New insights into the spliceosome by single molecule
fluorescence microscopy
Aaron A Hoskins
1,2,*
, Jeff Gelles
3
and Melissa J Moore
1,2
Splicing is an essential eukaryotic process in which introns are
excised from precursors to messenger RNAs and exons ligated
together. This reaction is catalyzed by a multi-MegaDalton
machine called the spliceosome, composed of 5 small nuclear
RNAs (snRNAs) and a core set of $100 proteins minimally
required for activity. Because of the spliceosome’s size, its low
abundance in cellular extracts, and its highly dynamic
assembly pathway, analysis of the kinetics of splicing and the
conformational rearrangements occurring during spliceosome
assembly and disassembly has proven extraordinarily
challenging. Here, we review recent progress in combining
chemical biology methodologies with single molecule
fluorescence techniques to provide a window into splicing in
real time. These methods complement ensemble
measurements of splicing in vivo and in vitro to facilitate kinetic
dissection of pre-mRNA splicing.
Addresses
1
Department of Biochemistry and Molecular Pharmacology, University
of Massachusetts Medical School, 364 Plantation St., Worcester, MA
01605, USA
2
Howard Hughes Medical Institute, USA
3
Department of Biochemistry, Brandeis University, 415 South St.,
Waltham, MA 02453, USA
Corresponding authors: Hoskins, Aaron A (ahoskins@wisc.edu), Gelles,
Jeff (gelles@brandeis.edu) and Moore, Melissa J
(melissa.moore@umassmed.edu)
*
Present address: Department of Biochemistry, University of Wisconsin-
Madison, 433 Babcock Dr., Madison, WI 53706, USA.
Current Opinion in Chemical Biology 2011, 15:864–870
This review comes from a themed issue on
Molecular Imaging
Edited by Alanna Schepartz and Ruben L Gonzalez Jr.
Available online 5th November 2011
1367-5931/$ – see front matter
# 2011 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2011.10.010
Introduction
Removal of introns from nascent RNA transcripts (pre-
cursors to messenger RNAs or pre-mRNAs) is an essen-
tial step in eukaryotic gene expression. The enzyme
responsible for this process is the spliceosome, which
carries out intron excision via two energy-neutral trans-
esterification reactions: lariat intron formation and exon
ligation. Despite the straightforward nature of the chem-
istry, the spliceosome itself is an extraordinarily complex,
2–3 MDa machine composed of 5 uridine-rich small
nuclear RNAs (U1, U2, U4, U5 and U6 snRNAs) and
anywhere from 90 to >300 proteins [1,2]. The snRNAs
and a subset of the proteins form stable particles called
small nuclear ribonucleoproteins(snRNPs)thatconsti-
tute the largest building blocks of the spliceosome. Aided
by a plethora of more loosely associated proteins (‘spli-
cing factors’), the snRNPs interact with one another and
the pre-mRNA to complete each round of splicing via an
extraordinarily dynamic process. The current model for
spliceosome assembly involves step-wise association of
first U1, then U2, followed by U4/U6.U5 tri-snRNP and
the multi-protein Prp19 complex (NTC). Once all the
major pieces are in place, additional structural rearrange-
ments lead to U1 and U4 expulsion, catalytic activation,
lariat formation, exon ligation, spliced product release
and finally dissociation of the remaining components
(Figure 1). Amazingly, this entire sequence is believed
to occur anew on every intron in each pre-mRNA mol-
ecule, rendering each assembled and catalytically acti-
vated spliceosome a single-turnover enzyme.
As has been recently reviewed [3,4,5
,6], our current
understanding of spliceosome assembly is based largely
on the procession of stable complexes that form upon
addition of a simplified splicing substrate (i.e. two short
exons separated by an efficiently spliced intron) to an in
vitro splicing reaction. Spliceosomal components are
most commonly provided as an Saccharomyces cerevisiae
whole cell extract (WCE) or as a mammalian nuclear cell
extract (NCE). Stable complexes are resolved by native
polyacrylamide gel electrophoresis (PAGE) or by
density gradient centrifugation and can be purified by
affinity chromatography. In some cases, such purified
complexes retain the ability to carry out an individual
step in the overall process (see, e.g. [7,8]). Combined
with more than two decades of intensive genetic dis-
section of the yeast spliceosome, these biochemical
studies have yielded tremendous insight into the overall
composition, structure and operation of the spliceso-
some. Nonetheless, what was missing until recently
was any detailed kinetic information about the comings
and goings of individual components and related struc-
tural transitions.
At any single moment in a splicing reaction, different
spliceosomes are in different states and doing different
things. Averaging of these states and behaviors across an
entire population of molecules leads to significant loss of
information about molecular dynamics. A means around
this is to monitor the behavior of each spliceosome
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