Spliceosome assembly pathway

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  1. The spliceosomal machinery

    Unlike Group I and Group II introns, pre-mRNA introns are not able to self-splice without the assistance of trans- acting RNA or protein factors.

    Splicing of precursor messenger RNA takes place in the spliceosome, a large (~40S) RNA/protein macromolecular machine assembles de novo at each round of splicing. The spliceosomal machinery complex is formed from five ribonucleoprotein subunits, termed uridine-rich small nuclear nucleoproteins (snRNPs), transiently associated to more than 760 non-snRNPs splicing factors (RNA helicases, SR splicing factors, etc...).

    Each snRNP particle consists of a phylogenetically conserved small nuclear RNA molecule (abbreviated snRNA) complexed with a set of seven Sm or Sm-like proteins (Lsm), and several snRNP-specific proteins. Thus, the S. cerevisiae U1 snRNP contains ten specific proteins as opposed to three in mammals [Gottschalk A. et al., 1998], the U2 snRNP eleven associated proteins, and the U4/U6.U5 snRNP thirteen proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP [Stevens et al., 2001]. This latter is composed of the two U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Lsm proteins.

    The major spliceosomal snRNPs U1, U2, U4, U5 and U6 are responsible for splicing the vast majority of pre-mRNAs (so-called U2 introns). A group of less abundant snRNPs, U11, U12, U4atac, and U6atac, together with U5, are subunits of the so-called minor spliceosome that splices, only in metazoan (plant, insects and vertebrates), a rare class of pre-mRNA introns, denoted U12-type.

  2. Spliceosome assembly pathway: a stepwise or a modular affair ?

    The traditional view: A stepwise assembled complex

    According to this longstanding model, the spliceosomes are highly dynamic machines, building anew on each pre-mRNA substrate in a highly ordered pathway in vitro. Significantly, this order of assembly is conserved from yeast to mammals and even extends to the metazoan non-conventional U12 spliceosome. Thus, the spliceosomal assembly pathway is likely to reflect fundamental requirements in the construction of a catalytically active machine.
    This model was, until very recently, view as a dogma, and described in every undergraduate molecular biology and biochemistry text [Johnston N., The scientist, 2002, online publication]

    The modern view: A preassembled complex

    Recent work has brought new insights into the spliceosome assembly pathway in higher eukaryotes [Will & Lührmann, 2001]. This alternative model supports that U2 and U4/U6.U5 tri-snRNPs functionally associate with the pre-mRNA at an early stage of spliceosome assembly.
    (i) According to this model, the U2 snRNP is functionally associated with the pre-mRNA at the time of E complex formation in mammals or commitment complex formation in yeast, together with the U1 snRNP and in an ATP independent step [Das R. et al., Mol Cell, 2000]. This suggests that the initial steps of the major spliceosome assembly may be similar to those of the minor spliceosome assembly in which U11 and U12 snRNPs, bind simultaneously to the 5' splice site and branch site, respectively, under the form of a pre-assembled18S complex [Frilander MJ & Steitz JA, 1999].
    (ii) In addition, the U4/U6.U5 tri-snRNP probably also functionally associates with the pre-mRNA at a much earlier stage of spliceosome assembly than previously admitted. According to recent studies, the 5' splice site is also recognized by the U4/U6.U5 tri-snRNP, together with U1, at an early stage of spliceosome assembly (i.e. prior to A complex assembly in mammals or pre-spliceosome complex in yeast) and in an ATP-dependent manner even in the absence of a stable U2 snRNP/branch site interaction [Maroney et al., 2000]. This suggests that the yeast spliceosomal machinery may also consist in a large preassembled complex.

    This is probably the case. Indeed, Scott Stevens and John Abelson [Stevens et al., 2002] brought clear molecular evidence that spliceosome assembly in yeast probably proceed by binding of the pre-mRNA substrate to a preformed, endogenous, U1.U2.U4/U6.U5 penta-snRNP complex assembled from the five snRNA molecules and 76 proteins rather than via a stepwise addition of discrete snRNPs. In this modular model, the pre-mRNA interacts with the penta-snRNP as currently understood, with the 5' splice site base pairing with the U1 snRNA component, with the branching point interacting subsequently with the U2 snRNA, followed by exchange of the U1-5' splice site interaction for a U6 interaction, but all in the context of a 45S penta-snRNP particle which represents a functional precursor to yeast spliceosomes. When a discrete U1 snRNP (e.g., free U1 snRNP) functionally commits a pre-mRNA to splicing, a 30S U2.U4/U6.U5 tetra-snRNP complex is recruited as assembly proceeds. However, the penta-snRNP model remains the preferred spliceosomal assembly pathway in yeast.

    The notion of a preformed snRNP complex makes, from an in vivo point of view, more sense to molecular biologists. As John Abelson says, "Everything points to the fact that cell likes to work in these huge assemblages of molecules" [Johnson N., The scientist, 2002].

  3. Contribution to the pre-mRNA spicing reactions

    The pre-mRNA splicing reaction proceeds in two steps: 5' splice site cleavage and ligation of the intron's 5' end to the so-called branch site occur concomitantly in the first step, and 3' splice site cleavage (with the resulting excision of the intron) and ligation of the 5' and 3' exons take place in the second step. The snRNPs carry out a number of essential functions during this essential event. Via the interaction of their RNA and protein components with the pre-mRNA:

    1. They mediate the recognition and subsequent pairing of the 5' and 3' splice sites of an intron.

    2. In addition, they are responsible for creating the three-dimensional (3D) structure required for the formation of the spliceosomes' two actives sites, one for each of the two splicing reaction steps.

    3. Components of the snRNPs also appear to catalyse the two reactions leading to excision of the intron and ligation of the 5' and 3' exons.

    Having performed its task of exon ligation and mRNA production, the spliceosome disassembles before mRNA export. In addition, just as a machine is designed to perform repetitive tasks, the postcatalytic spliceosomal machinery must be reconfigured to allow a new round of splicing. The snRNP-bound lariat intron must be dissociated, allowing the lariat intron to be degraded and the snRNP to be recycled. Release of mRNA, disassembly, and recycling all necessarily involve extensive RNA:RNA rearrangements. The base pairing of U6 to the 5' splice site, U2 to branch point, and U5 to the exons must be severed. Moreover, the mutually exclusive pairings involving U2, U6 and U4 must be restored to their original conformations.

Selected References

More details on individual steps, structural rearrangements, and factors involved in the spliceosomal pathway can be found in the references cited below:

Last modified: Tue Feb 10 11:20:05 CET 2004