Trends in Biochemical Sciences
ReviewThe spliceosome: a flexible, reversible macromolecular machine
Section snippets
The spliceosome snips and stitches RNAs
In eukaryotes, many genes are transcribed as precursors to messenger RNAs (pre-mRNAs) containing both introns and exons (see Glossary). To generate mature mRNAs, the introns must be snipped out and the exons stitched back together by the molecular tailor of the cell: the spliceosome. A fashion designer as well, the spliceosome can alter its snipping and stitching to produce many different mRNAs from a single bolt of pre-mRNA cloth. Such alternative splicing is particularly prevalent in
Purified spliceosomes and reaction reversibility
Many biochemical analyses require well-characterized enzymes and controlled conditions, making ‘purification’ one of the ‘Ten Commandments’ of enzymology [16]. With such a plethora of components to contend with, obeying this commandment was a seemingly insurmountable hurdle for spliceosome biochemists in the past. Recently, however, advances in affinity purification have made purifying active spliceosomes a reality, and several groups are now employing these methods to great effect.
The Lührmann
Probing the active site of an orthogonal spliceosome
The observation that spliceosomes can be toggled between two different catalytic states by simply varying the salt concentration suggests an extremely flexible active site. Query and coworkers have now developed a system wherein the extent of this active site flexibility can be probed in vivo using an orthogonal yeast spliceosome [36]. To construct their orthogonal spliceosome, the authors generated a second copy of U2 snRNA in yeast containing a highly mutated branch site binding region (e.g.
Dynamics of single spliceosomes
In the experiments described above, yeast genetics and in vitro splicing assays provided evidence for a conformationally flexible spliceosome with at least three reversible steps: 5′ SS cleavage, exon ligation and discard by Prp16 during 5′ SS cleavage. None of these methods, however, is capable of providing quantitative kinetic or thermodynamic information about any discrete step in the splicing reaction. What was needed were time-resolved in vitro assays that could follow the dynamics of
Microarrays and the importance of pre-mRNA identity
Most of the experiments described above were carried out using one to three model pre-mRNAs that splice well both in vivo and in vitro: RP51A (containing a shortened form of the intron in ribosomal protein gene RPS17A), ACT1 (containing the full-length intron from the actin gene) and UBC4 (a very short full-length intron in a ubiquitin conjugating enzyme gene). However, given the intimate roles of the pre-mRNA in binding to proteins and snRNAs and providing the reactive nucleophiles for
Concluding remarks
Far from being the series of static complexes connected by one-way arrows as often appears in textbook pictures of the spliceosome cycle, we now know that the spliceosome is an extraordinarily dynamic and flexible machine. As described above, new technologies are providing ample evidence for reversible interactions and chemistry during splicing, as well as a highly flexible active site heavily influenced by the pre-mRNA substrate. As has been previously postulated 35, 37, 45, these results
Acknowledgments
This work was supported by National Institutes of Health awards R00-GM086471 (A.A.H.) and R01-GM053007 (M.J.M). M.J.M. is a Howard Hughes Medical Institute investigator.
Glossary
- Alternative splicing
- the process by which the spliceosome can generate multiple mRNA isoforms from a single pre-mRNA. This is a fundamental mechanism for encoding genomic complexity in higher eukaryotes without an expansion in gene number.
- Branch point
- the nucleotide (usually an adenosine) in the branch site sequence that provides the nucleophile for 5′ SS cleavage and becomes the site of lariat formation. In yeast, the branch point is the 3′-most A (underlined) in the UACUAAC branch site
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