I.=) 1994 Oxford University Press Nucleic Acids Research, 1994, Vol. 22, No. 6 1029-1036 A group Ill twintron encoding a maturase-like gene excises through lariat intermediates Donald W.Copertino1 2+, Edina T.Hall2,§, Fredrick W.Van Hook2, Kristin P.Jenkins2 and Richard B.Hallick1,2* 1Departments of Biochemistry and 2Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA Received December 9, 1993; Revised and Accepted February 10, 1994 ABSTRACT The 1605 bp intron 4 of the Euglena gracilis chloroplast the closely related nonphotosynthetic euglenoid Astasia longa psbC gene was characterized as a group Ill twintron (8, 9). composed of an internal 1503 nt group Ill intron with Euglena chloroplast genes also contain 15 twintrons, introns- an open reading frame of 1374 nt (ycfl3, 458 amino within-introns, that are sequentially spliced (3). There are acids), and an external group Ill intron of 102 nt. twintrons with one intron internal to another intron (2, 6, 10), Twintron excision proceeds by a sequential splicing and complex twintrons with multiple internal introns (11; for pathway. The splicing of the internal and external group review, see 3). Group II and group III introns can occur as Ill introns occurs via lariat intermediates. Branch sites internal and/or external introns of twintrons. Excision of internal were mapped by primer extension RNA sequencing. introns occurs prior to excision of external introns. The excision The unpaired adenosines in domains VI of the internal of internal group IH introns of twintrons can proceed from and external introns are covalently linked to the 5' multiple 5' and 3' splice sites (6, 1 1). Internal introns are believed nucleotide of the intron via 2'-5' phosphodiester bonds. to interrupt functional domains of external introns, neccesitating This bond is susceptible to hydrolysis by the de- sequential splicing (3, 4). branching activity of the HeLa nuclear S100 fraction. The psbC gene of the Euglena gracilis chloroplast DNA, which The internal intron and presumptive ycfl3 mRNA encodes a 44 kDa protein component of the photosystem II accumulates primarily as a linear RNA, although a lariat reaction center (12, 13), contains an intron with an open reading precursor can also be detected. The ycfl3 gene frame (ORF) of 1374 nt (ycfl3, 458 codons ). It had previously encodes a maturase-like protein that may be involved been proposed that this intron is an atypical group II intron (14). in group Ill intron metabolism. We examined the splicing of the 1.6 kb intron by direct RNA sequencing, cDNA cloning and sequencing, and northern INTRODUCTION hybridization. This intron is a group III twintron, and ycfl3 is encoded within the internal group IH intron. Splicing of the The complete DNA sequence of the Euglena gracilis chloroplast internal and external group III introns occurs via lariat genome is known (1). The chloroplast genes ofEuglena contain intermediates, establishing a functional role for group HI domain 155 introns, including 74 group II introns. Many of the Euglena VI. group II introns are smaller than those found in other organisms because domains I -IV are abbreviated (2-4). All possess a MATERIALS AND METHODS distinct catalytic domain V, and domain VI, which includes the unpaired adenosine residue that initiates nucleophilic attack at cDNA cloning and sequencing the 5' splicejunction. Euglena chloroplast genes also contain 64 Primers were synthesized by either The University of Arizona introns of a unique class designated group I (S). Group III Biotechnology Center or The Midland Certified Reagent introns are abbreviated group II introns that have retained group Company. Chloroplast RNA (ctRNA) was isolated and II-like 5' and 3' boundary sequences and a domain VI-like isopropanol-fractionated from purified chloroplasts as described structure, but lack domains HI-IV (2, 6, 7). It is not known if (10). DNA sequence coordinates (see below) have been described the putative group HI domain VI has a functional role during (1; EMBL accession number X70810, release 37, version 18). splicing in lariat formation. In many group HI introns, the region Two synthetic deoxynucleotide primers complementary to the between the 5' boundary and domain VI resembles group II intron RNA-like strand (cDNA primers) at the psbC intron 4-exon 5 domain ID. Group III introns also occur in the plastid genes of boundary (5'-GGGGATCCGCTACATGAGCTTAATTTAG-3', *To whom correspondence should be addressed Present addresses: +Department of Neurobiology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037 and 1Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA 1030 Nucleic Acids Research, 1994, Vol. 22, No. 6 positions 20234-20253), and in psbC exon 5 (5'-GGAA- hybridization, and post-hybridization procedures were performed CAAAATGAGCTACTTC-3', positions 20300-20319) were as described (10). used to prime cDNA synthesis with total ctRNA as template (2). Another deoxyoligonucleotide 5'-GGTAATTCTAGACTTAT- RNase H digestion and debranching of RNA lariats AAATGTATCAGG-3' of the RNA-like strand (PCR primer) in 12 Atg of HMW ctRNA was dried with or without 500 ng of a psbC exon 4 (positions 18596-18624) was used to amplify the purified deoxyoligonucleotide primer 5'-CCTCGCGTATAAA- resulting cDNAs by the polymerase chain reaction (PCR) (Perkin- GTAAAGAGAG-3' complementary to the RNA-like strand in Elmer, Cetus) as described (10). The PCR product derived from psbC intron 4 at positions 19555- 19577. Pellets were the partially spliced psbC RNA was digested with Xba I and resuspended in 10 tl 25 mM Tris-HCl, pH 7.5; 1 mM EDTA; BamHI, gel-purified by crush and soak (15), and cloned into the 50 mM NaCl; heated at 68°C for 10 minutes, and slow cooled Xba I and BamHI sites of pBluescript KS - vector (Stratagene to 30°C. An equal volume of 2 x RNase H buffer (40 mM Cloning Systems). This plasmid DNA was designated pEZC 1041 Tris-HCl, pH 7.5; 20 mM MgCl2; 100 mM NaCl; 2 mM (Figure 1). The PCR product derived from the fully splicedpsbC DTT; 60 ,ug/ml BSA) with or without 0.5 units RNase H RNA was directly cloned into ddT-tailed pBluescript KS- (16). (Promega) was then added and the reactions were incubated at This plasmid DNA was designated pEZC 1091 (Figure 1). The 30°C for 1 hour. Reactions were terminated by adding 100 yl plasmid DNA pEZC1041.1 was generated by digesting stop mix (38.5 Atg/ml tRNA, 20 mM EDTA, 300 mM NaOAc). pEZC1041 with HincH and SmaI, releasing a 40 bp fragment The mixture was phenol extracted, and products were ethanol of the multiple cloning site that contains the EcoRI site, and precipitated. Control RNase H experiments were performed using religating the remaining plasmid DNA. To synthesize an external 2.2 x 105 dpm of transcript synthesized in vitro using T7 RNA group III intron-specific riboprobe, pEZC 1041.1 was linearized polymerase from pEZC2000 linearized with BamHI. This at the EcoRI site within the 5' region of the external intron transcript is 1688 nt in length (41 nt vector, 31 nt exon 3, 21 (position 18648). This plasmid DNA also contains 10 nt of the nt 5' portion of external intron, 1503 nt internal intron, 81 nt 3'-exon as part of the cDNA primer. The plasmid cDNA inserts 3' portion of external intron, and 11 nt exon 4). Transcription, were completely sequenced on both strands by dideoxy DNA purification, and self-splicing of the 887 nt yeast mitochondrial sequencing using the Sequenase kit (U. S. Biochemical). intron aI5,y in 0.5 M (NH4)2SO4 from pJD20 was done as described (19). 6 mg aliquots of HMW ctRNA and purified aI5,y Genonic cloning and sequencing lariat were treated with HeLa S100 nuclear extract for 1 hour Euglena gracilis chloroplast DNA (ctDNA) was isolated as as described (20). Reactions were subjected to electrophoresis described (17). The cDNA primer at the psbC intron 4-exon 5 through 4% polyacrylamide gels containing 8 M urea in 44.5 boundary and the PCR primer in exon 4 described above were mM Tris-borate, 44.5 mM boric acid, 1 mM EDTA (0.5x used to amplify the corresponding region of the chloroplast TBE), electroblotted to a Genescreen membrane, and either genomic DNA. 200 ng of ctDNA in 2 ,u of ddH20 was boiled exposed to film (in vitro controls) or hybridized with a riboprobe for 10 minutes, placed on ice, and amplified by PCR. 430 ng specific for the internal ycfl3-containing group HI intron as of cDNA primer and 400 ng of PCR primer were included in described above (see Figure 2). the amplification. Prior to amplification, a 2 minute 'hot start' at 80°C was done in the absence of Taq polymerase. The Taq Primer extension cDNA sequence analysis polymerase was then added and amplification preceeded for 30 Purified deoxyoligonucleotide primers 5'-CACTAATTTAA- cycles. The DNA was denatured at 940C for 1 minute, annealed TTAAACTACTAAC-3 ', 5'-GGAACAAAATGAGCTACT- at 42°C for 1 minute, and extended at 72°C for 2 minutes. The TC-3', and 5'-GAAACTTGAAAAATTTCATAAAAAATC-3' PCR product was then digested with Xba I and BamHI, gel- complementary to the RNA-like strand in psbC at positions purified by GENECLEAN (BIO 101), and cloned into the Xba 20202 -20225 (intron 4), 20300-20319 (exon 5), and I and BamHI sites of pBluescript KS-. The plasmid DNA was 18726 - 18745 (ycfl3), respectively, were 5' end-labelled using designated pEZC2000. The internal group III intron-specific polynucleotide kinase (BRL). 32P-labelled primer (1 x 107 dpm) sequences corresponding to positions 19154- 19587 were cloned was dried with 12 ,ug of total ctRNA or HMW ctRNA, or 2 tg by gel-purifying the 437 bp HindIllEcoRI fragment from sRNA and resuspended in 12 ,a1 of 200 mM KCl; 10 mM pEZC2000 using GENECLEAN, and ligating the fragment into Tris-HCl, pH 8.3 at 42°C.