Introduction
The primary transcripts of most eukaryotic genes are retained in the nucleus until noncoding intron sequences are removed by the spliceosome to produce translatable mRNA. For many genes, regulated splice-site choice stimulated by tissue-specific or developmental cues results in the synthesis of alternative protein products from the same primary transcript. A significant fraction of all human genetic diseases, including a number of cancers, are believed to result from deviations in the normal pattern of pre-mRNA splicing, yet how and why these deviations occur is not well understood. In this issue, two groups show that microbial natural products, identified by chemical genetics as potent antitumor compounds, specifically target the spliceosome to inhibit splicing and alter the pattern of gene expression1, 2. This discovery presents a unique opportunity for the development of a new class of therapeutic agents and provides a valuable experimental tool for the investigation of spliceosome assembly and function.
The spliceosome assembles in a stepwise fashion though a conserved series of ribonucleoprotein (snRNP) particle addition and rearrangement steps3 (Fig. 1a). Each snRNP particle is composed of one or more uridine-rich small nuclear RNAs and multiple proteins. Before their recruitment by the splicing apparatus, the abundant snRNP particles are stored in discrete subnuclear domains called speckles. Thought to be ribozymes, the snRNA components of the spliceosome act, at least in part, to promote the two transesterification reactions of splicing (Fig. 1b). snRNP proteins and other splicing factors are necessary to stabilize and resolve these interactions through the course of the spliceosome cycle4.
Figure 1: Assembly of the U2-dependent spliceosome and the chemistry of splicing.
(a) The spliceosome cycle. The snRNP addition, rearrangement and release steps associated with assembly of the major form of the mammalian spliceosome are illustrated. The initial pre-mRNP is shown marked with proteins bound at the branchpoint motif, CTRAY (where the 2'-hydroxyl of the underlined adenosine promotes pre-mRNA 5' splice site cleavage) (SF1), a pyrimidine-rich sequence, (Py)n, (U2AF65) and the terminal 3' splice site AG (U2AF35). The 5' splice site is indicated by the highly conserved GU dinucleotide. Three snRNPs are added in a single step with recruitment of the U4/U6–U5 tri-snRNP particle. This particle consists of the extensively base-paired U4 and U6 snRNAs and associated proteins bound to the U5 snRNP. (b) The two transesterification steps of splicing. In step 1, the 2'-hydoxyl of the branchpoint adenosine (asterisk) serves as the nucleophile for 5' splice site attack. In step 2, the 3'-hydroxyl of the 5' exon intermediate (asterisk) acts as the nucleophile for 3' splice site attack.
Full size image (56 KB)The U2 snRNP is recruited with two weakly bound protein subunits, SF3a and SF3b, during the first ATP-dependent step in spliceosome assembly. SF3b is composed of seven conserved proteins, SAP155, SAP145, SAP130, SAP49, SAP14a, SAP14b and SAP10 (ref. 5). SF3b directly facilitates U2 addition by binding the U2AF65–U2AF35 heterodimer associated with the 3' end of the intron. This is an important event, as stable U2 snRNP addition is often a regulated step in alternative pre-mRNA splicing. Although RNA cross-linking experiments place many SF3a and SF3b components in close contact with the pre-mRNA, the precise functions of the individual proteins remain largely obscure. In addition, it is unclear whether the weakly associated SF3a and SF3b factors function outside the context of the full U2 snRNP particle during the spliceosome cycle.
Thanks to the compounds reported here, these questions may now prove tractable. Through a combination of affinity enrichment, UV cross-linking and protein analysis by western blotting and mass spectroscopy, Kotake et al. show that derivatives of pladienolide B (Fig. 2a), an antitumor macrolide from Streptomyces platensis, bind the SAP130 protein of SF3b. To explore the compound's function in vivo, the authors used a fluorescently tagged probe for pladienolide to confirm that it was found in the same place as snRNP-enriched nuclear speckles and that RNA interference (RNAi) knockdown of SF3b components prevented this colocalization. Analysis of RNA and cDNAs derived from treated cells shows the accumulation of incompletely processed pre-mRNA with pladienolide addition, consistent with direct inhibition of splicing by impaired U2 snRNP function. Importantly, the binding of pladienolide derivatives to SF3b correlates well with the drug's effectiveness in inhibiting cell growth, linking this response to the observed splicing defect.
Figure 2: Blocking gene expression by targeting the spliceosome.
(a) Structures of pladienolide and spliceostatin A, described in this issue. (b) Impact of spliceosome inhibitors on pre-mRNA splicing. In normal cells, transcripts made in the nucleus (inner oval) are efficiently processed to remove intron sequences (white boxes). With drug treatment, splicing is inhibited and incompletely processed mRNA accumulates; some of this mRNA is then released into the cytoplasm. Some transcripts are degraded via nonsense-mediated decay (NMD), while other mRNAs that escape this control produce aberrant protein products. The exon-junction complex (EJC) stimulates NMD when translational termination codons are >50 upstream. The asterisk indicates the position of the first in-frame termination codon in mRNA form displayed.
Full size image (37 KB)In the second article, Kaida et al. show that spliceostatin A (Fig. 2a), a methylated derivative of the Pseudomonas sp. compound FR901464, also binds SF3b, most likely through SAP130 or SAP155, and impairs cellular pre-mRNA splicing. In principle, splicing inhibition might occur through direct impairment of spliceosome function or be an indirect consequence of altered gene expression of splicing factors or regulators within the treated cell. The authors address this issue by showing that spliceostatin A blocks the processing of pre-mRNA in cell-free splicing extracts. As the in vitro system is not sensitive to indirect effects on gene expression, the splicing defects observed must result from direct impairment of spliceosome assembly or activity. Consistent with this observation, drug addition alters the size and number of the snRNP-enriched nuclear speckles, much as others have observed when established splicing factors are inactivated by RNAi, antisense nucleic acids or antibodies (for instance, see refs. 6,7).
Incompletely spliced transcripts retain introns that typically contain in-frame translational termination codons (Fig. 2b). mRNAs with premature termination codons are generally destroyed through a cytoplasmic quality-control process known as nonsense-mediated decay (NMD). NMD efficiently occurs when the termination codons are present 50 or more nucleotides upstream of a splice junction marked by the splicing-dependent exon-junction complex (EJC). RNAs that escape NMD are likely to produce aberrant, often truncated proteins (Fig. 2b). Indeed, Kaida et al. show that splicing inhibition by spliceostatin A addition or SF3b subunit knockdown results in the accumulation of a truncated p27 cyclin-dependent kinase (CDK), termed p27*. Interestingly, although truncated, the stable p27* peptide retains biological activity and may contribute to the cell cycle arrest and antitumor properties associated with drug treatment.
Regardless of the specific role of p27*, these data support a primary role for SF3b in the retention of unprocessed pre-mRNA in the mammalian nucleus. Such a function is consistent with work done in the Saccharomyces cerevisiae system where mutations in several early-acting splicing factors, including U2 snRNP components, cause cytoplasmic leakage of pre-mRNA8. Indeed, the yeast SF3b particle physically associates with a three-protein complex called RES (for pre-mRNA retention and splicing), believed to be a marker for the nuclear retention of unprocessed pre-mRNA9, 10. Conceivably, yeast SF3b association with RES factors could contribute to this marking event even before stable U2 snRNP addition to the spliceosome. RES homologous proteins exist in mammals and include a protein very similar to the SAP14a subunit of SF3b9, 10. Such interspecies comparisons may guide further investigations into other factors that are important in mammalian pre-mRNA retention. And although it remains to be determined whether SF3b-RES interactions also occur in mammals, the spliceostatin A studies already suggest temporally distinct contributions of SF3b to initial nuclear retention of pre-mRNA and subsequent U2 snRNP-dependent steps in splicing.
The observations made here highlight the value of chemical genetics and the importance of natural products in refining our understanding of cell biology. This work raises several important questions regarding the nature of the SF3b-drug interaction and the cellular function of SF3b. For instance, do the pladienolide B– and spliceostatin A–type compounds bind identical, overlapping or distinct target sites? What functional groups on the drug compounds contribute to the apparent high degree of protein specificity? Do the drug interactions occur only in the context of the intact SF3b particle as suggested by Kotake et al.? Do the drug interactions interfere with SF3b complex assembly or stability, or with SF3b integration into the U2 snRNP particle, or do they directly interfere with productive U2 snRNP recruitment to the spliceosome? Are all splicing substrates equally susceptible to inhibition? Is p27* synthesis critical to the cytotoxicity and cell cycle arrest observed with spliceostatin A?
Natural and synthetic inhibitors of translation are among the most valuable antibiotics and have likewise proven important tools in genetic, biochemical and structural studies of ribosome assembly and function. The natural products described in the accompanying papers open the door for similar work on the eukaryotic spliceosome. Current and future studies will test their value for the treatment of cancer. Beyond this, it may prove possible to exploit species-specific distinctions in SF3b to adapt such compounds for treating parasitic and fungal infections or develop related compounds that modulate the splicing patterns of specific genes.
