryotic gene expression.Trends Biochem
Sci., 28:215-20. [abstract]
Nott A, Meislin SH, Moore MJ. (2003) A quantitative analysis
of intron effects on mammalian gene expression. RNA.
9:607-17. [abstract]
Jurica MS, Moore MJ. (2003) Pre-mRNA splicing: awash in
a sea of proteins. Mol Cell., 12:5-14. [abstract]
Nott A, Le Hir H, Moore MJ. (2004) Splicing enhances translation
in mammalian cells: an additional function of the exon junction
complex. Genes Dev. 18:210-22.
Jurica MS, Sousa D, Moore MJ, Grigorieff N. (2004) Three-dimensional
structure of C complex spliceosomes by electron microscopy.
Nat Struct Mol Biol. 11:265-9. [abstract]
Shibuya T, Tange TO, Sonenberg N, Moore MJ. (2004) eIF4AIII
binds spliced mRNA in the exon junction complex and is essential
for nonsense-mediated decay. Nat Struct Mol Biol.
11:346-51. [abstract]
Tange TO, Nott A, Moore MJ. (2004) The ever-increasing
complexities of the exon junction complex, Curr Opin
Cell Biol. 2004 Jun;16(3):279-84. Review.
Du H, Tardiff DF, Moore MJ, Rosbash M., (2004) Effects
of the U1C L13 mutation and temperature regulation of yeast
commitment complex formation, Proc Natl Acad Sci U
S A. 2004 Oct 12;101(41):14841-6. Epub 2004 Oct 1
Moore MJ., (2005) From birth to death: the complex lives
of eukaryotic mRNAs, Science. 2005 Sep 2;309(5740):1514-8.
Schroder PA, Moore MJ., (2005) Association of ribosomal
proteins with nascent transcripts in S. cerevisiae., RNA.
2005 Oct;11(10):1521-9.
Tange TO, Shibuya T, Jurica MS, Moore MJ., (2005) Biochemical
analysis of the EJC reveals two new factors and a stable
tetrameric protein core. RNA. 2005 Dec;11(12):1869-83.
Shibuya T, Tange TO, Stroupe ME, Moore MJ., (2006) Mutational
analysis of human eIF4AIII identifies regions necessary
for exon junction complex formation and nonsense-mediated
mRNA decay., RNA. 2006 Mar;12(3):360-74.
Stroupe ME, Tange TO, Thomas DR, Moore MJ, Grigorieff N.,
(2006) The three-dimensional arcitecture of the EJC core.
J Mol Biol. 2006 Jul 21;360(4):743-9. Epub 2006 Jun
5.
View Complete Publication List on PubMed:
Melissa Moore
Last reviewed: Jan. 24, 2007. E-mail comments
or questions to the webmaster.
