A Protein Interaction Domain Contacts RNA in the Prespliceosome

Molecular Cell 302

of the late buds and expression of specific genes (Mucchielli and Mitsiadis, 2000; Fraboulet et al., 2003). Epithelial budding is a fundamental early step in morphogenesis of many organs. What really governs the direction of the bud—invagination, such as in teeth and hair follicles, or evagination, such as in gustatory papillae—is poorly understood. Thus, the IKK␣ mutants provide an excellent tool with which to address this issue. The next challenge will be to unravel the forces behind the reversed polarized growth of the IKK␣ mutant incisors. A first indication was provided by Ohazama et al. (2004), who found changes in the expression levels and domains of some of the “usual suspects,” namely Shh, Wnt7b, and Notch1/2, after a screen of a number of candidate genes. These molecules are known to control polarized growth and cell-fate specifications in several developmental settings. The fact that in the IKK␣ mutants Notch2 expression was maintained in molars, but not in incisors during their budding, is of particular interest and may be linked to the normal budding of molar anlage in these mutants. Naturally, these new findings prompt new questions. What are the upstream regulators of this new function of IKK␣? How do they operate to integrate the Shh, Wnt, Notch, and other signalings? What is the role of the mesenchyme? What are the links between this signal

A Protein Interaction Domain Contacts RNA in the Prespliceosome SR proteins bind to exons and recruit the spliceosome via protein interactions mediated by an arginine-serinerich (RS) domain. In this issue of Molecular Cell, Shen et al. (2004) demonstrate that the RS domain of SR proteins contacts the pre-mRNA branchpoint, indicating that these domains participate in both protein and RNA interactions. The eukaryotic spliceosome is a marvelously complex molecular machine containing five small nuclear RNAs (snRNAs) and nearly 200 different proteins that participate together with the pre-mRNA in a series of highly choreographed maneuvers that culminate in exon ligation and intron excision (Jurica and Moore, 2003). In simple terms, spliceosome assembly can be divided into two steps: formation of the prespliceosome and subsequent creation of the complete spliceosome. The prespliceosome contains U1 small nuclear ribonucleoprotein particle (snRNP) bound to the 5⬘ splice, U2 snRNP associated with the branchpoint, and the 65 and 35 kDa subunits of U2 snRNP auxiliary factor (U2AF) bound to the pyrimidine tract and 3⬘ splice site, respectively (Figure 1). Although a great deal is understood about how the spliceosome assembles, there are still many unknown details of how splice site recognition is regulated. SR proteins are required for the splicing of all introns

transduction system and cell polarity and cell-cell adhesion? Given its accessibility to experimental manipulation, the embryonic tooth will certainly provide pointed answers. Amel Gritli-Linde Department of Oral Biochemistry Sahlgrenska Academy at Go¨teborg University SE-405 30 Go¨teborg Sweden Selected Reading Aradhya, S., and Nelson, D. (2001). Curr. Opin. Genet. Dev. 11, 300–306. Fraboulet, S., Kavvadia, K., Pourquie´, O., Sharpe, P.T., and Mitsiadis, T.A. (2003). Gene Exp. Patterns 3, 255–259. Hu, Y., Braud, V., Oga, T., Kim, K., Yoshida, K., and Karin, M. (2001). Nature 410, 710–714. Jernvall, J., and Thesleff, I. (2000). Mech. Dev. 92, 19–29. Miletich, I., and Sharpe, P.T. (2003). Human Mol. Genet. 12, R69–R73. Mucchielli, M.L., and Mitsiadis, T.A. (2000). Mech. Dev. 91, 379–382. Ohazama, A., Hu, Y., Schmidt-Ullrich, R., Cao, Y., Scheideret, C., Karin, M., and Sharpe, P.T. (2004). Dev. Cell 6, 219–227. Thesleff, I., and Mikkola, M. (2002). Science’s STKE, www.stke.org/ cgi/content/fullOC_sigtrans; 2002/131/pe22.

and modulate splice site selection (Graveley, 2000). All SR proteins have a similar architecture and contain an RNA binding domain and an arginine-serine-rich (RS) domain. SR proteins have both exon-dependent and exon-independent activities. The exon-dependent activities involve the binding of SR proteins to exonic sequences called splicing enhancers where they enhance exon inclusion. The exon-independent SR protein activities are required for splicing but are poorly characterized (Hertel and Maniatis, 1999). SR proteins function as homotypic protein interaction domains and have been shown to interact with U1-70K, a component of U1 snRNP, and U2AF35, both of which contain RS domains (Wu and Maniatis, 1993). It has also been shown that enhancer-bound SR proteins recruit U2AF to the upstream weak 3⬘ splice site in a manner that involves a direct interaction between the RS domains of the SR protein and U2AF35 (Wang et al., 1995; Zuo and Maniatis, 1996) (Figure 1A). However, a few reports have failed to observe enhancer-dependent U2AF recruitment (see Graveley, 2000, and Shen et al., 2004, and references therein). Artificial tethering experiments have shown that the RS domains of SR proteins, U2AF65, U2AF35, and U1-70K, or a synthetic RS domain consisting solely of alternating arginine and serine residues, are all sufficient to activate splicing (Graveley, 2000; Cartegni and Krainer, 2003; Philipps et al., 2003; Shen et al., 2004). The ability of the U2AF65 RS domain to activate splicing is particularly intriguing because this domain of U2AF65 was previously shown to contact the pre-mRNA branchpoint (Valca´rcel, et al. 1996). This led Shen et al. (2004) to ask whether the enhancer-bound RS domain could also interact with RNA.

Previews 303

Figure 1. Models for Enhancer-Dependent Spliceosome Assembly (A) Traditional enhancer model. The SR protein binds to the splicing enhancer and recruits U2AF via protein interactions between the RS domains of the SR protein and U2AF35. U2 snRNP is subsequently recruited by virtue of interactions between U2AF65 and components of U2 snRNP and by the interaction between the U2AF65 RS domain and the branchpoint (Valca´rcel et al., 1996). (B) Model of enhancer-dependent splicing incorporating the SR protein-branchpoint interaction. In this model, the RS domain of the enhancer-bound SR protein is shown interacting with both the U2AF35 RS domain and the branchpoint. (C) Multiple SR protein model. In this model, the enhancer-bound (exon-dependent) SR protein interacts with the U2AF35 RS domain. In addition, the RS domain of the protein carrying out the exonindependent SR protein activities is involved in contacting the branchpoint.

To do this, Shen et al. used a system in which the RS domain is tethered to a pre-mRNA by fusing it to the bacteriophage MS2 coat protein, an RNA binding protein (Graveley, 2000). However, to look specifically for RS domain-RNA interactions, they engineered a recognition site for the TEV protease and a FLAG epitope between the MS2 coat protein and the RS domain. These

fusion proteins were then used to initiate spliceosome assembly, and any proteins that bound to the pre-mRNA were crosslinked with UV light. Next, TEV protease was used to separate the MS2 coat protein from the RS domain, and the RS domain was immunoprecipitated with FLAG antibodies. Binding of the RNA to the RS domain was then monitored by SDS-PAGE and autoradiography. Interestingly, binding of the enhancer-bound RS domain to the pre-mRNA was observed in the prespliceosome, but not the complete spliceosome, on two different pre-mRNAs. Shen et al. also found that the RS domain very specifically contacts a 10 nucleotide region surrounding the branchpoint. From these experiments, Shen et al. conclude that the contact between the enhancer-bound RS domain and the branchpoint is the interaction that promotes spliceosome assembly—a proposal that requires a dogmatic shift in the models for how SR proteins activate splicing. Although the idea that enhancer-bound RS domains function by contacting the branchpoint is intriguing, there are several caveats. First, these new data do not rule out the possibility that enhancer-bound RS domains engage in functionally relevant protein interactions. These new data are equally consistent with a model in which the enhancer-bound RS domain promotes spliceosome assembly via a protein interaction-mediated mechanism (i.e., U2AF recruitment) and that the RS domain is simply positioned in the prespliceosome such that it interacts with the branchpoint (Figure 1B). It is conceivable that RS domains simultaneously engage in both protein and RNA interactions. Second, it remains possible that the protein contacting the branchpoint is different from the protein bound to the enhancer. In addition to their exon-dependent activites, SR proteins have additional exon-independent functions that are required for splicing (Hertel and Maniatis, 1999). Thus, it is possible that one molecule of the MS2-RS fusion protein binds to the enhancer and recruits U2AF, and that the RS domain of a second MS2-RS fusion protein, engaged in performing the exon-independent SR protein functions, contacts the branchpoint (Figure 1C). Finally, the most important thing that remains to be shown is whether the RS domain-branchpoint contact is the sole interaction that promotes spliceosome assembly. Unfortunately, because the RS domain is required for formation of the prespliceosome and formation of the prespliceosome is required for the RS domain to contact the branchpoint, distinguishing between these two possibilities may be a bit like figuring out whether the chicken or the egg came first. Experiments designed to test whether the enhancer-bound RS domain contacts proteins within the prespliceosome, and if so which proteins, would help to discriminate between these models. Nonetheless, these new results are very tantalizing and should lead to the development of new testable models for how SR proteins function.

Brenton R. Graveley Department of Genetics and Developmental Biology University of Connecticut Health Center 263 Farmington Avenue Farmington, Connecticut 06030

Molecular Cell 304

Selected Reading

Shen, H., Kan, J.L.C., and Green, M.R. (2004). Mol. Cell 13, this issue, 367–376.

Cartegni, L., and Krainer, A.R. (2003). Nat. Struct. Biol. 10, 120–125. Graveley, B.R. (2000). RNA 6, 1197–1211.

Valca´rcel, J., Gaur, R.K., Singh, R., and Green, M.R. (1996). Science 273, 1706–1709.

Hertel, K.J., and Maniatis, T. (1999). Proc. Natl. Acad. Sci. USA 96, 2651–2655.

Wu, J.Y., and Maniatis, T. (1993). Cell 75, 1061–1070.

Jurica, M.S., and Moore, M.J. (2003). Mol. Cell 12, 5–14.

Zuo, P., and Maniatis, T. (1996). Genes Dev. 10, 1356–1368.

Wang, Z., Hoffmann, H.M., and Grabowski, P.J. (1995). RNA 1, 21–35.

Philipps, D., Celotto, A.M., Wang, Q.Q., Tarng, R.S., and Graveley, B.R. (2003). Nucleic Acids Res. 31, 6502–6508.

The RITS Complex—A Direct Link between Small RNA and Heterochromatin

In a recent report, Moazed, Grewal, and colleagues (Verdel et al., 2004) characterize the RITS (RNAinduced initiation of transcriptional silencing) protein complex in fission yeast. They provide a sought-for link between the small RNA produced by the RNA interference machinery and heterochromatin components, suggesting a mechanism for how heterochromatin formation can be targeted in trans to specific chromosomal regions. RNA interference (RNAi) was initially thought of as a purely posttranscriptional process in which small (21–23 nucleotide) inhibitory RNA molecules (siRNA) trigger degradation of homologous mRNA by the RNase IIIlike enzyme, Dicer, with the help of the RNA induced silencing complex (RISC) containing Argonaute (Fire et al., 1998; Zamore et al., 2000; Bernstein et al., 2001). However, in 2002 it was demonstrated that centromeric silencing in fission yeast (Schizosaccharomyces pombe) requires components in the RNAi pathway and therefore in this case RNAi also acts at the transcriptional level to silence genes (Volpe et al., 2002). Subsequently, similar links between RNAi and chromatin modifications have also been established in plants (Arabidopsis)where silencing of genes and retrotransposons depend on RNAi (Zilberman et al., 2003), in Drosophila (Pal-Bhadra et al., 2002), and in the ciliate (Tetrahymena) where RNAi was shown to direct chromatin modifications and DNA elimination (Mochizuki et al., 2002). Hence it seems as though RNAi coupled to transcriptional regulation by chromatin modification could be quite widespread in eukaryotes. In fission yeast, Dicer (Dcr1), the RISC component Argonaute (Ago1), an RNA-dependent polymerase (Rdp1), and additional uncharacterized Csp gene products (centromere suppressor of position effect) (Ekwall et al., 1999) have been shown to be required for heterochromatin formation and silencing of centromeres (Volpe et al., 2002, 2003). In these mutants, long unprocessed transcripts from both strands of the centromere dg and dh sequences accumulate. The long transcripts are normally short lived since the RNAi pathway rapidly processes them to produce small siRNA. The siRNA produced by Dicer then somehow leads to formation of

heterochromatin, i.e., methylation of histone H3 at K9 by the methyl-transferase Clr4 and subsequent binding of the heterochromatin protein Swi6 over the dg and dh repeats. Conversely, in RNAi pathway mutants both H3 K9 methylation and Swi6 binding are lost. Since the role of Swi6 in centromere function is to mediate cohesin binding, the RNAi pathway mutants display a typical chromosome segregation defect, i.e., lagging chromosomes in anaphase and sensitivity to microtubule inhibitors (Provost et al., 2002; Volpe et al., 2003). Another role of RNAi in fission yeast is to direct localized repressive chromatin formation to genes in euchromatin. This was elegantly shown by expressing an inverted repeat RNA, which can form short hairpin RNA (shRNA) capable of targeting heterochromatin formation and cohesin binding in trans (Schramke and Allshire, 2003). The gene becomes methylated at H3 K9 and Swi6 protein binds locally, thus silencing the gene homologous to the shRNA. This type of RNAi-mediated gene repression is dependent on Ago1, Dcr1, Rdp1, Clr4, and Swi6 and is normally used to silence meiotic genes in mitotic fission yeast cells via nearby retrotransposable long terminal repeats (LTR) elements (Schramke and Allshire, 2003). One of the biggest challenges in the heterochromatin/ RNAi field is to understand how the siRNA produced by Dicer is able to direct heterochromatin formation in trans. In a recent issue of Science, Moazed and Grewal’s groups address this issue by purifying and characterizing a new protein complex called RITS (RNA-induced initiation of transcriptional silencing) (Verdel et al., 2004). RITS directly links the siRNA produced by Dicer to heterochromatin because it contains both a previously known chromodomain protein Chp1 that binds centromeres (Partridge et al., 2002) and the S. pombe Argonaute homolog, Ago1. The RITS also contains a previously uncharacterized protein (Tas3). In posttranscriptional RNAi silencing, Argonaute has an important role in the RISC complex, whereby it binds siRNA, thus mediating the contact with complementary mRNA required for its destruction by Dicer. The authors propose that RITS could play an analogous role by mediating targeting of specific regions of chromatin for heterochromatin formation. They showed that the siRNA molecules copurified with the RITS complex are homologous to centromeric dg and dh sequences. Furthermore, in Dicer mutants these siRNA molecules are absent from an inactive form of the RITS complex. In addition, they showed by chromatin immunoprecipitation that two RITS proteins, Ago1 and Chp1, bind centromeres, and that all three RITS proteins are required for methylation