Delay in Synthesis of the 3′ Splice Site Promotes trans-Splicing of the Preceding 5′ Splice Site

Molecular Cell, Vol. 18, 245–251, April 15, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.03.018

Delay in Synthesis of the 3ⴕ Splice Site Promotes trans-Splicing of the Preceding 5ⴕ Splice Site Terunao Takahara,1 Bosiljka Tasic,2 Tom Maniatis,2 Hiroshi Akanuma,1 and Shuichi Yanagisawa1,3,* 1 Department of Life Sciences Graduate School of Arts and Sciences The University of Tokyo Komaba, Meguro, Tokyo 153-8902 Japan 2 Department of Molecular and Cellular Biology Harvard University Cambridge, Massachusetts 02138 3 Department of Applied Biological Chemistry Graduate School of Agricultural and Life Sciences The University of Tokyo Yayoi, Bunkyo, Tokyo 113-8657 Japan

Summary Premessenger RNA (pre-mRNA) splicing can occur within an individual pre-mRNA (cis-splicing) or between separate pre-mRNAs (trans-splicing). Although a number of examples of mammalian trans-splicing have been reported, the molecular mechanisms involved are poorly understood. Here, we investigate the mechanisms of Sp1 pre-mRNA trans-splicing with human cells expressing modified Sp1 transgenes. We find that the presence of a long intron or the insertion of an RNA polymerase II pause site within an intron promotes trans-splicing. We also add examples of naturally occurring trans-splicing. We propose that Sp1 trans-splicing, and other examples of mammalian trans-splicing, are a consequence of low-frequency disruption of the normal mechanisms that couple transcription and splicing. Introduction Most mammalian pre-mRNAs consist of multiple exons and introns. Mature mRNA is generated by pre-mRNA splicing, which removes the introns and joins the adjacent exons. In the vast majority of mammalian premRNAs, the exons are joined only within an individual pre-mRNA (cis-splicing). However, in rare cases, an exon from one pre-mRNA can join to an exon from another pre-mRNA (trans-splicing). Examples of transsplicing in mammalian cells include certain highly abundant, viral pre-mRNAs and certain cellular premRNAs (Maniatis and Tasic, 2002). Among the cellular pre-mRNAs, trans-splicing has been observed between pre-mRNAs from the same gene (homotypic alternative trans-splicing) and pre-mRNAs from different genes (intergenic alternative trans-splicing). However, in all of these cases, exons that participate in trans-splicing also participate in cis-splicing, and the physiological relevance of the trans-spliced mRNAs has not been addressed. There have also been several reports of inter*Correspondence: [email protected]

Short Article

chromosomal trans-splicing in mammals, but none of the studies clearly demonstrates this phenomenon. Thus, at present, there is no evidence that the low levels of trans-splicing observed in mammalian cells lead to the production of proteins with essential functions (reviewed in Maniatis and Tasic, 2002). However, there are two cases in Drosophila where alternative trans-splicing (mod[mdg4] and lola) is, in fact, used to produce essential mRNAs and protein isoforms. In both of these cases, the loci are similarly organized, with four constitutively spliced (common) exons located at the beginning of each locus, and trans-splicing of downstream variable exons to the fourth common exon produces at least some alternative mRNAs, including interchromosomal hybrids (Dorn et al., 2001; Labrador et al., 2001; Horiuchi et al., 2003). It is possible that alternative trans-splicing evolved as a mechanism for generating protein diversity exclusively in organisms in which somatic chromosome pairing occurs, such as Drosophila. In these organisms, interchromosomal trans-splicing allows intragenic complementation and could therefore provide an evolutionary advantage. In mammals, where chromosome pairing does not occur in somatic cells, the efficiency of interchromosomal trans-splicing may be too low to be an effective mechanism for generating protein diversity (Tasic et al., 2002; Wang et al., 2002). It is important to distinguish these types of transsplicing from spliced leader addition trans-splicing, which is a constitutive process that provides 5# noncoding exons to most, if not all, pre-mRNAs in many invertebrates, but it does not lead to protein diversification (Davis, 1996). Our previous studies showed that Sp1 pre-mRNA can be spliced in trans to generate mRNA with the exon 3-2-3 arrangement in both human and rat cells (Takahara et al., 2000; 2002). In this case, and in other cases of mammalian trans-splicing, only certain exons engage in trans-splicing, whereas others splice only in cis. At present, the mechanism for exon choice in mammalian alternative trans-splicing is not understood. In this paper, we investigate the mechanisms involved in trans-splicing of the pre-mRNA encoding the mammalian transcription factor Sp1. Results and Discussion To initiate investigation of the molecular mechanism involved in the generation of trans-spliced Sp1 mRNA, stable transgenic HepG2 cell lines expressing a modified Sp1 gene (C1, C1 + 1.6 kb intron sequence [int], or C1 + 4.1 kb int) were established (Figure 1A). These transgenes contained the region from the Sp1 promoter to the fourth exon of the human Sp1 gene, but the size of the third intron was differently reduced from its original size (from 23 kbp to 203 bp, 1.6 kbp, or 4.1 kbp) by an internal deletion. We analyzed trans-spliced and cisspliced products in these stable cell lines by reverse transcription-PCR (RT-PCR), because we have pre-

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Figure 1. Shortened Third Intron Leads to Abolition of trans-Splicing between Sp1 pre-mRNAs (A) Schematic representation of the endogenous Sp1 gene and integrated transgenes (C1, C1 + 1.6 kb int, C1 + 4.1 kb int, C2, C2 + 1.6 kb int, and C2 + 4.1 kb int). The Sp1 gene promoter was located immediately upstream of exon 1. Unique restriction enzyme sites (EcoRI and XhoI) were created in the transgenes. Exons are shown as boxes. The polyadenylation signal (pA) is from bovine growth hormone gene (Goodwin and Rottman, 1992). (B) Expected structures of RT-PCR products derived from the endogenous Sp1 gene and the integrated transgenes. The origins of RTPCR products are identified by restriction enzyme digestion. The expected sizes of RTPCR products after restriction enzyme treatment are indicated on the right. (C) RT-PCR analysis of trans-splicing. Specific amplification of the exon 3-2, exon 1-2, or exon 3-4 junctions, or exon 3 alone was performed with total RNA from the wild-type (WT, lane 1), or the transformed HepG2 cells (lanes 2-7). The products derived from the transgene (T) and from the endogenous Sp1 gene (E) were distinguished using restriction enzymes shown on the right.

viously shown that RNase protection analysis was not a reliable method to detect the trans-spliced Sp1 mRNA (Takahara et al., 2002). We used different sets of primers, which allowed specific amplification of exon 3-2, exon 1-2 or exon 3-4 junctions, or exon 3 alone. Because two unique restriction sites (EcoRI and XhoI) were created in the transgenes (Figure 1A), products from the endogenous Sp1 gene and the transgenes were distinguishable by restriction enzyme digestion (Figure 1B). The trans-spliced product (containing the exon 3-2 arrangement) from the transgenes was not detected in the cell line having the C1 or the C1 + 1.6 kb int transgene, whereas the trans-spliced product from the endogenous Sp1 gene was detected in both cell lines (Figure 1C, lanes 2 and 3). Because the cis-spliced products from the transgenes were detected, the absence of trans-splicing using pre-mRNAs from the transgenes was not due to low expression of the transgenes or due to defects of the splice sites. We detected a low amount of a trans-spliced product from the C1 + 4.1 kb int transgene, which had a relatively longer third intron (lane 4). These results suggested two

possibilities. (1) The third intron of the endogenous Sp1 gene might contain sequences that stimulate transsplicing, and these sequences were eliminated by intron shortening. (2) The length of the third intron itself might influence the frequency of trans-splicing. To examine these possibilities, we attempted to generate a situation similar to the presence of a large intron. We established stable, transgenic HepG2 cell lines with a transgene (C2, C2 + 1.6 kb int, or C2 + 4.1 kb int), which was generated by a further deletion of the 3# splice site of the third intron and exon 4 (Figure 1A). Because termination of transcript occurs at some distance from the poly A site (Proudfoot et al., 2002), we expected that a fraction of pre-mRNAs was synthesized far beyond the poly A site. Deletion of the 3# splice site led to generation of significant amounts of the trans-spliced products (Figure 1C, lanes 5–7). Although deletion of the poly A site from the C2 transgene resulted in reduction of the mRNA level, the trans-spliced product from this transgene was detected, suggesting that trans-splicing with transcripts from the C2 transgene is independent of the presence of the poly A site (data not shown).

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Figure 2. Naturally Occurring trans-Splicing between pre-mRNAs from the Genes with a Long Intron or a Pausing Site for RNAPII in an Intron (A) Partial genomic structures of human genes including the Sp1 gene, the insulin receptor gene and the gene for AK000375 cDNA clone (left). Exons are shown as boxes. The sizes of exons and introns were not drawn on scale. The results of RT-PCR amplification with (+) or without (–) reverse transcription were shown with the sizes of PCR products (right). Structures of the PCR products were identified by DNA sequencing. (B) The genomic structure of the rat apolipoprotein A-I gene. The positions of the two arrest sites for RNAPII are indicated by arrows (Dallinger et al., 1999). The result of the RT-PCR amplification with (+) or without (–) reverse transcription is shown with the size of the product on the right. (C) Schematic representation of integrated transgenes (Apo and Apo-Darrest) that were generated with a genomic DNA fragment of rat apolipoprotein A-I gene and a polyadenylation signal of bovine growth hormone gene. In the Apo-⌬arrest construct, the pause site of RNAPII was replaced with an unrelated sequence (a dotted line). The products of RT-PCR amplification for the exon 3-2-3 and the exon 1-2-3-4 arrangements were shown with their sizes on the right.

Because transcripts from the C2 transgene, which had only an 84 bp long sequence of the third intron, were effectively trans-spliced, particular sequence in the third intron did not appear to be responsible for transsplicing of Sp1 pre-mRNAs. To address the possibility that long introns promote homotypic trans-splicing, we analyzed mRNAs from the human insulin receptor gene and the gene corresponding to the AK000375 cDNA clone (Figure 2A). RT-PCR followed by DNA sequencing revealed homotypic trans-spliced mRNAs containing exon 2-2 junction or

exon 9-8-9 arrangement from the human insulin receptor gene and AK000375 gene, respectively. In contrast, trans-splicing with the 5# splice site of a short intron was not found in insulin receptor mRNA and AK000375 mRNA (data not shown). The splice sites used in these cases of homotypic trans-splicing were the same ones that participated in cis-splicing. The amounts of these trans-spliced products were estimated to be less than 1% of the cis-spliced products by semiquantitative RTPCR analysis with different amplification cycles (data not shown). The ratio of cis- and trans-spliced products was comparable to that of cis- and trans-spliced Sp1 mRNAs in rat and human cells (Takahara et al., 2002; data not shown). In principle, long introns increase the time between the synthesis of the 5# and the 3# splice sites. The pausing of RNA polymerase II (RNAPII) in an intron would also be expected to delay 3# splice site synthesis. We therefore investigated homotypic trans-splicing of a pre-mRNA from the rat apolipoprotein A-I gene containing a pause site for RNAPII in a short third intron (Dallinger et al., 1999). A product was generated by RTPCR for specific amplification of trans-spliced mRNA (Figure 2B). By DNA sequence analysis, it was confirmed that the product had the exon 3-2-3 arrangement, reflecting trans-splicing with the 5# cis-splice site of the third intron and the 3# cis-splice site of the first intron. To examine whether the homotypic trans-splicing is really dependent on the presence of the pausing site, we generated two HepG2 cell lines. One had the rat apolipoprotein A-I gene (Apo transgene) and another had a modified apolipoprotein A-I gene, in which the sequence of the arrest site 2 was replaced with the unrelated sequence (Apo-Darrest transgene, Figure 2C). As shown in Figure 2C, trans-splicing with transcripts from the Apo transgene was detected, whereas trans-splicing with transcripts from the Apo-Darrest transgene was not detected. In both cell lines, cisspliced forms from transgenes were detected. These results strongly suggested that trans-splicing of the apolipoprotein A-I pre-mRNA was dependent on the presence of the pausing site of RNAPII. To further investigate the correlation between transsplicing and the pausing of RNAPII, we established a cell line expressing the C1-MAZBS transgene (Figure 3A). The transgene was generated by insertion of four copies of the binding site for a zinc finger MAZ protein (MAZBSx4) into the third intron of the C1 transgene. The MAZBSx4 DNA sequence has been shown to cause a mostly complete stop of RNAPII transcribing a template in vitro and pausing of RNAPII in vivo (Ashfield et al., 1994; Roberts et al., 1998; Yonaha and Proudfoot, 2000; Robson-Dixon and Garcia-Blanco, 2004). We also established a control cell line expressing the C1-A transgene in which an unrelated sequence similar in size to MAZBSx4 was inserted into the third intron (Figure 3A). By RT-PCR, we detected trans-splicing between pre-mRNAs from the C1-MAZBS transgene, but not from the C1-A transgene (Figure 3B, lanes 1 and 3). In addition, trans-splicing of C1-MAZBS pre-mRNAs was promoted by transient expression of the MAZ⌬NFLAG protein, which contained a C-terminal region of MAZ (157–477 aa) with a FLAG tag (lanes 2 and 4). Approximately 2-fold enhancement by MAZ⌬N-FLAG was

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repeatedly observed in independent experiments. We used only the C-terminal region of MAZ in this experiment, because a previous report suggested that this MAZ truncation had stronger pausing activity for RNAPII than the full-length protein (Ashfield et al., 1994). We also confirmed similar increased levels of MAZ mRNAs by RT-PCR and significant production of the MAZ⌬N-FLAG protein by Western blot analysis in the two cell lines (Figures 3C and 3D). In addition, chromatin immunoprecipitation (CHIP) assays with the antiRNAPII antibody demonstrated that the RNAPII density at the region 2 (the region immediately upstream of MAZBSx4 in the third intron) was elevated by the presence of MAZBSx4 (Figures 3E and 3F, lanes 1 and 3). The transient expression of the MAZ⌬N-FLAG protein further increased the RNAPII density at region 2 in the C1-MAZBS transgene, but not in the C1-A transgene (lanes 2 and 4). We conclude that pausing of RNAPII in the third intron promotes trans-splicing of pre-mRNAs from the C1-MAZBS transgene. Taken together, the present results strongly indicate that delay of the 3# splice site synthesis can trigger homotypic trans-splicing involving the preceding 5# splice site. This conclusion is consistent with previously reported cases in which the occurrence of trans-splicing correlates with the presence of a long intron (Flouriot et al., 2002; Tasic et al., 2002). Moreover, it has been reported that a particular 3# splice site in the adenovirus major late pre-mRNA trans-splices to 5# splice sites within large introns (>20 kb) of cellular pre-mRNAs in host cells (Kikumori et al., 2002). Recent studies showed involvement of transcription elongation rate in splicing control and coupling of transcription and splicing (de la Mata et al., 2003; Howe et al., 2003). On the basis of interaction of a yeast U1 snRNP-associated protein (Prp40) and RNAPII (Morris and Greenleaf, 2000), it has been also suggested that a newly synthesized 5# splice site is bound to the carboxyl terminal domain (CTD) of the largest subunit of RNAPII until the synthesis of the downstream 3# splice site in the cotranscriptional splicing (Goldstrohm et al., 2001; Maniatis and Reed, 2002). We detected a similar

Figure 3. Pausing Sites for RNAPII in an Intron Cause Homotypic trans-Splicing (A) Schematic representation of the C1-A and C1-MAZBS transgenes. Four copies of the MAZ binding site (MAZBSx4) or an unrelated sequence of similar length were inserted into the third intron of the C1 transgene. The unrelated sequence was obtained from the third intron of the Sp1 gene. Region 1 and 2 represent the two regions used in CHIP. (B) RT-PCR analysis with total RNA from the cell line expressing

the C1-A transgene or the C1-MAZBS transgene. The MAZ⌬NFLAG vector (+) or a control vector (–) was transfected. The products derived from the transgene (T) and from the endogenous Sp1 gene (E) were distinguished by restriction enzyme digestion, with enzymes shown on the right. (C) RT-PCR analysis of the total level of both endogenous and transiently expressed MAZ mRNAs. Similar results were also obtained from different cycles of PCR amplification. (D) Western blot analysis with anti-FLAG antibody. Only the transiently expressed MAZ⌬N-FLAG protein can be detected. (E) CHIP assays with the anti-RNAPII antibody. Regions 1 and 2 were amplified by PCR with specific primers. The relative DNA amounts of regions 1 and 2 in each cell lysate used for CHIP assays were verified by PCR amplification (input). Different numbers of PCR cycles were used for amplification of immunoprecipitated DNA and input DNA samples (see Experimental Procedures). (F) Relative density of RNAPII at the region 2. The value was estimated from the ratio of intensity of the band for region 2 to that for region 1. Mean ± SEM is given with the results of four independent CHIP assays including the result in (E). The ratio obtained from the C1-A cell line transfected with an empty vector was set to one.

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Figure 4. A Possible Model for Homotypic trans-Splicing In this model, two RNAPIIs are simultaneously transcribing a gene. Each 5# splice site associates with the CTD from the RNAPII that transcribed it. The probability that the 5# splice site will dissociate from the CTD while the intron is being synthesized is proportional to the time required for intron synthesis (if there are no RNAPII pausing sites in the intron, then it may be proportional to intron length). Once the 5# splice site dissociates from the CTD, it can reassociate either with the same CTD or with a CTD of another polymerase already carrying 3# splice site. In principle, more than one 5# splice site could associate with a single CTD, as it contains multiple heptad repeats (52 repeats in mammalian RNAPII). The two associated 5# splice sites could then compete for the single 3# splice site. trans-splicing would occur if the 5# splice site from exon 3 joins to the 3# splice site of exon 2, resulting in the formation of an mRNA with the exon 3-2-3 arrangement.

physical interaction between RNAPII and FBP21, a human homolog of yeast Prp40 (Bedford et al., 1998), in human cells, suggesting the CTD-mediated cotranscriptional splicing in both yeast and human cells (Figure S1 available online with the Supplemental Data). In addition, spliceosome appeared to form a ternary complex together with RNAPII and RNA through reversible interactions, because the amount of the FLAG taggedFBP21 coimmunoprecipitated with RNAPII was dramatically reduced by washing with a buffer containing a high concentration (1 M) of NaCl after RNase treatment (Figure S1). This is consistent with the recent finding that mammalian splicing factors p54nrb and PSF can directly associate with a 5# splice site and CTD of RNAPII (Emili et al., 2002; Kameoka et al., 2004). As proposed by Tasic et al. (2002), the disruption of this cotranscriptional splicing might lead to intergenic trans-splicing. Similarly, the disruption of the cotranscriptional splicing might cause homotypic trans-splicing in which the splice site pairing occurs between premRNAs from the same gene rather than from different genes (Figure 4). Such disruption could result from the dissociation of the 5# splice site from the CTD while the downstream intron is being synthesized. In principle, for cis-splicing, the longer the intron, the longer the time the 5# splice site must remain associated with the CTD while the intron is being synthesized. The prob-

ability that a given 5# splice site will dissociate from the CTD increases with the time required to synthesize the downstream intron. Thus, a given 5# splice site is more likely to dissociate from the CTD when a long intron or a pausing site of RNAPII is present. Recently, it has been also shown that the MAZx4 site induces not only pausing of RNAPII but also changes in the transcription elongation complex and splicing patterns (RobsonDixon and Garcia-Blanco, 2004). Therefore, traveling the large introns or pausing at the arrest site 2 of the apolipoprotein A-I gene or MAZx4 site might also influence the interaction between spliceosome on a 5# splice site and CTD of RNAPII through a change in RNAPII elongation complex. We added two examples of trans-splicing between pre-mRNAs containing a large intron. However, we could not detect the trans-spliced product involving E-cadherin pre-mRNA in spite of the large size of its second intron (65 kb) (data not shown). The exon 2-2 arrangement in E-cadherin mRNA, which we had expected, is out-of-frame. In the trans-splicing cases we report here, as well as in most cases previously reported, the exons in the trans-spliced products are joined in-frame. It is possible that we could detect only trans-spliced products that escaped the mRNA quality control system such as nonsense-mediated mRNA decay (Hentze and Kulozik, 1999). Although the reported trans-spliced products have an open reading frame, none of the trans-splicing events in mammals have been shown to be biologically relevant at the present time. It is possible that the mammalian trans-splicing cases reported thus far are a consequence of aberrant splicing, resulting in nonfunctional mRNAs that can be tolerated by the organisms due to their low abundance. The fact that most, if not all, mammalian trans-spliced mRNAs are not abundant and originate from pre-mRNAs that are more efficiently cis-spliced adds further strength to the argument that they are not biologically relevant. If splicing occurred after completion of pre-mRNA synthesis and if the 5# splice sites were not constrained by the coupling process, a high level of inappropriate splice site pairing could occur. Therefore, one function of the coupling between transcription and splicing might be to prevent trans-splicing of pre-mRNAs that normally engage in cis-splicing. Experimental Procedures Construction of Plasmids All plasmids used for stable transformation or transient expression were constructed on the basis of pcDNA3.1/Zeo(+) (Invitrogen), which contained the cytomegalovirus (CMV) promoter, the polyadenylation signal (pA) from bovine growth hormone gene, and the Zeocin resistance gene as a selection marker. For the plasmids for transformation, we initially constructed the pcDNA3.1/Zeo⌬CMV vector by elimination of the CMV promoter from pcDNA3.1/Zeo(+) through NruI and BamHI digestion, blunt-end creation, and selfligation. Then, the C2, C2 + 1.6 kb int, and C2 + 4.1 kb int plasmids were generated by insertion of a genomic DNA fragment of human Sp1 gene (positions –1338 to +3586, –1338 to +4958, or –1338 to +7453 relative to the transcription start site; Takahara et al., 2000) between EcoRI and XbaI sites of pcDNA3.1/Zeo⌬CMV, respectively. To create two unique restriction sites at positions from +1558 to +1563 and at positions from +3172 to +3177, we prepared the inserted DNA fragments by reconstruction of PCR-amplified frag-

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ments. The C1, C1 + 1.6 kb int, and C1 + 4.1 kb int plasmids were produced by inserting a genomic DNA fragment (positions +26345 to +26633) into BamHI and XbaI sites of the C2, C2 + 1.6 kb int, and C2 + 4.1 kb int plasmids, respectively. The C1-A plasmid was created by insertion of a genomic DNA fragment of Sp1 gene (positions +3587 to +3777) into the BamHI site of the C1 plasmid. To generate the C1-MAZBS plasmid, four copies of a MAZ binding site (Ashfield et al., 1994; Roberts et al., 1998) were introduced into the BamHI site in the third intron of the C1 plasmid. The Apo plasmid was produced by insertion of a genomic DNA fragment (positions – 466 to +1320 relative to the transcription start site) of rat apolipoprotein A-I gene between EcoRI and XhoI sites of pcDNA3.1/ Zeo⌬CMV. The Apo-⌬arrest plasmid was created by replacing the sequence (positions +976 to +1158) of rat apolipoprotein A-I gene with the sequence (positions +22279 to +22451 relative to the translation initiation site) of the human deoxycytidylate deaminase gene (Weiner et al., 1995) by the overlap-extension PCR method. To create the MAZ expression vector, pMAZ⌬N-FLAG, we obtained the cDNA by RT-PCR with RNA from HeLa cells and then inserted the cDNA into pcDNA3.1/Zeo(+) by synthetic DNAs encoding a double FLAG epitope tag. The sequences of all primers are shown in Table S1. The desired sequences of all constructed plasmids were verified by DNA sequencing. HepG2 Cell Transformation and RT-PCR Analysis To establish human cells expressing a modified Sp1 transgene or a rat apolipoprotein A-I transgene, plasmids for transformation were linearized and transfected into HepG2 cells by Lipofectin and Plus reagent (Invitrogen). The transfected cells were grown in the selection medium containing 300 ␮g/ml of Zeocin (Invitogen). For transient expression of the MAZ⌬N-FLAG protein in cells expressing the C1-A or C1-MAZ transgene, the pMAZ⌬N-FLAG plasmid was transiently transfected using Lipofectin and Plus reagent. We estimated the transfection efficiency at approximately 40% by a GFP expression vector. RNA was isolated from cells with Trizol reagent (Invitrogen) and was reverse transcribed by Superscript II (Invitrogen) with oligo(dT)18 or a gene-specific primer T5. PCR amplifications were performed with Takara Ex Taq (Takara Shuzo, Tokyo, Japan) by cDNA corresponding to 0.2 ␮g total RNA. For amplification of the exon 3-2 alignment in Sp1 mRNA, 22 cycles of the first PCR and the 30 cycles of nested PCR was performed. Detection of the exon 1-2, exon 3-4, and exon 3 structures were achieved by 25 cycles of PCR. Amplified fragments were digested with EcoRI and XhoI to identify the origin of the PCR products. The expression level of MAZ mRNA was analyzed by performing 35 cycles of PCR. The same result was obtained with 28–33 cycles of amplification. The sequences of primers used and RT-PCR analyses for insulin receptor, AK000375, and apolipoprotein A-I mRNA are described in Supplemental Data. CHIP Assay CHIP assays were performed essentially as previously described (Kadener et al., 2002). A detailed procedure is described in the Supplemental Data. The intensity of bands was quantified with light capture (AE-6962, ATTO, Japan), and enrichment was calculated by using the following fomula: R = (Region2/Region1)C1-MAZ / (Region2/Region1)C1-A.

Supplemental Data Supplemental Data include Supplemental Experimental Procedures, one figure, and one table and are available with this article online at http://www.molecule.org/cgi/content/full/18/2/245/DC1/.

Acknowledgments We thank for Dr. T. Maeda (The University of Tokyo) for providing encouragement to T.T. T.T. was supported by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists.

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