Trends in Cell Biology
Volume 19, Issue 8, August 2009, Pages 375-384
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Review
The spliceosome: a self-organized macromolecular machine in the nucleus?

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In higher eukaryotes, the vast majority of protein-coding genes contain introns that must be removed from precursor mRNA (pre-mRNA) with great precision by the spliceosome. Spliceosomes are massive RNA–protein macromolecular machines with >100 distinct components that assemble onto nascent transcripts and are released from the mRNA after splicing. A large and longstanding body of biochemical evidence indicates that the spliceosome is constructed de novo for each round of splicing in an ordered piece-by-piece stepwise assembly. More recently, exciting advances in intracellular imaging approaches are providing new clues to understand the dynamic organization of this and other multi-component molecular machines in the nucleus. The emerging data suggest that stochastic interactions between spliceosomal proteins originate intermediate complexes that localize in the close vicinity of active genes, thus contributing to increase the local availability of the individual pieces required for assembly of functional spliceosomes on newly synthesized pre-mRNA.

Introduction

Splicing of precursor mRNAs (pre-mRNAs) is no trivial task. The vast majority of human genes contain introns with an average size of 1000–2000 base pairs, approximately ten times the size of protein-coding exons [1]. Excision of introns with single-nucleotide precision relies on the spliceosome, one of the largest and most elaborate macromolecular machines in the cell [2]. The building blocks of this multi-megadalton entity that sediments as a 60S complex are the U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs), each of which consists of a specific small nuclear RNA (snRNA) and associated proteins. Spliceosomal snRNPs make additional interactions with >100 distinct non-snRNP proteins 3, 4.

In vitro, transcripts generated by RNA polymerase II are spliced within 15–60 min [5], whereas the average in vivo excision time for an intron seems to be <3 mins 6, 7. Accurate spliceosome assembly on pre-mRNA is thus much more efficient in the living cell nucleus than in nuclear extracts. However, precisely how spliceosomal components are efficiently recruited and assembled onto nascent transcripts in living cells remains unclear and subject to debate. In this review, we discuss how recent applications of advanced fluorescence microscopy technologies are contributing new clues to understanding the dynamic organization of spliceosome assembly in the nucleus.

Section snippets

Current models for spliceosome assembly

Over two decades of in vitro studies in yeast, invertebrate and mammalian systems support the textbook view that spliceosome assembly follows a carefully orchestrated stepwise pathway, building anew on each pre-mRNA substrate as each new intron emerges from the transcriptional machinery by the ordered, stepwise assembly of discrete snRNP particles and proteins, and then disassembling before the next round of splicing 8, 9, 10.

According to the classical sequential assembly model (Figure 1), the

FRET microscopy reveals spliceosomal assembly intermediates in the nucleus

Spliceosomal components are not distributed homogeneously throughout the mammalian cell nucleus. Microscopy assays have shown that the vast majority of known spliceosomal components accumulate in a characteristic speckled pattern in the nucleus and spliceosomal snRNPs additionally concentrate in Cajal bodies (CBs) 27, 28. EM analysis of thin sections from mammalian cell nuclei reveal two distinct structures associated with the nuclear speckles: granular regions termed interchromatin granule

Lessons from other macromolecular machines in the nucleus

In addition to pre-mRNA splicing, other nuclear processes such as transcription initiation by RNA polymerases I and II and DNA repair by nuclear excision involve the assembly of large multi-component molecular machines. Different models for how these machines assemble onto their substrates have been considered, ranging from dynamic stepwise assembly of subunits to interaction of stable pre-assembled whole units (holocomplexes) [39].

Recent direct observation of transcription factor action on

Is the spliceosome a sequentially assembled, self-organized machine?

Advances in live-cell imaging provided unprecedented insight into the dynamic properties of proteins that make up cellular structures. Based on these observations, Misteli 54, 55 proposed that self-organization is a general mechanism by which macromolecular structures in the nucleus are formed and maintained. Self-organization refers to the spontaneous emergence of structures out of the interactions among its multiple components. In vitro splicing assays did demonstrate that spliceosomes can

Concluding remarks

Within the past 10 years, tools have become available to measure molecular kinetics in living cells. How nuclear proteins assemble on DNA and RNA to form macromolecular machines has crucial implications for the regulation of gene and genome functions. Contrasting with the concept that nuclear machines are pre-assembled and engage on their substrates in a single step, the alternative model of sequential assembly implies that any step in the process might be controlled, thus enabling rapid and

Acknowledgements

We thank our colleagues J. Braga and M. Gama-Carvalho for stimulating discussions and critical reading of the manuscript. Our laboratory is supported by grants from Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-GMG/69739/2006) and the European Commission (LSHG-CT-2003–503259 and LSHG-CT-2005–518238).

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