4750–4754 Nucleic Acids Research, 2000, Vol. 28, No. 23 © 2000 Oxford University Press

Mitochondrial of myxomycetes terminate with non-encoded 3′ poly(U) tails Tamara L. Horton and Laura F. Landweber*

Departments of Ecology and Evolutionary Biology, , Princeton University, Princeton, NJ 08544, USA

Received July 26, 2000; Revised and Accepted October 10, 2000

ABSTRACT even been detected on the ends of many unedited and partially edited pre-mRNAs in Trypanosoma brucei, and are thought to We examined the 3′ ends of edited RNAs from the be added by a rampant terminal uridyl transferase activity myxomycetes Stemonitis flavogenita and Physarum operating on editing intermediates (14). polycephalum using a modified anchor PCR Physarum polycephalum is a myxomycete, or plasmodial approach. Surprisingly, we found that poly(A) tails slime mold, that is amenable to cellular study in the laboratory. are missing from the cytochrome c oxidase subunit 1 The production of functional mitochondrial transcripts for mRNA (coI) from both species and the cytochrome c almost all of P.polycephalum’s messenger and structural RNAs oxidase subunit 3 mRNA (cox3)fromP.polycephalum. requires several types of RNA editing. Many single cytidine Instead, non-encoded poly(U) tails of varying length insertions, a small number of uridine and mixed dinucleotide were discovered at the 3′ ends of these transcripts. insertions, and a few instances of cytidine to uridine base These are the first described examples of 3′ poly(U) conversions modify the RNA sequences. For instance, the tails on mature mRNAs in any system. cytochrome c oxidase subunit 1 (coI)mRNAiseditedbyinsertion of 59 Cs, a single U and three mixed dinucleotides. Four C to U conversions are also found in this transcript (15). INTRODUCTION Here, we present our discovery of a new form of RNA processing that alters myxomycete mitochondrial transcripts. RNA molecules are routinely processed by splicing, editing We show that P.polycephalum and Stemonitis flavogenita both ′ and 3 , resulting in transcripts that contain have non-encoded 3′ poly(U) tails added to edited mito- information not encoded by the DNA genome. In eukaryotic chondrial mRNAs. The unusual tails on mRNAs that have also ′ nuclear genes, 3 end processing consists of mRNA cleavage undergone editing suggest a possible connection between these followed by the addition of a poly(A) tail by a group of factors, types of RNA sequence change. including poly(A) polymerase and the C-terminal domain of RNA polymerase II (1). Some prokaryotic, mitochondrial and chloroplast mRNAs also have poly(A) tails. While poly- MATERIALS AND METHODS adenylation of eukaryotic nuclear genes enhances stability and translation initiation (2), in prokaryotes and chloroplasts poly- Cultures adenylation provides a signal for rapid RNA degradation (3,4). Freeze-dried cultures of S.flavogenita (24714) were obtained In mitochondrial systems, polyadenylation has a variety of from the American Type Culture Collection, and grown on functions: creating stop codons on most human mitochondrial half-strength cornmeal agar plates into full size plasmodia. transcripts (5), possibly signaling for translation initiation in Plates of P.polycephalum plasmodia were obtained from trypanosomes (6), and stimulating quick degradation of a plant Carolina Biologicals. mitochondrial mRNA (7) and some trypanosome RNAs (8). Isolation of nucleic acids Adenosine is not the only nucleotide found in unencoded 3′ tails. Kinetoplastids, unicellular eukaryotes with extensive RNA and DNA were extracted from the slime mold plasmodia uridine insertional/deletional editing in their mitochondria, by use of Trizol reagent from Life Technologies. RNA was have 3′ polyuridine tails on guide RNAs (gRNAs), a population treated with DNase (Promega); DNA was treated with RNase A of mitochondrial RNAs involved in editing (9). These non- (Sigma). Nucleic acids were then extracted with phenol/ encoded poly(U) tails may aid the editing complex in holding chloroform, ethanol precipitated, and resuspended in 10 mM Tris, together the broken halves of the purine-rich mRNA during the pH 7.4, 0.1 mM EDTA. cleavage stage of the editing process (10,11), or might denature secondary structure in the regions of the mRNAs being edited Artificial RNA tailing, reverse transcription and PCR of cDNA (12). The other class of non-messenger RNA encoded by the GTP tails were added to total RNA by yeast poly(A) kinetoplastid mitochondrial genome, the ribosomal RNAs, polymerase as described (16) in the presence of 0.5 mM GTP. also have non-encoded 3′ poly(U) tails (13). Poly(U) tails have Reverse transcription was performed using SuperScript II

*To whom correspondence should be addressed. Tel: +1 609 258 1947; Fax: +1 609 258 1682; Email: [email protected] Present address: Tamara L. Horton, Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Nucleic Acids Research, 2000, Vol. 28, No. 23 4751 reverse transcriptase from Life Technologies, and primer UXR′C12 on S.flavogenita RNA, and TXRC12 for P.polycephalum RNA (primer sequences listed below). The nested PCR of S.flavo- genita coI cDNA was performed for 20 cycles with primer CUAUXR′ andprimercoi551st,followedby20cycleswith CUAUXR′ and primer coi561st. Physarum polycephalum coI cDNA was amplified in 37 cycles of PCR, with primers 3PPcoiF and TXR. Physarum polycephalum cox3 cDNA was amplified in 40 cycles of PCR with primers 3PPcox3F and TXR. Physarum polycephalum mitochondrial LSU cDNA was amplified in a nested PCR of 20 cycles with primers 3PLSUF1 and TXR, followed by 25 cycles with primers 3PPLSUF and TXR. Physarum polycephalum nuclear SSU cDNA was amplified in 37 cycles of PCR with primers 3PPSSUF and TXR. Amplification of DNA Figure 1. Poly(U) tails on S.flavogenita coI cDNA. The 3′ untranslated region of S.flavogenita coI cDNA clones are aligned with directly sequenced DNA Walking PCR of S.flavogenita DNA was performed as PCR product (GenBank accession no. AF239222). The alignment begins with described (17). The single strand amplification was 40 cycles nucleotide 1805 (T) in the GenBank record, and the inferred stop codon is under- with primer coi551b. The second PCR was 22 cycles with lined. Dotted regions within clone sequences indicate identity with the DNA ′ sequence. The regions with white letters inside a black box indicate poly(U) tails. primer coi561st and UXR C12. The third PCR was 25 cycles The gray shaded regions designate the primer used for reverse transcription. Slight with primer coi581st and CUAUXR′. A single clone was variation in the length of poly(G) regions of reverse transcription primers may be obtained by this method, the plasmid isolated and sequenced. due to minor imperfections in the primer pool, or PCR slippage through the Primer stem-ptR was designed at the 3′ most extreme of this homopolymeric region. The final base of the primer is a non-C anchor base, intended to direct annealing location of this primer to the most 5′ end of a clone sequence, then used in a 40 cycle PCR with primer potentially long poly(G) run during reverse transcription. Non-primer-derived coi551st. The PCR product was precipitated and directly nucleotides beyond the poly(U) tails (black letters on a white background sequenced with primer coi551st and stem-ptR. within the clone sequences) are probably artifacts of the tailing proceedure, as explained in the Results. Cloning, purification and sequencing PCR products were cloned with the TOPO TA cloning kit from Invitrogen. Plasmids were purified with the High Pure Plasmid Isolation Kit from Boehringer-Mannheim/Roche. Both strands G tail to the RNA (16), then reverse-transcribed and amplified of all plasmids were sequenced at the Princeton University from the introduced tail into the coding region. SynSeq facility. Sequence analysis of six cloned cDNA fragments revealed Primer sequences that the 3′ end of S.flavogenita coI mRNA has a conventional termination codon (UAA), followed by a 24 nt untranslated region (Note that D designates a 1:1:1 mixture of A, G, and T.) and a homopolymeric tail of 20–31 nt (Fig. 1). Surprisingly, the UXR′C12 (5′-CUACUACUACUACTCGAGAATTCCCCCCCCCCCCD-3′) TXRC12 (5′-CATCATCATCATCTCGAGAATTCCCCCCCCCCCCD-3′) tail is not composed of poly(A), as expected, but consists CUAUXR′ (5′-CUACUACUACUACTCGAGAATT-3′) primarily of uridines. We recovered the DNA sequence in this TXR (5′-CATCATCATCATCTCGAGAATT-3′) region by a walking PCR approach (17), and found that the coi551st (5′-TTGTTAGCAAATGATTATCG-3′) poly(U) tail is not encoded in the genomic sequence (Fig. 1). ′ ′ coi551b (5 -biotin-TTGTTAGCAAATGATTATCG-3 ) Didymium nigripes coI cDNA clones also terminate with coi561st (5′-TACATTTCCTTTAACTGTTGC-3′) 3PPcoiF (5′-CGCCGTATTCCAGATTATCCTGATGC-3′) similar poly(U) tails (data not shown), although we did not 3PPcox3F (5′-CATGCTCCTTTCTCTATTTCTGATGG-3′) determine the corresponding DNA sequence. 3PLSUF1 (5′-TCTGTCTAGTACGAAAGGACTGG-3′) To expand our survey of the distribution of this type of 3PPLSUF (5′-TGAGCTGTTTGCGCACGCTCATTCGC-3′) ′ ′ poly(U) tailing in a myxomycete with a greater number of 3PPSSUF (5 -GTAAAACGAGTGCTTGAACAAGGCGTCC-3 ) ′ stem-ptR (5′-TAAGTAAATGCAGTAACATTTG-3′) published mitochondrial gene sequences, we amplified the 3 region of several P.polycephalum RNAs by the same modified anchor PCR technique. We examined the termini of two edited RESULTS mitochondrial mRNAs, an edited mitochondrial structural While investigating the distribution and types of RNA editing RNA, and a non-edited nuclear structural RNA. We found that in myxomycetes, we attempted to recover the 3′ end of the coI the non-encoded poly(U) tail is a common feature of the edited mRNA of S.flavogenita by anchor PCR, a technique that relies mitochondrial mRNAs in both species. on the presence of a 3′ poly(A) tail (18). Though sequences Although both species share the presence of a 3′ poly(U) tail obtained by this method extended to near the 3′ end of the on the coI transcripts, P.polycephalum’s coI tails are shorter predicted coding region, the sequences lacked a stop codon. than those of S.flavogenita. Physarum polycephalum’s coI tails RT and PCR products were not full length, due to annealing of are only 9–25 nt long, and vary in their start site on the RNA our modified poly(T) primer to the A-rich sequences still within over a 33 nt region (Fig. 2). In seven clones analyzed, only one the coding region. To circumvent the problems associated with tail contained a single cytidine residue, as compared to two out traditional anchor RT and PCR of a transcript of such a high A/T of six S.flavogenita clones, which contain one and three content (70%), we used poly(A) polymerase to add an artificial cytidines apiece. Another P.polycephalum clone contained a 4752 Nucleic Acids Research, 2000, Vol. 28, No. 23

Figure 3. Poly(U) tails on P.polycephalum cox3 cDNA. The 3′ untranslated region of P.polycephalum cox3 cDNA clones are aligned with corresponding ′ DNA sequence (GenBank accession no. AF084526). The first nucleotide of Figure 2. Poly(U) tails on P.polycephalum coI cDNA. The 3 untranslated the alignment corresponds to nucleotide 3368 (T) in the GenBank record, and region of P.polycephalum coI cDNA clones are aligned with DNA sequence the inferred stop codon is underlined. Annotation as in Figure 1. from Jonatha Gott (personal communication). Numerical notation is continuous with GenBank accession no. L14779. The inferred stop codon is underlined. Annotation as in Figure 1.

We also amplified the terminal region of a structural RNA, the mitochondrial large subunit rRNA (23S), which is edited by 52 C insertions and five dinucleotide insertions (19). guanidine residue amidst the uridine run. When the P.poly- Analysis of six clones corresponding to the 3′ end of the mito- cephalum tail sequences are aligned with their corresponding chondrial LSU transcript revealed only a few U residues: two regions on the mitochondrial genomic DNA sequence transcripts terminated with one U each, and one ended with (J.M.Gott, personal communication), the Us are not encoded in three Us (Fig. 4A). These ‘tails’ were added over a 7 bp region, the genomic copy. The cDNA sequences agree with nuclease though one of the clones that lacked any apparent U tail ended protection experiments that imply that the end of the P.poly- 30 bases downstream of the end of the earliest-terminating cephalum coI mRNA is ~50 bases downstream of the stop codon clone. Some clones had small poly(A) tails, but these could be (L.M.Visomirski-Robic and J.M.Gott, personal communication). artifacts of the artificial tailing process, as described above. RNA editing adds 32 Cs and a single UC dinucleotide to the To ascertain whether the tails were unique to mitochondrially cytochrome c oxidase subunit 3 (cox3)mRNAinP.poly- encoded transcripts, which had undergone RNA editing, we cephalum (19). Five out of six clones of the 3′ end of cox3 also examined the 3′ end of the P.polycephalum 18S nuclear- terminate with poly(U) tails (Fig. 3). The tails range from 12 to encoded small subunit (SSU) rRNA. No nuclear transcripts in 37 bases in length, with the starting point of the tails spanning P.polycephalum are known to exhibit editing. Six clones of the a 21 base region. The tails in these clones were composed SSU transcript displayed no evidence of poly(U) tails on the SSU uniformly of U residues. We note that for all of the P.poly- rRNA (Fig. 4B). These sequences also serve as a negative control cephalum cDNA transcripts, some clones contain a non-encoded for the poly(G) tailing and reverse transcription, as both A/G-rich sequence just upstream of the 3′ primer. These A/G treatments were performed singly for the total P.polycephalum stretches are probably caused by a few contaminating adenosines RNA sample. Detection of different RNA transcripts both with in the poly(A) polymerase-catalyzed G tailing reaction, since and without 3′ poly(U) tails by this method strengthens our yeast poly(A) polymerase catalyzes the addition of adenosine assertion that the poly(U) tails are naturally present on the twice as efficiently as guanosine (16). If the artificial tail were mitochondrial RNA transcripts. contaminated with a few As, then the 3′ anchor primer (TXRC12) would anneal slightly downstream of the real beginning of the artificial tail, resulting in the presence of these DISCUSSION A residues interspersed with poly(G) tails. The fact that these Although poly(A) tails are present on nuclear mRNAs of A/G regions are found in common on all P.polycephalum P.polycephalum (20), and common on mitochondrial mRNAs sequences, even those without poly(U) tails (see below), in some organisms, they are not known to be universally supports this explanation, and confirms a real difference present, and have not been directly detected on mitochondrial between poly(U)-tailed and non-poly(U)-tailed transcripts. RNAs of myxomycetes. We have found that at least one Nucleic Acids Research, 2000, Vol. 28, No. 23 4753

of poly(A) tails in other organisms. The tails may be actively targeted exclusively to mRNAs. In eukaryotic nuclear systems, where poly(A) tails are only found on mRNAs, their absence on rRNAs is explained by transcription of messenger and structural RNA by different polymerases, where pol II is activelyinvolvedinthetailaddition(1).Inmitochondrial systems, a single polymerase produces all transcripts. It is unclear how addition of poly(A) tails is restricted to mRNAs in mitochondria; in fact, a few mitochondrial RNAs with 3′ poly- adenylation have been detected in Plasmodium falciparum, mosquitoes and mammals (24). It is possible that some funda- mental difference in sequence or secondary structure of the P.polycephalum mitochondrial large subunit rRNA does not promote its termination by poly(U), or that another process specifically removes tails after addition. The poly(U) tail sequences in this study vary somewhat in length and overall composition; a few tails include cytidine and guanine residues. A previous study of edited trypanosome cDNAs concluded that though PCR-induced mutation occurred at a low frequency overall, the PCR error within long T homonucleotide stretches was much higher than the other regionsofthetemplate.Infact,innearly3kbofanalyzed sequence, 11 of 14 total PCR-induced mutations occurred in homo(T) regions, with the majority of these in the longest T stretch. Most mutations consisted of a single T insertion or deletion per poly(T) run, probably due to polymerase slippage, and there was also one T to C transition (25). However, even if Figure 4. Physarum polycephalum mitochondrial large subunit ribosomal most of the variation we observed within the mononucleotide RNA (mit LSU) cDNA and nuclear small subunit ribosomal RNA (SSU) runs of myxomycete poly(U) tails is real, similar or greater cDNA. (A) Physarum polycephalum mitochondrial large subunit ribosomal mixed base composition has been noted in some poly(A) RNA (mit LSU) cDNA clones are aligned with the DNA sequence (GenBank sequences in organelles. The poly(A) rich 3′ tail sequences of accession no. AF080602). The first position of the alignment corresponds to nucleotide 2783 (T) in the GenBank record. (B) Physarum polycephalum spinach chloroplast genes contain ~25% guanosines and a nuclear small subunit ribosomal RNA (SSU) cDNA clones are aligned with combined 5% uridines and cytosines (3). Trypanosome the DNA sequence (GenBank accession no. X13160). The alignment begins poly(A) tails also contain occasional U insertions, which may with nucleotide 1950 (G) in the GenBank record. Annotation as in Figure 1. be added either by the RNA polymerase or by the terminal (The extreme ends of the lower clones’ reverse transcription primer sequences have been omitted for clarity.) uridyl transferase (TUTase) activity present in these organisms (26). This study demonstrates that RNA processing of mitochondrial mRNA transcripts in myxomycetes includes not only RNA editing, but also 3′ polyuridylation. All of the mRNA transcripts S.flavogenita mitochondrial mRNA and two P.polycephalum in which we found 3′ poly(U) tails were also processed by mitochondrial mRNAs are not polyadenylated, but rather editing (21). To discern whether these two unique forms of polyuridylated. With the exception of the incompletely edited RNA processing are related, one might examine the 3′ end of a pre-mRNAs of kinetoplastid mitochondria (14), these are the mitochondrial mRNA uninvolved in RNA editing. However, first described poly(U) tails on mRNA transcripts in any all currently known P.polycephalum mitochondrial mRNAs organism. Interestingly, both kinetoplastids and myxomycetes require editing (19), and the editing process is extremely share RNA editing of mitochondrial transcripts, albeit by very efficient (27). Thus, while mysteries abound in the organelles of dissimilar and presumably independently evolved mecha- myxomycetes, further conclusions await progress in molecular nisms. However, in contrast to the poly(U)-tailed kinetoplastid techniques, such as isolation of non-tailing or non-editing pre-mRNAs, which appear to be transient intermediates in the mutants and the facile transformation of mitochondria. editing process, the myxomycete poly(U)-tailed transcripts appear to be mature mRNAs, as they show complete editing in the 3′ region that was amplified and sequenced (21). Because ACKNOWLEDGEMENTS insertional editing in myxomycetes is believed to be We gratefully acknowledge many helpful discussions with cotranscriptional, proceeding from the 5′ to the 3′ end of the Dennis Miller and Jonatha Gott, and thank Jonatha for transcript (22,23), the mRNAs are probably completely trans- generously supplying us with clones of P.polycephalum coI latable. and unpublished downstream DNA sequence. We also than The absence of clearly defined poly(U) tails on mito- Catherine Lozupone for providing technical assistance. T.L.H. chondrial large subunit rRNA may mean that the tails perform was supported in part by a National Science and Engineering translation-related functions for mRNAs analogous to the roles Graduate Fellowship. This work was supported in part by 4754 Nucleic Acids Research, 2000, Vol. 28, No. 23

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