The Role of Intron Structures in trans-Splicing and Cap 4 Formation for the LeishmaniaSpliced Leader RNA
1999; Elsevier BV; Volume: 274; Issue: 27 Linguagem: Inglês
10.1074/jbc.274.27.19361
ISSN1083-351X
AutoresNancy R. Sturm, David A. Campbell,
Tópico(s)Viral Infections and Immunology Research
ResumoA 39-nucleotide leader istrans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that inLeishmania tarentolae spliced leader RNA 5′ modification is influenced by the primary sequence of stem-loop II, the Sm-binding site, and the secondary structure of stem-loop III. The sequence of stem-loop II was found to be important for cap 4 formation and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem-loop II was detrimental totrans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore splicing, this result contrasts the findings in the kinetoplastidLeptomonas, where mutations that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-binding site and the structure of stem-loop III and was inhibited by incomplete 3′ end processing. The critical nature of the L. tarentolaeSm-binding site is consistent with its essential role in theAscaris spliced leader RNA, whereas inLeptomonas mutation of the Sm-binding site and deletion of stem-loop III did not affect trans-splicing. A pathway forLeishmania spliced leader RNA processing and maturation is proposed. A 39-nucleotide leader istrans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that inLeishmania tarentolae spliced leader RNA 5′ modification is influenced by the primary sequence of stem-loop II, the Sm-binding site, and the secondary structure of stem-loop III. The sequence of stem-loop II was found to be important for cap 4 formation and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem-loop II was detrimental totrans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore splicing, this result contrasts the findings in the kinetoplastidLeptomonas, where mutations that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-binding site and the structure of stem-loop III and was inhibited by incomplete 3′ end processing. The critical nature of the L. tarentolaeSm-binding site is consistent with its essential role in theAscaris spliced leader RNA, whereas inLeptomonas mutation of the Sm-binding site and deletion of stem-loop III did not affect trans-splicing. A pathway forLeishmania spliced leader RNA processing and maturation is proposed. spliced leader tagged SL ADP-ribosylation factor-like polymerase chain reaction reverse transcriptase transversion wild type base pair kilobase pair nucleotide(s) small nuclear RNA Kinetoplastid nuclear gene expression is dependent on thetrans-splicing process. The common substrate for alltrans-splicing reactions is the spliced leader (SL)1 RNA, also known as the mini-exon derived RNA, whose first 39 nt constitute the 5′ ends of both mono- and polycistronically synthesized mRNAs (1Agabian N. Cell. 1990; 61: 1157-1160Abstract Full Text PDF PubMed Scopus (287) Google Scholar). The polycistronic pre-mRNAs require trans-splicing to acquire the specialized “cap 4” structure on the SL RNA. The cap 4 consists of a 7mG attached to the first nucleotide (2Sutton R.E. Boothroyd J.C. Mol. Cell. Biol. 1988; 8: 494-496Crossref PubMed Scopus (19) Google Scholar), in addition to methylation of the first four and sixth nucleotides of the SL RNA (3Perry K.L. Watkins K.P. Agabian N. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8190-8194Crossref PubMed Scopus (128) Google Scholar, 4Freistadt M.S. Cross G.A.M. Robertson H.D. J. Biol. Chem. 1988; 263: 15071-15075Abstract Full Text PDF PubMed Google Scholar, 5Bangs J.D. Crain P.F. Hashizume T. McCloskey J.A. Boothroyd J.C. J. Biol. Chem. 1992; 267: 9805-9815Abstract Full Text PDF PubMed Google Scholar). These modifications are made to the primary SL RNA and spliced onto the mRNA as part of the 39-nt exon. The cap 4 may have roles in mRNA trans-splicing, transport, stability and translation. The SL RNA contains two functional domains as follows: the exon and the intron or snRNA-like domain (6Sharp P.A. Cell. 1987; 50: 147-148Abstract Full Text PDF PubMed Scopus (31) Google Scholar). The exon sequence is conserved among 38 different members of the order Kinetoplastida (7Ullu E. Tschudi C. Günzl A. Smith D.F. Parsons M. Molecular Biology of Parasitic Protozoa. IRL Press at Oxford University Press, Oxford1996: 115-133Google Scholar). Positions 1–9 and 20–39 of the exon are nearly identical, whereas positions 10–19 are relatively heterogeneous and characteristically A/T-rich. This conservation cannot be ascribed to internal promoter location inLeishmania (8Agami R. Aly R. Halman S. Shapira M. Nucleic Acids Res. 1994; 22: 1959-1965Crossref PubMed Scopus (42) Google Scholar, 9Saito R.M. Elgort M.G. Campbell D.A. EMBO J. 1994; 13: 5460-5469Crossref PubMed Scopus (58) Google Scholar), as found in Ascaris (10Hannon G.J. Maroney P.A. Ayers D.G. Shambaugh J.D. Nilsen T.W. EMBO J. 1990; 9: 1915-1921Crossref PubMed Scopus (35) Google Scholar). It was surprising that mutations within positions 20–39 permitted accurate trans-splicing in Leishmania tarentolaeand did not lower splicing efficiency (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) because these results contrasted with findings in Leptomonas (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar). Thus, the results in L. tarentolae more closely resemble the findings in worms as follows: in Ascaris, exon sequences are not necessary for trans-splicing in vitro (13Maroney P.A. Hannon G.J. Shambaugh J.D. Nilsen T.W. EMBO J. 1991; 10: 3869-3875Crossref PubMed Scopus (28) Google Scholar); inCaenorhabditis elegans, length, primary sequence, and composition of the SL are not critical parameters for essential embryonic function, although certain nucleotides may be essential forin vivo splicing of the SL1 RNA (14Xie H. Hirsh D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4235-4240Crossref PubMed Scopus (13) Google Scholar, 15Ferguson K.C. Rothman J.H. Mol. Cell. Biol. 1999; 19: 1892-1900Crossref PubMed Google Scholar). The primary sequence of the SL RNA intron is not conserved among the trypanosomatids (7Ullu E. Tschudi C. Günzl A. Smith D.F. Parsons M. Molecular Biology of Parasitic Protozoa. IRL Press at Oxford University Press, Oxford1996: 115-133Google Scholar); however, the secondary structure is consistent (16Bruzik J.P. Van Doren K. Hirsh D. Steitz J.A. Nature. 1988; 335: 559-562Crossref PubMed Scopus (144) Google Scholar). This structure has been confirmed by physical-chemical and enzymatic studies (17LeCuyer K.A. Crothers D.M. Biochemistry. 1993; 32: 5301-5311Crossref PubMed Scopus (77) Google Scholar, 18Harris Jr., K.A. Crothers D.M. Ullu E. RNA (NY). 1995; 1: 351-362PubMed Google Scholar) and examined by mutagenesis (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar). An equivalent structure is also conserved in the nematode SL RNAs (16Bruzik J.P. Van Doren K. Hirsh D. Steitz J.A. Nature. 1988; 335: 559-562Crossref PubMed Scopus (144) Google Scholar,19Nilsen T.W. Shambaugh J. Denker J. Chubb G. Faser C. Putnam L. Bennett K. Mol. Cell. Biol. 1989; 9: 3543-3547Crossref PubMed Scopus (68) Google Scholar). The intron contains a putative Sm-binding site (16Bruzik J.P. Van Doren K. Hirsh D. Steitz J.A. Nature. 1988; 335: 559-562Crossref PubMed Scopus (144) Google Scholar), an element found in the small nuclear RNAs of higher eukaryotes but apparently lacking in all U-RNAs of kinetoplastids (20Mottram J. Perry K.L. Lizardi P.M. Luhrmann R. Agabian N. Nelson R.G. Mol. Cell. Biol. 1989; 9: 1212-1223Crossref PubMed Scopus (98) Google Scholar) except U5 RNA (21Dungan J.M. Watkins K.P. Agabian N. EMBO J. 1996; 15: 4016-4029Crossref PubMed Scopus (50) Google Scholar, 22Xu Y. Ben-Shlomo H. Michaeli S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8473-8478Crossref PubMed Scopus (45) Google Scholar). The Sm-binding element is required for SL RNA trans-splicing in Ascaris (23Hannon G.J. Maroney P.A. Yu Y.T. Hannon G.E. Nilsen T.W. Science. 1992; 258: 1775-1780Crossref PubMed Scopus (48) Google Scholar) but not in Leptomonas (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar). We demonstrated recently that the T tract downstream of the SL RNA gene is a transcription termination element and that staggered T tract termination products are processed via nucleolytic cleavage to the base of stem-loop III (24Sturm N.R. Yu M.C. Campbell D.A. Mol. Cell. Biol. 1999; 19: 1595-1604Crossref PubMed Scopus (59) Google Scholar). The signals for 3′-processing begin in the Sm-binding site at position 76 and include the structure, but not content, of stem-loop III. Studies in Leptomonas seymouridemonstrated that mutation of a variety of elements in the intron was acceptable for trans-splicing (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar), whereas inLeptomonas collosoma the loop portions of stem-loops II and III were tolerant to insertions but not to replacement with theTrypanosoma brucei intron (25Goncharov I. Xu Y. Zimmer Y. Sherman K. Michaeli S. Nucleic Acids Res. 1998; 26: 2200-2207Crossref PubMed Scopus (12) Google Scholar). By contrast, the bulge of stem-loop II was critical for trans-splicing inAscaris (26Denker J.A. Maroney P.A. Yu Y.T. Kanost R.A. Nilsen T.W. RNA (NY). 1996; 2: 746-755PubMed Google Scholar). In this paper we report that methylation of nucleotides in the cap 4 structure of the Leishmania SL RNA is influenced by formation of stem-loop III, the Sm-binding site, and specific sequences in stem-loop II. The methylation of the cap 4 structure correlates with correct 3′ end formation; defects in 3′ end processing and cap 4 formation result in failure of the mutated SL RNA to undergotrans-splicing. However, correct maturation of the SL RNA is not sufficient to obtain a positive splicing phenotype since mutation of nucleotides in the stem I region of the intron can also result in loss of function. Our data from L. tarentolae broadly reflect the results obtained in vitro in the nematodeAscaris, where nucleotides in stem-loop II and the Sm-binding site are necessary for splicing. Our data broadly contrast the results obtained in Leptomonas, where the structure and not the primary sequence of stem-loop II was necessary for splicing, and where the Sm-binding site and stem-loop III were not required for splicing. A model summarizing the features of the LeishmaniaSL RNA involved in maturation and trans-splicing is presented. Mutagenesis was performed using the Sculptor Mutagenesis kit (Amersham Pharmacia Biotech) or using PCR to generate mutagenized DNA fragments for subcloning into the transfection vector. Mutated fragments were cloned for transfection into a pX plasmid (27LeBowitz J.H. Coburn C.M. McMahon-Pratt D. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9736-9740Crossref PubMed Scopus (190) Google Scholar) containing an SL RNA gene (9Saito R.M. Elgort M.G. Campbell D.A. EMBO J. 1994; 13: 5460-5469Crossref PubMed Scopus (58) Google Scholar). Transfections were performed by electroporation as described (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). RNA was purified using TriZOL reagent (Life Technologies, Inc.) and was electrophoresed through 1.1% agarose-formaldehyde, blotted, and hybridized as described previously (28Sturm N.R. Kuras R. Büschlen S. Sakamoto W. Kindle K.L. Stern D.B. Wollman F.-A. Mol. Cell. Biol. 1994; 14: 6171-6179Crossref PubMed Scopus (41) Google Scholar). Quantitation was performed using a PhosphorImager (Molecular Dynamics). To assay fortrans-splicing of mutant-SL RNAs, complementary oligonucleotides were hybridized to Arl mRNA (29Sturm N.R. Van Valkenburgh H. Kahn R. Campbell D.A. Biochim. Biophys. Acta. 1998; 1442: 347-352Crossref PubMed Scopus (8) Google Scholar) and extended by Moloney murine leukemia virus-reverse transcriptase (RT) to produce templates for PCR analysis (RT-PCR (30Sturm N.R. Simpson L. Cell. 1990; 61: 871-878Abstract Full Text PDF PubMed Scopus (89) Google Scholar)) with a second, SL-specific oligonucleotide. The following oligonucleotides were used: Arl(−)68, 5′-TGCGGATCGCCTTCTGGCCACC; LtSL5′RI, 5′-GGGAATTCGCTTTCAACTAACGCTAT; 30/39–5′HI, 5′-GGGATCCTGTATCAGTTTCAGCCT. Amplification products were run in 1.5% agarose gels, denatured, blotted, and hybridized with 28/39-tag oligonucleotide (5′-ACTTCCTCGAGGCTGAA) to detect the exon tag or wild-type (5′-CAATAAAGTACAGAAACTGA) exon sequence (corresponding to positions 20–39). The expected product size is ∼300 bp. 5′ cap 4 methylation was assayed by primer extension of total RNA with an exon tag-specific oligonucleotide (28/39-tag) as described previously (9Saito R.M. Elgort M.G. Campbell D.A. EMBO J. 1994; 13: 5460-5469Crossref PubMed Scopus (58) Google Scholar, 11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To localize specific elements within the intron of the SL RNA that play a role in the process of trans-splicing, a systematic mutagenesis approach was adopted (Fig. 1). To differentiate mutated, episomally derived SL RNA from the endogenous WT SL RNA population, an exon tagged at positions 28 and 30–39 (28/39), which was previously shown to trans-splice accurately and efficiently (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), was used as a molecular tag (tSL RNA) for detection by hybridization. A series of linker scan (CTCGAGCTCA) mutations in the tSL RNA gene was created for transfection to L. tarentolae. Two mutations in the 40–49 region were created as follows: a mutant with alterations in bases 43 and 44 (43/44 = GTversus TG in WT) tested a postulated SL RNA-U6 snRNA interaction (31Xu G.L. Wieland B. Bindereif A. Mol. Cell. Biol. 1994; 14: 4565-4570Crossref PubMed Scopus (27) Google Scholar), and a second mutant, altered at positions 42–48 (42/48), changed all but the splice donor site with the linker scan sequences. Subsequent intron mutations continued from position 50 (52/59) and proceeded through the end of the intron. Three mutations lay downstream of the mature 3′ end of the SL RNA transcript (position 96) and were included to identify potential adjacent expression elements (100/109, 110/119, 120/129; WT sequence not shown). Analysis of total RNA from the transfectants demonstrated tSL RNA (∼96 nt) in all the samples by low resolution formaldehyde-agarose gel blotting (Fig. 2 A). A broadened size range from the wild-type (WT) 96 nt to at least 175 nt was noted for the 100/109 tSL RNA, consistent with discrete higher molecular weight bands visible in higher resolution gel analyses (24Sturm N.R. Yu M.C. Campbell D.A. Mol. Cell. Biol. 1999; 19: 1595-1604Crossref PubMed Scopus (59) Google Scholar). The presence of tagged precursor SL RNA indicated that all the mutants have the potential to trans-splice. An artifactual transcript (∼1.45 kb) that accumulated in each sample provided an internal control for transfection, should stability be disrupted. The presence of the exon tag in a range of high molecular weight RNA species (500 nt to 9 kb) in the tSL, 43/44, 100/109, 110/119, and 120/129 samples suggested that active trans-splicing was occurring in these transfectants (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Splicing of 43/44 is consistent with results from a similar study in Leptomonas (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar). Conversely, splicing of the tagged exon was impaired in the 42/48, 52/59, 62/69, 70/79, 80/89, and 90/99 mutants, where only substrate tSL RNA and the artifactual transcripts accumulated. The levels of accumulating tSL RNAs varied relative to the artifactual transcripts and the episomally encoded drug selectable marker mRNA NEO (data not shown). The 52/59 mutant in particular showed an increased accumulation of substrate molecules relative to other non-splicing mutants. In addition, trans-splicing was assayed by RT-PCR (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 29Sturm N.R. Van Valkenburgh H. Kahn R. Campbell D.A. Biochim. Biophys. Acta. 1998; 1442: 347-352Crossref PubMed Scopus (8) Google Scholar) to detect low levels of splicing (Fig. 2 B). When “total” SL primer (i.e. will amplify from both WT and tSL exons) was used in the amplification, all samples showed the positive control WT amplification products, but the 28/39-tag oligonucleotide hybridized only to the tSL, 43/44, 100/109, 110/119, and 120/129 products, consistent with the total RNA blot analysis. However, using a tSL-specific primer for amplification, some level of splicing was detected in all but the 70/79 mutant. These experiments included WTL. tarentolae RNA, no reverse transcriptase, and no RNA reactions as negative controls for contamination and dependence on the use of RNA templates. Furthermore, a promoter knockout in combination with tSL (−67/−58 + tSL (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar)) was used as a control for spurious PCR amplification; this cell line resulted in an artifactual ∼1.45-kb transcript containing the 28/39-tag sequence but no mature tSL RNA (shown in Fig. 5 B) and did not yield a tSL PCR product. Previously determined phenotypes for cap 4 and 3′ end formation (24Sturm N.R. Yu M.C. Campbell D.A. Mol. Cell. Biol. 1999; 19: 1595-1604Crossref PubMed Scopus (59) Google Scholar) are also indicated in Fig. 2. Thus, trans-splicing was adversely affected in mutants 42/48, 52/59, 62/69, 80/89, and 90/99 and appeared to be abolished in mutant 70/79. Structural analyses of the SL RNA predict three stem-loop structures and a single-stranded region containing the Sm-binding site (Ref. 17LeCuyer K.A. Crothers D.M. Biochemistry. 1993; 32: 5301-5311Crossref PubMed Scopus (77) Google Scholar; Fig. 1). Previously, it was demonstrated that stem-loop I is not required for trans-splicing in L. tarentolae(11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Because trans-splicing was reduced or abolished in mutants 52/59, 62/69, 70/79, 80/89, and 90/99, we considered the effects of mutations in stem-loop II, the Sm-binding site, and stem-loop III on SL RNA trans-splicing with regard to the structural or sequence elements. The mutations described below are organized with regard to both these elements and the linker scan mutation results in the following order: structural features of stem-loop II, fine analysis of the 70–81 region which includes part of stem-loop II and the Sm-binding site, and features of stem-loop III. Two mutations, 52/59 and 62/69, disrupted stem-loop II (Fig. 3 A) and were not efficiently trans-spliced (Fig. 2). To address the importance of stem-loop II, 52/59 was further mutated to restore base pairing (52/59 + 65/72; Fig. 3 A); this replaced the stem structure but with a different sequence content than WT. A further mutation was designed (42/48 + 77/80) to restore a possible extension of stem-loop II in the 45–48 region, which was disrupted by mutations 42/48 and 70/79 (Fig. 3 A). 77/80 was also tested for independent effects due to its disruption of the conserved Sm-binding site. Neither of the compensatory base pairing mutations restoredtrans-splicing (Fig. 3 B). 77/80 alone or in combination with 42/48 resulted in extended, heterogeneous 3′ end formation (data not shown) consistent with the 70/79 phenotype (24Sturm N.R. Yu M.C. Campbell D.A. Mol. Cell. Biol. 1999; 19: 1595-1604Crossref PubMed Scopus (59) Google Scholar), whereas 52/59 and 52/59 + 65/72 possessed correct 3′ ends (data not shown). 42/48 + 77/80, 52/59 + 65/72, and 77/80 showed undermethylated cap 4 structures (data not shown), as did 42/48, 52/59, and 70/79 (24Sturm N.R. Yu M.C. Campbell D.A. Mol. Cell. Biol. 1999; 19: 1595-1604Crossref PubMed Scopus (59) Google Scholar). Both the structure and sequence content of stem-loop II are thus important features in the maturation of the SL RNA precursor. The structure alone is not sufficient to direct either cap 4 methylation or splicing. The stem-loop II extension structure may play an intermediate role in the splicing pathway, but it is not sufficient to restore processing or splicing. Because 70/79 altered most of the Sm-binding site and resulted in no trans-splicing and defects in both 5′ and 3′ end formation, we examined the area in finer detail. A 2-bp transversion (TV) series was created from position 70 to 81; in addition, 70/79 and 75/81 TV mutations were made (Fig.4 A). It should be noted that 70/71 and 72/73 comprise part of stem-loop II (see Fig. 1) and that 74/75 TV may extend the Sm sequence (AAUCUUUUGG). The total RNA of these transfectants revealed a variety of phenotypes for trans-splicing and methylation. By formaldehyde-agarose gel analysis, only the 74/75-tSL RNA was an efficienttrans-splicing substrate, with low levels of splicing evident in 72/73 (Fig. 4 B). The presence of tSL RNA but lack of the 1.45-kb artifact RNA in mutants 76/77, 70/79 TV, and 75/81 suggested additional increased stability phenotypes. Primer extension revealed an intriguing gradient of SL RNA cap 4 methylation in the 70/71, 72/73, and 74/75 mutants (Fig. 4 C), which showed low (5%), medium (40%), and normal (75%) methylation, respectively, and were trans-spliced proportional to their methylation state. Thus, as a component of the Sm-binding consensus, A75 does not appear to be an essential nucleotide in Leishmania; alternatively, the 74/75 mutation is a biologically acceptable extension of the Sm site. Mutations 80/89 and 90/99 disrupted stem-loop III (Fig. 5 A) and resulted in 3′-extended, undermethylated tSL RNAs (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) that were not efficientlytrans-spliced. To examine further the role of stem-loop III structure, a series of mutations was created that disrupted and then replaced 1 or 3 bp of the stem and that altered the loop sequence (Fig.5 A). The single base disruptions were expected to disrupt only one rung of the stem, and thus lead to a minor size difference in the tSL RNA, whereas the triple base mutations were designed to disrupt the stem completely. Formaldehyde-agarose gel analysis of total RNA showed that the single base pair-disrupted SL RNAs (83, 96) could be trans-spliced, but the triple base pair-disrupted SL RNAs (83/85, 94/96) were nottrans-spliced efficiently (Fig. 5 B). The base pairing compensated SL RNAs (83 + 96, 83/85 + 94/96) were both efficient splicing substrates. The 88/91 loop mutation SL RNA appeared to be spliced with lowered efficiency based on the reduced mRNA smear relative to the tSL RNA substrate levels. These experiments show that the structure, but not the primary sequence, of stem-loop III is necessary for trans-splicing. Primer extension analysis indicated that the cap 4 methylation patterns (Fig. 5 C) correlated with the levels oftrans-splicing. 83, 96, and 83 + 96 were methylated efficiently and trans-spliced relative to the abundance of free tSL RNA (Fig. 5 B) and by total SL RT-PCR assays; 83/85 and 94/96 showed less than 5% methylation and had splicing that was only detectable by the mutation-specific RT-PCR assay (data not shown). In mutant 83/85 + 94/96, the tSL RNA cap 4 was methylated to WT levels, thus the compensating mutations, which restored stem-loop III, also restored a structural signal for the cap 4 methylase. 88/91 showed approximately 50% methylation (Fig. 5 C) and displayed reduced splicing (Fig. 5 B); the intron tag previously used to follow SL RNA transcription (9Saito R.M. Elgort M.G. Campbell D.A. EMBO J. 1994; 13: 5460-5469Crossref PubMed Scopus (58) Google Scholar) was inserted into this loop and does not interfere with cap 4 methylation of tagged SL RNA (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Thus, methylation is linked to the formation of a minimum of 4/5 bp stem in stem-loop III. We have made a series of mutations in the region downstream of the exon in the SL RNA gene to examine effects ontrans-splicing. We have assayed for the ability of mutated SL RNA to trans-splice, and we have correlated this with correct cap 4 formation, transcription termination, and 3′ end processing as determined here and elsewhere (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 24Sturm N.R. Yu M.C. Campbell D.A. Mol. Cell. Biol. 1999; 19: 1595-1604Crossref PubMed Scopus (59) Google Scholar). A summary of nine phenotypes associated with the intron mutations is presented in TableI. In the majority of mutants, reduction or loss of trans-splicing correlates with defects in cap 4 methylation and 3′ end maturation. Where cap 4 methylation and 3′ end formation are WT, primary catalytic elements may have been mutated (e.g. 42/48).Table ISummary of mutant phenotypestrans-Splicing5′ end, cap 43′ endMutants, +tSLAgaroseRT-PCRstlpIIIaNucleolytic processing to base of stem-loop III.T trackbStaggered T track termination products.+++++++++++++++tSL (≅WT), 43/44, 110/119, 120/129, 74/75, 83, 83/85 + 94/96, 106/107cSee Ref. 24.++++++++−100/109, 102/107,cSee Ref. 24. 104/107cSee Ref. 24.+++++++++++88/91+++++++++72/73−++++++++++42/48−+−++++++52/59, 62/69, 52/59 + 65/72, 70/71−+−−+++80/89, 90/99, 83/85, 94/96−−−++++80/81−−−−+++70/79, 75/81, 76/77, 78/79, 80/81The symbols used are: +++, WT activity; +, reduced activity; −, ≤5% activity.a Nucleolytic processing to base of stem-loop III.b Staggered T track termination products.c See Ref. 24Sturm N.R. Yu M.C. Campbell D.A. Mol. Cell. Biol. 1999; 19: 1595-1604Crossref PubMed Scopus (59) Google Scholar. Open table in a new tab The symbols used are: +++, WT activity; +, reduced activity; −, ≤5% activity. The mutant phenotypes have allowed us to evaluate structures and elements that may be important for SL RNA maturation and to propose a possible pathway for discrete processing steps in L. tarentolae (Fig. 6). In this model, the T tract functions as a transcription termination element (24Sturm N.R. Yu M.C. Campbell D.A. Mol. Cell. Biol. 1999; 19: 1595-1604Crossref PubMed Scopus (59) Google Scholar). The Sm-binding site and stem-loop III structure are required to allow precise 3′ end maturation. Formation of the mature 3′ end, along with elements within stem-loop II, are required for cap 4 synthesis, as is the 10-29 region of the exon (11Sturm N.R. Fleischmann J. Campbell D.A. J. Biol. Chem. 1998; 273: 18689-18692Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Nucleotides within the intron region of stem I are likely to be required for splicing catalysis. Transcription of the SL RNA gene in vivo terminates in a T tract of greater than six T residues (Fig. 6, step 1). Mutation of the Sm-binding site (e.g. 76/77) and stem-loop III (e.g. 90/99) yielded mutants with defects in the nucleolytic formation of the mature 3′ end of the SL RNA, demonstrating a cooperative function between these two elements (step 2). Structure rather than primary sequence of the stem-loop III stem was required for trans-splicing. Consistent with this, an 8-nucleotide insertion in the loop of stem-loop III in L. collosoma did not affect trans-splicing (25Goncharov I. Xu Y. Zimmer Y. Sherman K. Michaeli S. Nucleic Acids Res. 1998; 26: 2200-2207Crossref PubMed Scopus (12) Google Scholar); however, in L. seymouri deletion of stem-loop III resulted in an actively trans-spliced and normally methylated SL RNA (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar). Mutants that do not terminate accurately due to the disruption of their downstream T tract show an intermediate cap 4 phenotype (e.g. 100/109) that we interpret as indicative of a temporal order of 3′-processing (step 2) followed by cap 4 methylation (step 3). Methylation alone is not sufficient to confer splicing potential, since the 42/48 mutant is normally methylated but a marginal trans-splicer (a phenotype similar to the Leptomonas Δstl II mutant (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar)). This suggests that the intron region of stem I may be involved in catalytic steps oftrans-splicing (step 4). Positions 43/44 are not implicated in the 42/48 splicing defect. Nucleotides 42 + 45–48 may interact with other splicing entities such as SLA1 RNA (32Roberts T.G. Sturm N.R. Yee B.K. Yu M.C. Hartshorne T. Agabian N. Campbell D.A. Mol. Cell. Biol. 1998; 18: 4409-4417Crossref PubMed Scopus (41) Google Scholar) or U5 snRNA (21Dungan J.M. Watkins K.P. Agabian N. EMBO J. 1996; 15: 4016-4029Crossref PubMed Scopus (50) Google Scholar, 22Xu Y. Ben-Shlomo H. Michaeli S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8473-8478Crossref PubMed Scopus (45) Google Scholar). Nucleotides +7 and +8 of the intron (equivalent to positions 46–47 in L. tarentolae) in L. collosoma can be mutated without affecting trans-splicing (22Xu Y. Ben-Shlomo H. Michaeli S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8473-8478Crossref PubMed Scopus (45) Google Scholar). The essential nature of the Sm-binding site for in vivo trans-splicing in L. tarentolae agrees with data fromAscaris, where in vitro studies showed that the Sm-binding site was required for SL RNA trans-splicing (23Hannon G.J. Maroney P.A. Yu Y.T. Hannon G.E. Nilsen T.W. Science. 1992; 258: 1775-1780Crossref PubMed Scopus (48) Google Scholar). We are aware of the limitations in comparing in vitro trans-splicing assays with in vivo splicing phenotypes (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar). We generally interpret lack of splicing phenotypes as due to splicing catalysis or, when they are detected, to maturation-related defects, but at this level of analysis our studies cannot exclude other explanations, for example impaired nucleus-cytoplasm-nucleus shuttling of the SL RNA. In contrast, a Leptomonas Sm-binding site mutant (“sub-Sm” (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar)) that closely approximated a non-splicing, 3′-extended L. tarentolae counterpart (78/79) was viable for ribonucleoprotein assembly and splicing. Splicing in the 74/75 mutant, which has a transversion of the A of the Sm site, may reflect flexibility within the conserved Sm-binding site, as found in the U5 snRNA of Saccharomyces (33Jones M.H. Guthrie C. EMBO J. 1990; 9: 2555-2561Crossref PubMed Scopus (64) Google Scholar). An additional experimental difference to be considered between the two studies in trypanosomatids is that the exon tag in L. tarentolae consisted of 11 mutated nucleotides, whereas that in Leptomonas consisted of one mutated nucleotide. The contradictory results for Sm-binding site and stem-loop III in the kinetoplastids may be informative in interpreting our results as follows: given that stem-loop III does not contain primary sequence necessary for trans-splicing inLeptomonas, our non-splicing phenotypes may be secondary effects (e.g. additional 3′-extended sequences may inhibit the folding of stem-loop II). As indicated by mutant 70/71, elements in stem-loop II are required for the intron component of cap 4 formation. In Leptomonas, deletion or substitution of stem-loop II above the bulge position did not affect cap 4 methylation (12Lücke S. Xu G.L. Palfi Z. Cross M. Bellofatto V. Bindereif A. EMBO J. 1996; 15: 4380-4391Crossref PubMed Scopus (33) Google Scholar), suggesting that some of the methylation phenotypes that we observed may be secondary effects due to interference with secondary or tertiary structure formation within the SL RNA itself or between the SL RNA and other splicing components. Similar to L. tarentolae, nucleotides in stem-loop II of theAscaris SL RNA (positions 39–42 and 61–65) are essential for trans-splicing and include a single nt (U, position 62) bulge (26Denker J.A. Maroney P.A. Yu Y.T. Kanost R.A. Nilsen T.W. RNA (NY). 1996; 2: 746-755PubMed Google Scholar). Consistent with the L. tarentolae results and contrasting the Leptomonas results, deletion of nucleotides 59–68 in stem-loop II of the Leishmania amazonensis SL RNA (Δ1) resulted in either inefficient or no trans-splicing (8Agami R. Aly R. Halman S. Shapira M. Nucleic Acids Res. 1994; 22: 1959-1965Crossref PubMed Scopus (42) Google Scholar). In this study we have identified how various structures within the intron of the SL RNA are interdependent in 3′ end formation and cap 4 methylation, and we provide a possible pathway to describe the processing steps. We distinguish among trans-splicing negative mutants that are defective for discrete steps in SL RNA maturation and a mutant that may be affected in catalytic steps. These and subsequent mutants will facilitate studies on the intracellular trafficking of SL RNA, the identification of newtrans-spliceosomal proteins and protein-RNA interactions, and allow testing of new models of interactions with other splicing RNA/ribonucleoproteins. We thank Steve Beverley for the pX plasmid, T. Guy Roberts, and Michael C. Yu for stimulating discussions, and Doug Black, Larry Feldman, and Dan Ray for critical reading of the manuscript.
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