Functional reconstitution of U6 snRNA in nematode cis- and trans-splicing: U6 can serve as both a branch acceptor and a 5′ exon

Cell, Vol. 75, 1049-1059,

December

17, 1993, Copyright

0 1993 by Cell Press

Functional Reconstitution of U6 snRNA in Nematode Cis- and Trans-Splicing: U6 Can Serve as Both a Branch Acceptor and a 5’ Exon Yi-Tao Yu, Patricia A. Maroney, and Timothy W. Nllsen Center for Molecular Parasitology Department of Molecular Biology and Microbiology Case Western Reserve University School of Medicine Cleveland, Ohio 44106

Summary Maturation of nuclear pre-mRNAs in nematodes requires both cis- and trans-splicing. Both processing pathways involve analogous two-step phosphotransfer reactions and both are dependent upon the integrity of U6 snRNA. We have developed a functional re constitution assay to assess the U6 snRNA sequence requirements for cis- and trans-splicing. Branch formation between the splicing substrates and U6 snRNA was observed. The frequency of this event was greatly enhanced when a highly conserved sequence in U6 snRNA was altered by mutation. In cis- and transsplicing reactions reconstituted with this mutant U6 snRNA the liberated exon of U6 proceeded through the second step of splicing using the appropriate splice acceptor sites. These results demonstrate covalent interactions between a U snRNA required for splicing and a splicing substrate, and they provide evidence for an unexpected degree of catalytic flexibility within the spliceosome. Introduction Splicing of nuclear pre-mRNA in eukaryotic cells results in the precise removal of intervening sequences. In most pre-mRNA splicing reactions (cis-splicing), exons to be joined are encoded as part of a single molecule. lntron removal requires the participation of five small nuclear ribonucleoprotein particles (snRNPs)(Ul, U2, U4, U5, and U6) and a large number of proteins as yet undetermined (for recent reviews see Green, 1991; Guthrie, 1991; Moore et al., 1993). Cis-splicing proceeds through two successive transesterification reactions (Moore and Sharp, 1993) and takes place within a large macromolecular complex known as the spliceosome. An additional nuclear pre-mRNA processing pathway, termed spliced-leader (SL) addition trans-splicing, occurs in a variety of lower eukaryotes, including the trypanosomatid protozoans and nematodes (for recent review see Nilsen, 1993). In SL addition trans-splicing, pre-mRNAs acquire their 5’ terminal exon from a small RNA (the SL RNA) that shares features in common with spliceosomal U snRNAs. Trans-splicing proceeds through a two-step reaction pathway that generates products and intermediates directly analogous to those produced in the two-step cis-splicing reaction. Furthermore, splice donor and acceptor sites used in trans-splicing conform to consensus sequences used in cis-splicing. Both cis- and trans-

splicing require some snRNP cofactors in common; targeted RNAase H digestion has shown that intact U2, U4, and U6 snRNPs are required for trans-splicing in trypanosomes and nematodes (Tschudi and Ullu, 1990; Hannon et al., 1991). However, trans-splicing may not require the entire complement of cis-spliceosomal U snRNAs. In trypanosomes, which carry out only trans-splicing, homologs of Ul and U5 snRNAs have not been identified, leading to the speculation that these snRNAs, whose role is identification and juxtaposition of splice sites in cis-splicing, might not participate in trans-splicing (for discussion and references, see Nilsen, 1993; Steitz, 1992). Of the U snRNAs required for splicing, U6 snRNA is by far the most conserved, both with respect to length and primary sequence (Brow and Guthrie, 1988). A large body of evidence indicates that U6 plays multiple roles in the splicing pathway. Intensive mutational analyses in yeast and vertebrate cis-splicing systems have identified nucleotides critical for the first and/or second catalytic steps of splicing (Fabrizio et al., 1989; Madhani et al., 1990; Vankan et al., 1990, 1992; Madhani and Guthrie, 1992; Fabrizio and Abelson, 1992; Wolff and Bindereif, 1992,1993; Wolff et al., 1993; Datta and Weiner, 1993). Furthermore, cross-linking analyses both in yeast and mammalian systems have indicated that there is an intimate association between U6 snRNA and intron sequences near the 5’ splice site (Sawa and Shimura, 1992; Sawa and Abelson, 1992; Wassarman and Steitz, 1992; E. Sontheimer and J. A. Steitz, personal communication). The extraordinary conservation of U6 coupled with the remarkable phenotypes obsenred with some mutations in its sequence have led to the conclusion that this U snRNA is central to and may participate in the catalysis of splicing (for discussion and references, see Guthrie, 1989,199l; Steitz and Steitz, 1993; Weiner, 1993.) Here we have used a functional reconstitution system and mutagenesis to assess the role of U6 snRNA in nematode c/s- and trans-splicing. Surprisingly, we find that mutation of a highly conserved sequence in the central domain of U6 immediately 5’ of a region in U6 previously shown to be in proximity to a 5’ splice site in yeast (Sawa and Abelson, 1992) causes U6 to be used as a splicing substrate in both cis- and trans-spliceosomes. These results indicate that U8 can function as a branch acceptor and even as a 5’ exon when mutations prevent it from correctly positioning the 5’ splice site within the spliceosome. Results Functional Reconstitution of U6 snRNA Ascaris lumbricoides extracts were digested with micrococcal nuclease to deplete endogenous snRNAs (see Experimental Procedures), a treatment that rendered the extracts inactive in c/s- and trans-splicing (Figure 1, lanes 2). Following inactivation of the nuclease, both splicing activities were restored by inclusion of a low molecular

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TRANS CIS --cumd-tinM --cuKN-in

Figure I. Reconstitution of Trans- and Cis-Splicing with Synthetic U6 snRNA Labeled trans- and cis-splicing substrates (Hannon et al., 1991) were incubated for 2 hr in A. lumbricoides extract, which was either mock treated (lanes 1) or digested with micrococcal nuclease (lanes 2-5). Lanes 2 contained no added RNA. In lanes 3, extracts were supplemented with total 4-6s A. lumbricoides RNA. Lanes 4 were supplemented with 4-6s RNAdepleted of U6 snRNA by oligonucleotide affinity. In lanes 5, extracts were supplemented with UG-depleted 4-6s RNA and 46 ng of synthetic U6 snRNA. The positions of trans- and cis-splicing products and intermediates are indicated schematically. Lane M contained labeled restriction fragments (Hpall digest of pBR322). Splicing reactions were assembled and analyzed by denaturing polyacrylamide electrophoresis as described in Experimental Procedures.

weight fraction (4-8s) of total A. lumbricoides RNA (Figure 1, lanes 3). If this 4-8s fraction was depleted of U8 snRNA, splicing activity was reduced to a low background level (Figure 1, lanes 4) that could be enhanced to near control levels by inclusion of synthetic U8 snRNA (Figure 1, lanes 5). The fact that the same synthetic U8 snRNA supported cis- and trans-splicing excluded the possibilitythat specific variants of U8 were required for each processing reaction and permitted a parallel analysis of the U8 sequence requirements for both reactions. For this analysis, site-directed mutagenesis was used to introduce consecutive four base block substitutions throughout the A. lumbricoides U8 snRNA coding sequence. In every block mutation (25 in all), each base was changed to its complementary base (Figure 2). Each mutant template was then transcribed, and the altered U8 snRNAs were tested for their ability to reconstitute either cis- or trans-splicing. The phenotypesof selected mutantsare shown in Figure 3, where reconstitution reactions were analyzed on both 4% and 8% polyacrylamide gels. The lower percentage gel permits increased resolution of apparent intermediates in trans-splicing, while the higher percentage gel enhances the aberrant migration of molecules containing unusual structures. Mutants 11 and 12 failed to support either cis- or trans-splicing. These mutants are predicted

to disrupt both the stem I U4-U8 interaction and the helix I U2-U8 interaction (Madhani and Guthrie, 1992; see Figure 2). Two block mutants (9 and 10) lay in the universally consenred ACAGAG box located in the central domain of U8. Alteration of the 3’-most AG of this sequence as part of a four base substitution (mutant 10) resulted (as it does both in vivo and in vitro in yeast and in vitro in mammalian systems [Fabrizio and Abelson, 1990; Madhani et al., 1990; Wolff et al., 19931) in a strong block to the second step of cis- and trans-splicing (Figure 3, lanes 10). By contrast, mutation of the first four bases of the sequence (mutant 9) had a relatively minor effect on splicing (Figure 3, lanes 9). Interestingly, in trans-splicing, both mutations in the ACAGAG box caused the appearance of a novel RNA species that did not comigrate with any of the predicted intermediates or products (Figure 3, lanes 9 and 10, asterisk under 8% Trans). Reconstitution with a U8 in which the sequence AAUU eight bases upstream of the ACAGAG box was altered to UUAA (mutant 8) displayed an unusual phenotype in both c/s- and trans-splicing. With the cis-splice substrate, the efficiency of splicing was reduced and a new RNA species with greatly retarded mobility appeared (Figure 3, lanes 8, asterisk under Cis). The phenotype of mutant 8 was more dramatic in trans-splicing. Here, normal splicing essentially was abolished and a slowly migrating RNA accumulated. This RNA had a different mobility than that predicted for the Y-branched exon intermediate (compare lanes 8 and 10 in Figure 3, 4% Trans). The 4% gel also revealed the presence of unexpected two-thirds intermediates in trans-splicing reactions reconstituted with mutant 3 (bases 12-15) or wild-type U8 snRNAs; four intermediates were evident with mutant 3, and two intermediates were resolved in wild-type reactions (Figure 3,4% Trans, lanes wt and 3). To determine which molecules contained branches, individual RNAs were excised from gels such as those shown in Figure 3 and incubated with HeLa cell SlOO extract that contains debranching enzyme (Ruskin and Green, 1985). By the criteria of debranching, all of the structured molecules (i.e., those that migrated above the substrate or those that showed altered mobility on 4% versus 8% gels) contained 2’-5’linkages (data not shown). In particular, debranching of the lariat intermediate generated in cis-splicing reactions yielded a linear RNA with the expected size. Surprisingly, debranching of the slowly migrating RNA observed in cis-splicing reactions reconstituted with mutant 8 (Figure 3, lanes 8, asterisk under Cis) yielded an RNA that comigrated with the substrate, a result that suggested that this branched molecule was composed of two separate RNAs. As expected, debranching of all of the apparent two-thirds intermediates generated in trans-splicing reactions yielded linear molecules that comigrated with the substrate. Debranching of the RNAs of unusual mobility generated in trans-splicing reactions reconstituted with mutants in the ACAGAG box (Figure 3, lanes 9 and 10, asterisk under 8% Trans) yielded molecules that migrated faster than the substrate, indicating that these RNAs may have been formed by internal branch point attack.

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The sequence of A. lumbricoides U6 snRNA is drawn with predicted base pairing to either U2 snRNA (Hausner et al., 1990; Datta and Weiner, 1991; Wu and Manley, 1991; Madhani and Guthrie, 1992) or U4 snRNA (Steitz et al., 1966). As described (see text) 25 consecutive four base block mutations (indicated by numbers and slashes) were made. In every block, each nucleotide was changed to its complementary base. The invariant ACAGAG sequence (see text) in U6 and presumptive branch point recognition sequence in U2 are in bold letters.

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Trans- and cis-splicing reactions as described in Experimental Procedures were reconstituted with wild-type and mutant U6 snRNAs as indicated. Upon completion of incubations. aliquots of each reaction were analyzed on 4% and 6% denaturing polyacrylamide gels (indicated). Minus lanes, no added RNA; lanes M, as in Figure 1. The asterisk (6% Trans, lanes 9 and 10) indicates the position of RNAs of unusual mobility described in the text. The asterisks (6% and 4% Cis, lanes 6) mark the positions on these gels of the slowly migrating RNA described in the text. The position of trace labeled U6 in reconstitution reactions is indicated (U6). The positions of splicing intermediates and products are indicated schematically.

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U6 Figure 4. Reconstitution of Trams-Splicing with Selected U6 snRNAs Using Labeled SL RNA Rather Than Labeled Acceptor Pre-mRNA Reconstitution reactions similar to those shown in Figures 2 and 3 were carried out with the indicated U6 snFlNAs. Mutant 15 (not shown in Figure 3) disrupts the stem II M-U6 interaction (see Figure 2). Incubations contained 250,000 cpm (- 10 ng) labeled SL RNA (Maroney et al., 1990) and 50 ng of unlabeled trans-splice acceptor. Lanes C, nondepleted RNA; minus lanes, no added U6 snRNA; lane M, as in Figure 1. Positions of trams-splice intermediates and products are indicated schematically. The unmarked band visible in all lanes results from nonspecific trapping of labeled SL RNA.

Analysis of Reconstitution of Tram-Splicing Using Labeled SL RNA Because trans-splicing involves the participation of two substrate molecules (the SL RNA and the acceptor premRNA), it is possible to assay the respective contributions of each molecule to intermediates and products produced in trans-splicing reactions. Therefore, we carried out transsplicing assays in which the SL RNA was labeled instead of the acceptor pre-mRNA (Figure 4). in most cases, the results with labeled SL RNA paralleled those seen with labeled acceptor. in particular, no trans-splicing was observed in reactions reconstituted with mutant 12, and mutation of the 3’AG of the ACAGS box showed a strong block to the second step of the reaction (Figure 4, lane 10). However, with labeled SL RNA, the aberrantly migrating speciesobserved in labeled acceptor reactions reconstituted with mutants 9 and 10 (see Figure 3) were not evident. Thus, these molecules do not contain SL RNA sequence. The structure and sequence contents of these molecules

U6

Figure 5. Incorporation of Labeled U6 snRNAs into Splicing Intermediates and Products Reconstitution reactions similar to those shown in Figure 4 were carried out with the indicated U6 snRNAs labeled to high specific activity (see Experimental Procedures). Each reaction contained 500,000 cpm (20 ng) of labeled U6 and 50 ng of unlabeled trans- or cis-splice substrates. Incorporation of U6 radioactivity into RNAs a, b, c, a’, and b’ was dependent upon the presence of a splice substrate (data not shown). The sequence composition of these RNAs is discussed in the text. The unmarked band visible in all lanes results from nonspecific trapping of labeled U6.

remain to be characterized in detail, and they will not be discussed further here. Most significantly, in labeled SL RNA assays, background levels of intermediates and products were observed in reactions reconstituted with mutant 6 (Figure 4, lane 6). Furthermore, in these assays only a subset of the Y intron and exon intermediates was labeled. in reactions reconstituted with wild-type UGsnRNA, oneof two intermediates was labeled and in reactions reconstituted with mutant 3, only 1 of 4 branched intermediates contained SL sequence (compare Figures 3 and 4). These observations indicated that the intermediates with unusual migration observed in labeled acceptor assays could not have resuited from alternative branching to the SL RNA and therefore were generated by reactions that did not involve branching to the SL RNA. Taken together with the results of the debranching experiments described above, this analysis indicated that, in reconstitution reactions, branched molecules were generated that contained RNA(s) that were not expected to interact covalently with splicing substrates.

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Figure 6. Sites of Branch Formation Trans-Splice premRNA Substrate

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(A) Slowly migrating RNAs (RNAs a; see Figure 5) produced in labeled U6 reconstitution reactions were excised from gels such as those

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shown in Figure 5 (trans) and incubated either without (minus lanes) or with (plus lanes) HeLa cell SlOO extract as described in Experimental Procedures. 3a, 3b, and 3c correspond to RNAs of slowest, intermediate, and fastest mobility of RNAs a (Figure 5) generated in reactions reconstituted with labeled mutant 3 U6 snRNA. RNAs released upon

incubation were sized with respect to the mobility of labeled restriction enzyme fragments (lane M) and a sequencing ladder of a cDNA clone of known sequence (CTAG). The numbers (in nucleotides) correspond lo the bases indicated by asterisks. Intermediates produced in reactions with wild-type and mutant 3 U6 snRNAs debranch inefficiently, presumably because they do not contain AG branches (see text). The faint band running across the entire gel corresponds lo a low level of intact U6 snRNA present in the excised gel slices. (B) A schematic representation of sites of branching to various U6 snRNAs. As discussed in the text, debranching, primer extension, and RNA fingerprinting were used to map branch points. Where the exact site of branch point formation has not been established, arrows indicate the most likely site of branching, and the extent of potential ambiguity is marked by a solid line. The numbers above or below each arrow indicate the particular U6 used; 6 + 10 is a double four base substitution mutant, while 6 + CC is mutant 6 with the Iwo downstream guanosines (G28 and G28) changed to cytosines.

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generated when trans-splicing was reconstituted with wildtype U6 snRNA. These results strongly suggested that the unusual branched molecules observed in reactions reconstituted with several different U6 snRNAs resulted from branch point attack upon U6 snRNA itself. These experiments predicted that branched molecules might be observed if labeled U6 snRNA instead of labeled splicing substrates was used in reconstitution reactions.

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To test this prediction directly, we prepared a number of labeled U6 snRNAs and performed reconstitution reactions using unlabeled trans- and cis-splicing substrates (Figure 5). Strikingly, incorporation of labeled U6 sequence into slowly migrating species (Figure 5, Trans and Cis, RNAs a and a’) was observed when either cis- or trams-splice substrates were used; incorporation of U6 radioactivity in each case was dependent upon the presence of a splicing substrate (data not shown). As shown in Figure 5, molecules labeled by wild-type, mutant 6, and mutant 3 U6 snRNAs in trans-splicing assays accounted for all of the intermediates that were present in labeled acceptor but absent in labeled SL RNA reactions; similar labeling of intermediates was observed when the cissplicing substrate was used (Figure 5; see below). No incorporation was detected with mutants 12 or 15, both of which do not reconstitute splicing activity. The fact that labeled U6 could be directly incorporated into splicing intermediates and products (see below) enabled us to use a variety of approaches to map the site(s) of branch point attack on U6, including debranching, primer extension, and two-dimensional RNAase Tl fingerprinting. A debranching experiment is shown in Figure 6. After treatment with HeLa cell Sl 00 extract, discrete fragments of U6 were released from each molecule. The size of these fragments was used to estimate the positions of branching in wildtype as well as mutant 3 and mutant 6 U6 snRNAs. To map the sites of branching more accurately and to establish (in trans-splicing) which nucleotide(s) initiated branching, we carried out primer extension and two-dimensional RNAase Tl fingerprint analyses. These experiments showed that two adjacent adenosines 16 nt and 19 nt upstream of the trans-splice acceptor site were used with equal frequency as branch points to U6, the same branch point adenosines that are used in trans-splicing to the SL RNA (Hannon et al., 1990). This analysis also showed that the phosphodiester bond between G26 and G29 of mutant 6 (Figure 6B) was cleaved during branch formation. These nucleotides lie immediately adjacent to and downstream of the sequence altered in mutant 6. The site of branching to wildtype U6 snRNA has not been established unambiguously by fingerprint analysis. However, primer extension and debranching indicate that, in this RNA, the most likely site of branching is between the U and A residues in the UUAA sequence altered in mutant 6. In mutant 3, three sites of branching are observed, including one that coincides with the site in wild-type and two others that are located within the 5’stem loop of U6 (Figure 6B). In both cis- and trans-splicing reactions reconstituted with labeled mutant 6, molecules that have significantly faster mobility than the Y intron and exon branched species are generated (see Figure 5, lanes 6). Reverse transcription (RT) followed by polymerase chain reaction (PCR) has established that the more intense band observed in the trans-splicing reaction (Figure 5, lane 6, RNA c) represents a spliced product in which the liberated 5’ exon of U6 is ligated to the exon in the trans-splice substrate at the appropriate position. The fainter band running just above this spliced product (see Figure 5, lane 6, RNA b) represents the Y intron product of the second step of

trans-splicing. Thus, in these reactions both steps of splicing occur. With the cis-splice substrate, the appropriate spliced product also is generated (as determined by RTPCR), although it is not visible on the gel in Figure 6 because it comigrates with intact U6 snRNA (data not shown). The band evident on the gel (Figure 5, lane 6, Cis, RNA b’) represents the Y intron product generated in the second step of a splicing reaction in which the 5’exon of U6 is joined to the downstream exon of the cis-splice substrate. Branching to U6 snRNA in Tram-Splicing Does Not Require a 5’ Splice Site The sequence changed in mutant 6 lies immediately upstream of a sequence in U6 that in yeast has been shown

Figure 7. Branch Formation to U6 snRNAs the Presence of the SL RNA

Does

Not Depend

upon

Cis- (lanes 1 and 2) and trans-splicing (lanes 3-10) were reconstituted with 4-6s RNA as in Figure 1. In lanes 2, 4, and 5-10, this RNA was depleted of the SL RNA by oligonucleotide affinity chromatography (see Experimental Procedures). In lanes 5-10, U6 snRNA also was depleted. Reactions in lanes 57, and 9 were supplemented with synthetic SL RNA. In addition, reactions in lanes 5 and 6 were supplemented with wild-type U6 snRNA and reactions in lanes 7 and 6 were supplemented with mutant 6. In reactions shown in lanes 5-6, the reconstituting U6 was lightly labeled (see Experimental Procedures) and is visible on the autoradiogram (U6). Reactions in lanes 9 and IO contained 20 ng of mutant 6 U6 snRNA labeled to high specific activity and unlabeled trams-splice acceptor (see Figure 5). The autoradiogram of lanes 1-6 was intentionally overexposed to highlight the region of thegel containing the Y exon and intron intermediates in trans-splicing. The arrowheads indicate the position of intermediates containing SL RNA sequence, and the asterisks indicate the position of intermediates containing U6 sequence. SL RNA and UG-containing intermediates in lanes 5 and 6 are not well resolved because of the use of synthetic SL RNA; the intermediate visible in lane 6 does not contain SL sequence (data not shown).

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Participation

of U6 in Splicing

be in proximity to a 5’ splice site (Sawa and Abelson, 1992; see Discussion). Thus, it seemed possible that the effect of mutant 6 was to alter the geometry of a 5’ splice site-U6 interaction such that U6, rather than the 5’ splice site, was presented to the 2’ hydroxyl of the branch point adenosine. If this were the case, branching to U6 would be expected to require a functional 5’ splice site. Because removal or inactivation of the 5’ splice site in a cis-splice substrate causes the cis-splice substrate to become a trans-splice substrate (P. A. M., Y.-T. Y., and T. W. N., unpublished data), the following experiments were carried out only with the trans-splice substrate. To inactivate the 5’ splice site in trans-splicing reactions, we took two approaches. We either masked the 5’ splice site of the SL RNA with a P’O-methyl oligoribonucleotide complementary to nucleotides 22-42 of the SL RNA or removed the SL RNA from the 4-6s pool (along with U6) by affinity selection prior to reconstitution. Masking of the 5’ splice site of the SL RNA effectively inhibited trans-splicing but, as expected, had no effect on cis-splicing (data not shown). Similarly, depletion of the SL RNA prior to reconstitution greatly reduced trans-splicing but did not effect cissplicing (Figure 7). Neither masking nor depletion had any quantitative or qualitative effect on the appearance of U6containing molecules (intermediates and products) in reactions reconstituted with either wild-type, mutant 3, or mutant 6 U6 snRNAs (Figure 7; data not shown). We concluded from these experiments that splicing to U6 snRNA did not require participation of the SL RNA. Consequently, branching to U6 did not result from an altered 5’ splice site-U6 interaction.

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Discussion Participation of U6 snRNA in Splicing Reactions We have shown that branch formation occurs between cis- and trans-splicing substrates and three distinct U6 snRNAs (wild-type, mutant 6, and mutant 3). In addition, we have observed branching to mutant 10, a double mutant (6 + lo), and a U6 snRNA in which the two guanosines downstream of the UUAA in mutant 6 (6+ CC) were altered to cytosines (Figure 66; data not shown). No branching is seen to U6 snRNAs that do not reconstitute splicing (Figure 5; data not shown). In every case in which branching is observed, the site of branch point formation is highly specific and characteristic for the U6 snRNA analyzed. The sites of branch point formation are dispersed within the 5’ stem loop and the 5’ region of the central domain of U6 (Figure 66); these sites do not bear any meaningful homology to conventional 5’ splice sites. Clearly, the phosphodiester bonds in U6 cleaved during branch formation must be correctly oriented for attack by the 2’ hydroxyl of the branch point adenosine and thus, by definition, must be at the catalytic site for the first step of splicing. In the case of mutant 6, in which branch formation occurs to aguanosine, we find it remarkable that the liberated fragment of U6 proceeds through the second step of splicing. Here, the free 3’ hydroxyl produced upon branch formation must be appropriately positioned within the active

site that catalyzes the second step of splicing (exon ligation and release of the branched intron). Thus, by inference, appropriate anchoring and positioning of the 5’exon of U6 must be achieved. In this regard, in all splicing reactions studied to date, alignment and tethering of the 5’ exon is achieved via base pairing to a guide sequence (IGS in group I, ESS[s] in group II, and U5 snRNA in nuclear pre-mRNA splicing [for recent review see Steitz and Steitz, 19931). At present, we do not know whether a guide is necessary for juxtaposing the U6 exon with the 3’ splice site. It is tempting to speculate that the higher order structure of U6 itself might be sufficient to position its own exon within the active site(s) for both steps of splicing. If U6 is in a highly constrained structure (i.e., where the 5’ stem loop and central domain are in close apposition) within the spliceosome, it could explain why different branch points are selected when distinct U6 snRNAs are used in reconstitution. We suggest that each RNA (mutant 3, mutant 6, and wild type) adopt subtly different structures, each of which brings specific phosphodiester bonds into proximity of the 2’ hydroxyl of the branch point adenosine. When wild-type and mutant 3 U6 snRNAs are used, spliced products are not observed. We assume that products are not evident in these reactions because branching is not to a guanosine, and non-AG branches proceed ineff iciently or not at all through the second step of splicing (reviewed by Moore et al., 1993). Reconstitution Analysis: Implications for Spliceosome Assembly and Catalysis As discussed in detail above, we have observed branch formation between splicing substrates and a number of U6 snRNAs (both wild type and mutant). The extent of branch formation in each case is not affected by the presence or absence of the SL RNA (Figure 7; data not shown). Taken together, these observations support two unexpected conclusions. First, the SL RNP is not an obligatory determinant of trans-spliceosome assembly, and, second, catalytic activation of the spliceosome can and does take place in the absence of an exogenous splice donor site. These conclusions are surprising in light of the extensive studies in yeast and vertebrate cis-splicing systems, in which it is well established that commitment to splicing (and initiation of assembly of a productive spliceosome) requires the binding of Ul snRNP to the 5’ splice site (Michaud and Reed, 1991; Jamison et al., 1992; reviewed by Rosbash and Seraphin, 1991). While there is some difference in detail, a functional collaboration between 5’ and 3’ cis-splice sites is established early in spliceosome assembly, and in neither the yeast nor the vertebrate systems are any splicing reactions observed when 3’ splice sites alone are incubated in extracts (for recent discussion and references, see Michaud and Reed, 1993). The fact that catalysis occurs in the absence of a 5’ splice site suggests a plausible explanation for the obsewed branching to wild-type and mutant 3 U6 snRNAs, both of which support normal levels of splicing. We suggest that, in the reconstituted system, spliceosomes that lack the SL RNA can form. At some frequency, these spliceosomes become catalytically active, presumably by

Cell 1056

Figure 6. Schematic Representation of the Effect of Mutant 6 As discussed in the text, it seems likely that the AAUU sequence element mediates 5’ splice site positioning through with the substrate (stippled box on the left). In mutant 6, this interaction is disrupted and the branch point adenosine bond linking two guanosines immediately downstream of the AAUU sequence element (see text).

an exchange of U6-U4 pairing for l&U2 pairing (Madhani and Guthrie, 1992) and the branch point attacks the nearest phosphodiester bond (see above). We have not previouslyobserved branching to U6 snRNA in intact extracts, although we have not used highly sensitive assays to look for such events. Therefore, it seems likely that the frequency of assembly of trans-spliceosomes lacking the SL RNA is increased (or dependent) upon prior treatment of the extract with micrococcal nuclease. We speculate that, in reconstitution assays, a factor that facilitates complete spliceosome assembly may be limiting. Alternatively, the balance of reconstituting RNAs, or their ability to assemble, may not recapitulate the situation in the intact extract. It is clear that branch point attack on U6 formation does not depend upon some characteristic specific to the synthetic U6 RNA used in reconstitution (the synthetic U6 has a different 5’ end, has a different 3’ end, and may be undermodified with respect to endogenous U6; see Experimental Procedures), because branch formation is readily observed in reactions reconstituted with endogenous U6 either present in nondepleted RNA or affinity purified from extracts (Figure 7, lane 3; data not shown). Based upon the foregoing discussion, it seems likely that the pathway of trans-spliceosome assembly differs markedly from our current view of cis-spliceosome assembly. It also is possible that the complete trans-spliceosome may be quite distinct from the cis-spliceosome in U snRNP constituents. In this regard, several lines of evidence (albeit indirect) suggest that the fully assembled transspliceosome may lack Ul and perhaps U5 snRNPs (for recent discussion see Nilsen, 1993; Steitz, 1992; Baserga and Steitz, 1993). It is now well established that the role of these snRNPs in cis-splicing is splice-site recognition

as yet unclear interactions attacks the phosphodiester

and juxtaposition (reviewed by Steitz, 1992). In a spliceosome lacking Ul , U5, or both, these functions would have to be carried out by other factors. We suggest that U6 snRNA may be the major and perhaps only snRNP responsible for 5’ splice site recognition in trans-splicing. If true, it would be anticipated that a mutation in U6 snRNA that effects 5’ splice site recognition (mutant 6; see below) would have a much more severe phenotype in transsplicing than in cis-splicing. The amplified effect in transsplicing of certain other mutants (i.e., mutants 9 and 10) also might be rationalized if the trans-spliceosome has a looser structure resulting from the absence of one or more snRNPs present in the cis-spliceosome. Our data suggest that U6 can participate in splicing reactions in both the cis- and the trans-spliceosome. However, it is important to note that we cannot at present exclude the possibility that U6 interactions observed with the cis substrate take place in the context of trans-spliceosomes formed infrequently on the cis substrate acceptor site. Mutant 6: Implications for 5’ Splice Site Recognition by U6 snRNA Recently, several lines of experimental evidence have revealed that the central domain of U6 contacts intron sequences at or near the 5’ splice site (Sawa and Shimura, 1992; Sawa and Abelson, 1992; Wassarman and Steitz, 1992; E. Sontheimer and J. A. Steitz, personal communication; C. Lesser and C. Guthrie, personal communication). These results have led to proposals that U6 snRNA maybe involved in 5’splicesite recognition and positioning during splicing (Sawa and Abelson, 1992; Wassarman and Steitz, 1992; C. Lesser and C. Guthrie, personal communication). In trans-splicing assays reconstituted with mutant

DirIir;t

Participation

of U6 in Splicing

6, the SL RNA is absent from splicing intermediates and products. At least two possible interpretations could account for this dramatic phenotype. First, the mutation could alter the structure of U6 in a manner that permits access of a specific U6 sequence to the catalytic center of the spliceosome. In this interpretation, U6 sequence would occupy the position normally occupied by the 5’ splice site, effectively occluding the 5’ splice site from the catalytic center. Alternatively, the sequence altered in mutant 6 could participate directly in positioning the 5’ splice site through higher order RNA-RNA interactions (Figure 6). Regardless of which interpretation is correct, it is clear that inappropriate positioning or absence of 5’ splice site leads to the use of U6 as surrogate splice substrate. Even though three of four of the nucleotides altered in mutant 6 (AAUU) are highly conserved (Madhani et al., 1990) and, with the exception of certain fungal U6 snRNAs (C. Guthrie, personal communication), are phylogenetitally invariant, previous mutational analyses in both yeast and vertebrate cis-splicing systems have failed to reveal a functional role for this sequence element (Madhani et al., 1990; Fabrizio and Abelson, 1990; Vankan et al., 1992). However, in these cases the maximum alteration of the AAUU sequence was 2 nt; a mutation in which all four bases are changed has yet to be tested in other systems. We also note that the phenotype of mutant 6 in nematode cis-splicing assays is milder than that observed in transsplicing (see above). Thus, a comparable phenotype to the one we have described may have escaped detection in reconstitution assays in other organisms. It is interesting that the AAUU sequence element lies immediately upstream of a region in yeast U6 that was shown to be in close proximity to intron sequences (+4 to +6) of a 5’ splice site (Sawa and Abelson, 1992). If the AAUU element is involved in 5’splice site positioning aswe suggest, the interactions detected by Sawa and Abelson (1992) via UV cross-linking could be anticipated. In mammalian extracts, usingtwodifferent cross-linking approaches (psoralen and site-specific substitution with thiouridine), Steitz and colleagues have detected interactions between the ACAGAG box in U6 and intron sequences comprising the 5’ splice site (Wassarman and Steitz, 1992; E. Sontheimer and J. A. Steitz, personal communication). Furthermore, genetic analysis in yeast indicates that the sequences within the ACAGAG box play a role in dictating the site of 5’splice site cleavage (C. Lesser and C. Guthrie, personal communication). Several lines of experimental evidence suggest that the ACAGAG box must be near or part of the catalytic center of the spliceosome (see Fabrizio and Abelson, 1990, 1992; Madhani et al., 1990; reviewed byGuthrie, 1991). ItistemptingtospeculatethattheAAUU sequence element in some way facilitates correct positioning of the 5’ splice site within the catalytic center, whereupon interactions are established between the substrate and the ACAGAG sequence both prior to (Wassarman and Steitz, 1992) and immediately following (E. Sontheimer and J. A. Steitz, personal communication) the first catalytic step (branch formation and 5’ splice-site cleavage) of splicing.

Forward and Reverse Splicing Prior to the results reported here, covalent interactions between spliceosomal snRNAs and splicing substrates have not been observed, although such interactions have been inferred. Specifically, the demonstration of premRNA-like introns in U6 snRNA (Tani and Ohshima, 1969, 1991) and more recently in U2 snRNA (Takahashi et al., 1993) has led to the hypothesis that these introns arose through rare instances of reverse spliceosomal splicing, in which sites of insertion reflected positions in the snRNA that were closely juxtaposed to the splicing substrate within the catalytic center (for discussion see Brow and Guthrie, 1989; Guthrie, 1989, 1991; Tani and Ohshima, 1991). This hypothesis is supported by the fact that several sites of insertion correspond to conserved sequences in U6 that are exquisitely sensitive to mutation. A notable exception to this correlation is the presence of an intron in the central domain of the Rhodotorula hasegawae U6 (Tani and Ohshima, 1991). Remarkably, and probably not coincidentally, the site of insertion of this intron appears to be identical to the site of branch formation to the Ascaris wild-type U6, and thus lies within the AAUU sequence altered in our mutant 6 (Figure 6B). While our results strengthen the correlation between functionally signficant sequences in U6 and sites of intron insertion, it is important to note that the reactions we have studied are not related to reverse splicing. Neither the intermediates nor the products of reactions involving U6 are those predicted from reverse splicing. Rather, they reflect a forward splicing reaction in which a site in U6 at the catalytic center proceeds through both chemical steps of splicing.

Preparation

of Micrococcal

Nuclease-Digested

Extracts

Splicing extracts were prepared from developing Ascaris embryos (64 cell) as previously described (Hannon et al., 1990). Prior to the dialysis step, 1 mM CaCk and 15,000-16,000 U of micrococcal nuclease (Worthington Biochemicals) were added toa7.5 ml extract and mixed gently in a Dounce homogenizer. The mixture was brought to 30°C and incubated for 15 min. Following inactivation of the nuclease by addition of 4 mM EGTA the extract was dialysed for 4 hr against a buffer containing 1 mM EGTA, 0.2 mM EDTA, 50 mM KCI, 1 mM dithiothreitol, 20% glycerol, and 20 mM Tris-HCI (pH 7.9). Following nuclease treatment, residual SL, Ul , U2, U4, U5, and U6 RNAs were quantitated by primer extension. Comparison with an equal aliquot of RNA prepared from a nondigested extract showed that with the exception of U5, greater than 95% of the RNAs were degraded; less than 30% of U5 was degraded.

Preparation

of RNA and Affinity

Depletion

RNA for reconstitution was prepared from undeveloped Ascaris eggs. Total cellular RNA (2 ml [5-6 mg]) was heated to 95OC for 5 min, cooled on ice, and then layered on 5%-20% (w/w) sucrose gradients in a buffer containing 1OOmM NaCI, 1mM EDTA, 0.5% sodium dodecyl sulfate, and 10 mM Tris-HCI (pH 7.5) (Weber et al., 1976). Gradients were centrifuged for 23 hr at 25,000 rpm in an SW26 rotor (Beckman). RNA from individual fractions was visualized on polyacrylamide gels stained with ethidium bromide. Fractions enriched for snRNAs (4-65) were pooled and aliquots were titrated for their ability to restore splicing activity to extracts that had been digested with micrococcal nuclease. To deplete the 4-6s RNA of U6 snRNA or the SL RNA, streptavidin beads (Bethesda Research Laboratories, Incorporated) were washed three times with WB-250 buffer (1 mM dithiothreitol, 250 mM KCI, 10

Cdl 1058

mM Tris-HCI [pH 8.01). Following preblocking with 150 ug of tRNA, 150 ug of glycogen, and 100 pg of bovine serum albumin per 50 nl of packed beads (2 hr at 4OC), beads were washed three times with 1 ml of WE-250 buffer and were incubated with a 5’ biotinylated oligo (5 ug of oligo per 50 pl of packed beads) in WE-250 buffer. After prebinding to the oligo, the beads were washed five times with 1 ml of WE250 buffer, mixed with the 4-8s RNA fraction, and rotated for two hr at 32V. The supernatant was separated from the beads by microcentrifugation and extracted twice with phenol-chloroform, and the RNA was recovered by ethanol precipitation. U6 snRNA was depleted using an oligodeoxynucleotide, 5’-GCCATGCTAATCTTCTCTG-3’ (USa) complementary to bases 37-55 of A. lumbricoides U6 snRNA. SL RNA was depleted using an oligodeoxynucleotide complementary to bases 22-42 (5’.GAGCTGAAACACGGAATTACC-3’). Oligodeoxynucleotides contained a modifiable Cl2 linker and were biotinylated with caproylamidobiotin-N-hydroxysuccinimide ester as recommended by the supplier (Bethesda Research Laboratories, Incorporated). Primer extension analysis showed that >98% of U6 snRNA, SL RNA, or both, was depleted by this technique (data not shown).

Spllclng

Reactions

and Substrates

Splicing reactions (10 ul) contained 5 nl of extract, 60 mM KCI, 4.2 mM MgC&, 2 mM ATP, 20 mM creatine phosphate, 10% glycerol, 2 mM dithiothreitol, 10 mM Tris (pH 7.9) 3% polethylene glycol, 250 ng poly(A), and the splicing substrates as indicated in each figure legend. The quantity of 4-8s RNA (see above) required to restore maximal splicing activity was determined by titration and was approximately equal to the amount of small RNAs present in 5-10 ul of nondigested extract. The amount of synthetic U6 snRNA required also was determined by titration. In general, the optimal amount of synthetic U6 was -40 ng. Conditions were identical for cis- and trans-splicing in both nuclease-treated and mock-treated extracts. Incubations were for 2 hr at 30°C. After digestion with proteinase K, RNAs were recovered by phenol-chloroform extraction and ethanol precipitation. Aliquots of each reaction were analyzed on 5% polyacrylamide 8 M urea gels unless otherwise indicated. Cis- and trans-splicing substrates (described by Hannon et al., 1991) were uniformly labeled with [a-=P]guanosine triphosphate, and 50,000 cpm (- 2 ng) were used in each reaction. In some reconstitution reactions, as indicated, U6 was labeled to low specific activity (- 20 cpml ng) to monitor its stability. In other reactions, U6 snRNA was labeled to high specific activity (25,000 cpmlng) as indicated in the figure legends.

Site-Directed

Mutagenesis

The A. lumbricoides U8 snRNA gene under control of the phage T3 promoter (J, Shambaugh and T. W. N., unpublished data) was subcloned into pt3luescript M13(+) (Stratagene) and used to transform duf ung Escherichia coli (CJ 236). The plus strand of the template was rescued and subjected to site-directed mutagenesis as described (Vera and Messing, 1987; Kunkel et al., 1987). The first 3 nt of the synthetic U6 RNA are altered compared with endogenous U6 (GUU to GGGAA). This change was introduced to increase efficiency of transcription by T3 RNA polymerase. The synthetic U6 also contains three additional C residues at its 3’ end that result from linearizing the transcription template with Smal. Alteration of the bases at the 5’or 3’ ends had no effect on reconstitution activity (data not shown).

Debranchlng

wlth HeLa S-100

Extract

Debranching reactions were carried out essentially as described by Ruskin and Green (1985). Individual RNAs, as described in the text, were isolated from gels and incubated at 30°C for 15 min in 25 pl reactions containing 0.12 mM EDTA, 0.6 mM dithiothreitol, 60 mM KCI, 12% glycerol, 12 mM Tris-HCI (pH 7.9) and 2.5 pl of HeLa cell S-100 extract (Dignam et al., 1983).

Acknowledgments Y.-T. Y. and P. A. M. contributed equally to this work. Shambaugh and J. Denker for help with the sequencing

We thank J. of mutants

and J. Shambaugh for help with illustrations. We also thank E. Sontheimer, J. A. Steitz, A. Bindereif, C. Lesser, andC. Guthrieforcommunicating results prior to publication and J. Gott. D. McPheeters, J. Wise, H. Madhani, and J. Bruzik for helpful comments on the manuscript. This work was supported by Public Health Service grants GM31528 and Al-28799, by the John D. and Catherine T. MacArthur Foundation, and by an award from the Burroughs Wellcome Fund. Received

August

13, 1993; revised

November

2, 1993.

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