The EMBO Journal vol.9 no.4 pp.1237 - 1244, 1990

Multiple domains of U 1 snRNA, including U 1 specific protein binding sites, are required for splicing

Jorg Hamm, Nina A.Dathan, Daniel Scherly and lain W.Mattaj European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900 Heidelberg, FRG Communicated by I.W.Mattaj

Domains of Ul snRNA which are functionally important have been identified using a splicing complementation assay in Xenopus oocytes. Mutations in, and deletions of, all three of the hairpin loop structures near the 5' end of the RNA are strongly deleterious. Similarly, mutation of the Sm binding site abolishes complementation activity. Analysis of the protein binding properties of the mutant Ul snRNAs reveals that three of the functionally important domains, the first two hairpin loops and the Sm binding site, are required for interaction with Ul snRNP proteins. The fourth functionally important domain does not detectably affect snRNP protein binding and is not evolutionarily conserved. All of the deleterious mutations are shown to have similar effects on in vivo splicing complex formation. Key words: RNA processing/Ul snRNA/Ul snRNP/ Xenopus oocytes

Introduction U small nuclear ribonucleoproteins (U snRNPs) are subunits of the spliceosome, the complex in which introns are removed from messenger RNA precursors by the process of splicing (Birnstiel, 1988). The major snRNPs consist of one (U1, U2, U5) or two (U4, U6), snRNAs, at least seven common proteins and a variable number of unique proteins (Luhrmann, 1988; Reddy and Busch, 1988; Bach et al., 1989). Ul snRNP, which is the subject of this paper, contains at least three unique proteins called Ul 70K, Ul A and U1 C. The structure of the Ul snRNP has been analysed by a number of methods including nuclease digestion of intact RNPs (Epstein et al., 1981; Liautard et al., 1982) or of RNPs from which one or more of the unique proteins had been removed (M.Bach, A.Krol and R.Luhrmann, in preparation), in vitro assembly in whole cell or nuclear extracts (Hamm et al., 1987, 1988; Patton et al., 1987, 1988; 1989; reviewed by Parry et al., 1989), and, more recently, by direct binding studies using U 1 RNA and RNP proteins synthesized in vitro (Query et al., 1989; Scherly et al., 1989; Surowy et al., 1989). The results obtained by these various methods are, in general, in very good agreement and are summarized below. In order to bind U 1 snRNA the common U snRNP proteins require a single stranded region, the Sm binding site, which lies immediately 5' of the fourth and final hairpin loop (see Figure 1). The unique proteins appear to bind nearer the 5' end of the RNA. Oxford University Press

Two of the human proteins, U 1 70K and Ul A, when assayed in isolation, bind to the first and second hairpin loops of Ul RNA respectively. Protein -protein interactions clearly also play a role in particle assembly. For example, a mutant RNA lacking the second hairpin, and therefore the binding site for the Ul A protein, can assemble in extracts of either Xenopus eggs or HeLa cell nuclei into an RNP which is immunoprecipitable with anti-U1 A antisera, suggesting that the RNA binding site for Ul A is not essential for its association with the RNP. Functional studies in vitro have shown that intact UsnRNPs are required for splicing (reviewed by Steitz et al., 1988). The RNA moiety of the RNP has been shown to play an important role in this function. Genetic complementation studies in cultured mammalian cells suggest a base pairing interaction between the 5' end of Ul and the 5' splice junction which, at least in part, is responsible for the definition of the site of pre-mRNA cleavage (Zhuang and Weiner, 1986; Aebi and Weissman, 1987). In vitro and in vivo experiments in Saccharomyces cerevisiae indicate that both the interaction of Ul snRNP with pre-mRNA (Legrain et al., 1988; Ruby and Abelson, 1988; Seraphin and Rosbash, 1989) and the choice of the cleavage site at the 5' splice junction (Seraphin et al., 1988; Siliciano and Guthrie, 1988) may be more complex in yeast than are the analogous events in vertebrates (see Discussion). Early work demonstrated that partially purified U1 snRNPs could bind to the 5' splice junction of a ,3-globin pre-mRNA intron in a manner which was sensitive to proteolysis (Mount et al., 1983). This suggested that the proteins of the U 1 snRNP also play a role in 5' splice site recognition. The Sm binding site, through which the common U snRNP proteins bind to Ul, has been shown to be required for the nuclear localization of Ul snRNA and for the hypermethylation of its 5' cap structure, suggesting a role for the common U snRNP proteins in these processes (reviewed in Mattaj, 1988). More direct evidence for a role of Ul snRNP proteins in splicing has not been available. This paper presents the results of an extensive functional dissection of Ul snRNA. Methods for the destruction of endogenous Xenopus oocyte U snRNAs and their functional replacement by the microinjection of U snRNA (Pan and Prives, 1988) or of genes encoding U snRNAs (Hamm et al., 1989; Pan and Prives, 1989) have been established. This latter method has been used to examine the functional activity of mutant Xenopus U1 snRNPs. Four domains of Ul RNA which have strong effects on splicing complementation are identified. Three are shown to correspond to sites required for stable protein assembly into U 1 snRNPs. The effects of mutation of the different domains on splicing complex assembly in vivo is also examined, leading to the conclusion that none of the transcripts bearing deleterious mutations is significantly able to compensate for the lack of wild-type Ul snRNA in spliceosome assembly.

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Fig. 1. The Ul mutants. The secondary structure of Ul RNA is shown. The positions of the structural elements deleted or altered in the AAA-AE series (Hamm et al., 1987) are listed and are indicated by large capital letters. AA, AB and AC delete entire hairpin loop structures while AD and AE alter the sequences of the Sm binding site and the fourth hairpin loop respectively. The positions of the nucleotides mutated in AA3, AB1 and ACI are indicated, and the sequences altered in these and the other AA1-AA6 mutants are listed. AA3Bl and AA3C1 are double mutants in which the B1 and A3 or C1 and A3 mutations were combined.

Results U 1 mutants defective in splicing complementation A model of the secondary structure of Ul snRNA is presented in Figure 1. The positions of large and small deletion and substitution mutations (AA, AB, AC, AD, AE, AAl -AA6, AB1, AC1) are shown. Mutants AA-AE and AAI -AA6 have been described previously (Hamm et al., 1987, 1988; note that the AA3 mutant used in this study is mutated at positions 27, 29 and 30 while that used previously was mutated at positions 27 and 29 only). In mutants ABI and AC 1 the loop sequences of stem -loop structures B and C were altered. All of the mutations used in this study were introduced into a Xenopus Ul snRNA gene (Zeller et al., 1984) by site directed mutagenesis. For functional analysis wild type (wt) or mutant U 1 snRNA genes were coinjected with U 1-5', an oligonucleotide complementary to the 5' end of Ul RNA previously shown to inhibit splicing in vitro and in vivo (Black et al., 1985; Pan and Prives, 1988; Hamm et al., 1989). The precursor RNA pBS Adl (Konarska and Sharp, 1987) was transcribed in vitro and microinjected into Xenopus oocyte nuclei 16-24 h after injection of a mixture of oligonucleotide and DNA. Within 60 min of microinjection the Adl precursor is efficiently spliced (Figure 2, lower panels, lanes To and T60). Prior microinjection of U1-5', which results in the RNase H-mediated removal of the 5' end of Ul (Figure 2, upper panel, the cleaved product is labelled Ul *) inhibits Adl splicing (Figure 2, lower panel, lane C). Coinjection of U2 DNA with U 1-5' does not affect this inhibition while coinjection of Ul DNA restores both splicing (lanes U2 wt and Ul wt) and the amount of intact Ul RNA (upper panel, lane Ul wt) due to the accumulation of transcripts from the injected wt Ul genes. All of the deletion mutants were transcribed and accumulated to similar levels on coinjection with U 1-5' (Figure 2, upper panel; note that AE migrates very close to the RNase H degradation product of U1). The ability of the different mutants to complement the splicing deficiency was tested. AA and AD

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were completely inactive (Figure 2, lower panel), while AE

restored splicing activity almost completely. AB and AC gave rise to a very low level of complementation. This level was, however, very reproducible in repeated experiments (data not shown, but see Figure 4B). The fact that injection of U 1-5' does not not lead to a complete inhibition of Adl splicing has been discussed previously (Hamm et al., 1989). Effects of point mutations The AA, AB and AC mutations tested are large deletions. Although they remove single hairpin structures their effects on Ul snRNP structure are likely to be extensive. The next series of mutant RNAs tested therefore carried smaller, localized, alterations. AA1 -AA6 have been previously described (Hamm et al., 1988). ABI and ACI change bases in the loops of the B and C hairpins respectively (Figure 1). Upon microinjection AAl, AA2 and AA4 complement Adl splicing as well as wt Ul (Figure 3B). AA5, AA6 and AA3 are progressively less able to complement splicing, with AA3 being indistinguishable from the negative control, wt U2 (Figure 3B). Due to sub-optimal resolution of the intron product band in this experiment the results are more easily seen by analysis of the ligated exon products (E12). All of the RNAs are transcribed and accumulate to similar levels (Figure 3A, the reduced level of AA3 transcripts seen in this experiment was unusual, see for example, Figure 4). The effects of these mutations on splicing complementation correlates extremely closely to their effects on the assembly of Ul specific proteins into Ul snRNPs in vitro (Hamm et al., 1988; see below). The complementation activity of the ABI and ACI mutant were next tested either alone or in combination with AA3. The mutants were all transcribed and accumulated similarly to wt Ul (Figure 4A). As expected, the double mutants and AA3 alone were completely negative (Figure 4B, lanes A3, A3B1 and A3C1). The single ABI and AC1 mutants complemented to a very limited extent (Figure 4B) in a manner indistinguishable from the large AB and AC deletion

Multiple domains of U1 snRNA are required for splicing

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Multiple domains of U1 snRNA, including U1 specific protein binding sites, are required for splicing.

Domains of U1 snRNA which are functionally important have been identified using a splicing complementation assay in Xenopus oocytes. Mutations in, and...
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