The spliceosome: a flexible, reversible macromolecular machine

doi:10.1016/j.tibs.2012.02.009

Trends in Biochemical Sciences

Volume 37, Issue 5, May 2012, Pages 179-188

https://doi.org/10.1016/j.tibs.2012.02.009 Get rights and content

With more than a hundred individual RNA and protein parts and a highly dynamic assembly and disassembly pathway, the spliceosome is arguably the most complicated macromolecular machine in the eukaryotic cell. This complexity has made kinetic and mechanistic analysis of splicing incredibly challenging. Yet, recent technological advances are now providing tools for understanding this process in much greater detail. Ranging from genome-wide analyses of splicing and creation of an orthogonal spliceosome in vivo, to purification of active spliceosomes and observation of single molecules in vitro, such new experimental approaches are yielding significant insight into the inner workings of this remarkable machine. These experiments are rewriting the textbooks, with a new picture emerging of a dynamic, malleable machine heavily influenced by the identity of its pre-mRNA substrate.

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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Trends in Biochemical Sciences

ReviewThe spliceosome: a flexible, reversible macromolecular machine

Section snippets

The spliceosome snips and stitches RNAs

Purified spliceosomes and reaction reversibility

Probing the active site of an orthogonal spliceosome

Dynamics of single spliceosomes

Microarrays and the importance of pre-mRNA identity

Concluding remarks

Acknowledgments

Glossary

Cell

Mol. Cell

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Biophys. J.

Cell

J. Biol. Chem.

Methods Enzymol.

Mol. Cell

Mol. Cell

Mol. Cell

Mol. Cell

J. Mol. Biol.

Mol. Cell

Mol. Cell

Cell

Expansion of the eukaryotic proteome by alternative splicing

Nature

Guide to the Human Genome

A computational analysis of sequence features involved in recognition of short introns

Proc. Natl. Acad. Sci. U.S.A.

Recognition of atypical 5′ splice sites by shifted base-pairing to U1 snRNA

Nat. Struct. Mol. Biol.

The spliceosome: the most complex macromolecular machine in the cell?

Bioessays

Where in the cell is the minor spliceosome?

Proc. Natl. Acad. Sci. U.S.A.

Cross-talk in transcription, splicing and chromatin: who makes the first call?

Biochem. Soc. Trans.

Review
The spliceosome: a flexible, reversible macromolecular machine