Tripartite chimeric pseudogene from the genome of rice blast fungus Magnaporthe grisea suggests double template jumps during long interspersed nuclear element (LINE) reverse transcription
© Gogvadze et al.; licensee BioMed Central Ltd. 2007
Received: 15 August 2007
Accepted: 08 October 2007
Published: 08 October 2007
A systematic survey of loci carrying retrotransposons in the genome of the rice blast fungus Magnaporthe grisea allowed the identification of novel non-canonical retropseudogenes. These elements are chimeric retrogenes composed of DNA copies from different cellular transcripts directly fused to each other. Their components are copies of a non protein-coding highly expressed RNA of unknown function termed WEIRD and of two fungal retrotransposons: MGL and Mg-SINE. Many of these chimeras are transcribed in various M. grisea tissues and during plant infection. Chimeric retroelements with a similar structure were recently found in three mammalian genomes. All these chimeras are likely formed by RNA template switches during the reverse transcription of diverse LINE elements.
We have shown that in M. grisea template switching occurs at specific sites within the initial template RNA which contains a characteristic consensus sequence. We also provide evidence that both single and double template switches may occur during LINE retrotransposition, resulting in the fusion of three different transcript copies. In addition to the 33 bipartite elements, one tripartite chimera corresponding to the fusion of three retrotranscripts (WEIRD, Mg-SINE, MGL-LINE) was identified in the M. grisea genome. Unlike the previously reported two human tripartite elements, this fungal retroelement is flanked by identical 14 bp-long direct repeats. The presence of these short terminal direct repeats demonstrates that the LINE enzymatic machinery was involved in the formation of this chimera and its integration in the M. grisea genome.
A survey of mammalian genomic databases also revealed two novel tripartite chimeric retroelements, suggesting that double template switches occur during reverse transcription of LINE retrotransposons in different eukaryotic organisms.
Reverse transcription is one of the key processes that shape eukaryotic genomes. At least 40% of mammalian DNA was formed through reverse transcription [1–3]. This phenomenon was discovered when Temin and Baltimore purified and characterised retroviral RNA-dependant DNA polymerase (reverse transcriptase, RT), which catalyzes the synthesis of complementary DNA on RNA template . Afterwards, RT sequences were found in very diverse genetic elements, termed retroelements (REs). All REs are transposable elements that proliferate through their RNA intermediates by using self-encoded or exogenous RT to synthesise the DNA copy of the element to be inserted into the host genome.
Retroelements carrying their own RT genes are autonomous REs that are classified into two major groups: long terminal repeat (LTR) containing elements, and non LTR retrotransposons . Autonomous non LTR REs are generally assigned to LINEs, long interspersed nuclear elements. Among the REs, only LINEs are thought to be able to provide their RT enzyme for the proliferation of non autonomous REs . LINEs have been found in essentially all eukaryotic DNAs [3, 7]. LINE insertions are flanked by 10–20 bp long duplications called target site duplications (TSD). LINEs also contain an oligo (A) or microsatellite A-rich sequences at their 3' termini. Another LINE distinguishing feature is their frequent 5'-truncation. These truncations likely result from LINE RNA abortive reverse transcription, when RT dissociates from its RNA template before having completed full cDNA copy synthesis .
The full-sized LINE (+) RNA is both a transpositional RNA intermediate and the template for protein synthesis . LINE transposition is known to proceed in several steps including Pol II transcription of an active element, reverse transcription of the RNA formed with the self-encoded RT, and integration of the cDNA into a new position within the genome . Due to the so-called 'cis-preference', the enzymatic machinery of a retrotransposition-competent LINE predominantly transposes its own copies . However, LINEs are capable of transposing other sequences, mostly non autonomous REs termed short interspersed nuclear elements (SINEs), but also cDNAs from different types of cellular RNAs, thus forming processed pseudogenes . Recently we have shown a new property of the LINE reverse transcriptional machinery that is able to form bipartite chimeric elements during reverse transcription in mammalian and fungal genomes [13–17]. These elements are composed of DNA copies from cellular transcripts either directly fused to each other or more frequently fused to the 3' part of a LINE retroposon.
This model of chimera formation was further supported by results obtained using experimentally controlled retrotransposition of human L1 LINE element in vitro  and in vivo . Interestingly, it has been recently postulated that RT templates jump from LINE RNA to host genomic DNA facilitating integration, thus, being normally required for successful LINE retrotransposition [20, 21]. In addition to the generation of chimeric retrogenes, template switching events during LINE reverse transcription could give rise to chimeric SINE elements  and to mosaic LINE structures. These events likely result from RNA recombination between different LINE templates [8, 21, 23, 24].
More recently, two tripartite chimeric retroelements, each consisting of fused copies of three human RNAs, have been found in the human genome sequence. Formation of such tripartite retrogenes might result from double RNA template switching events during LINE retrotransposition . However, no proof was provided for this concept, as both triple elements were inserted into A or AT-rich genomic sequences, making it impossible to identify direct repeats flanking the integrated element. In this report, we provide direct evidence for in vivo double template-switching in the genome of the rice blast fungus Magnaporthe grisea. We identified one tripartite chimeric retroelement in this fungal genome and showed that it is flanked by identical non-satellite 14 bp-long direct repeats. We also identified two similar tripartite chimeric retroelements in the mouse genome and found that WEIRD, the major component of fungal bipartite chimeric retroelements, is a non-coding sequence highly expressed in various M. grisea tissues and during plant infection. We have shown that template switching does not occur in M. grisea at random sites of the template RNA as thought for mammalian chimera formation ), but occurs at hot spots located downstream of specific sequence motifs. Lastly, this study allowed the identification of novel bipartite chimeric retroelements in M. grisea.
Results and discussion
Characterization of fungal chimeric retroelements
Expression of WEIRD and MGL relatively to housekeeping genes Ilv5 and Ef1
0 hours infection
8 hours infection
24 hours infection
1.6 ± 0.5
1.7 ± 0.8
1.0 ± 0.4
2.5 ± 0.8
35. 2 ± 7.8
20.6 ± 5.2
35.0 ± 6.5
61.2 ± 12.2
2.8 ± 0.9
8.9 ± 2.0
1.3 ± 0.7
2.4 ± 0.4
60.2 ± 14.5
108.2 ± 14.3
44.0 ± 6.6
58.2 ± 10.9
Novel non-canonical retropseudogenes from the genome of M. grisea
Apart from the previous 31 MINEs, we identified one novel bipartite chimeric retroelement with a WEIRD sequence fused to 5'-truncated Mg-SINE. This WEIRD-MgSINE retroelement was flanked by 12 bp-long direct repeats (figure 2B, Additional file 1). Mg-SINE is a non-autonomous SINE-family retroelement . It likely utilizes a "molecular mimicry" to MGL for its proliferation. Indeed, the 3' end of Mg-SINE is similar to the 3' end of MGL retrotransposon used for priming reverse transcription [29, 30]. This property of Mg-SINE sequence likely results in the capture of Mg-SINE transcripts by the MGL retrotranspositional apparatus.
We have also identified a novel chimera composed of three elements (figure 2C, Additional file 1). Its 5' component is a full length WEIRD fused to a 5'-truncated Mg-SINE that is itself fused to a 5'-truncated MGL. The tripartite chimeric retroelement is flanked by 14 bp-long direct repeats showing that it was integrated into the M. grisea genome as a single chimeric retrogene likely formed by two successive template RNA switches during the reverse transcription of MGL.
Overall, this survey of M. grisea genome showed that most chimeric retrogenes containing MGL sequences are represented by MINEs(WEIRD-MGL), although we also identified another type of bipartite chimeras (WEIRD-MgSINE) and a triple chimera (WEIRD-MgSINE-MGL).
Several fungal chimeric retrogenes are transcribed
MINE-A, -B and -C expression relatively to housekeeping genes Ilv5 and Ef1
4.4 ± 1.6
70.1 ± 14.5
4.4 ± 0.2
71.2 ± 5.1
1.4 ± 0.1
24.1 ± 8.5
2.1 ± 0.4
20.0 ± 0.7
4.7 ± 0.5
44.6 ± 1.7
2.8 ± 0.3
26.4 ± 1.0
infection 24 hours
10.4 ± 1.0
154.3 ± 17.8
4.3 ± 1.24
64.4 ± 10.2
3.1 ± 0.6
42.7 ± 7.4
Tripartite chimeric retroelement: evidence for double template switching model
Previously, two tripartite chimera-like elements were identified in human DNA [16, 18] (Fig. 4B). One of them was inserted in an expanded microsatellite locus, while the other was flanked by AT-rich sequences. As a result, the identification of direct repeats that should normally flank the chimera was not possible for these elements (Additional file 2). However, a survey of mammalian genomic databases allowed us to identify two new tripartite chimeric retrogenes flanked by perfect 15- and 16 bp-long direct repeats in the mouse genome (Fig. 4C, Additional file 2). These chimeras were composed of (5' to 3') B2 SINE, U6 snRNA and L1 LINE elements. Finding tripartite chimeric retroelements displaying considerable structural similarities in such evolutionary distinct organisms as mammals and fungi suggests that double template switches during LINE reverse transcription is probably not the exceptional case, but might be a conserved mechanism of eukaryotic DNA rearrangement.
Template switching occurs at specific positions within template RNAs in M. grisea
Representative chimerization sites found in M. grisea
It remains unclear why the RT dissociates from the template RNA exactly before the GCC triplet. It is known for many RTs that the enzyme processivity depends greatly on the nucleotide composition of the template RNA. It can be hypothesized that this GCC motif, in particular when located in a hairpin, is a "difficult place" for the MGL RT, inducing a reverse-transcriptional pause or even the RT to dissociate or to jump on another RNA template.
Finally, it should be mentioned that the above chimerization mechanism does not include any kind of specific nucleotide basepairing between the nascent cDNA and the second RNA template. Indeed, we were not able to find extended- or micro-homologies between the templates around the chimerization sites.
In this paper, we demonstrate that several forms of chimeric retroelements are present in the Magnaporthe grisea genome. For the first time, we provide evidence that a triple chimera was generated in vivo and integrated into the M. grisea genome. The most probable mechanism for the formation of this chimera is template switching during LINE-mediated reverse transcription. Therefore, bipartite and tripartite chimeric retroelements likely result, respectively, from single and double template RNA switches during reverse transcription. Several chimeric retroelements are transcribed in M. grisea. The major fungal chimera components are MGL LINE retrotransposon and WEIRD. WEIRD does not encode for a protein and is not a transposable element. It is expressed at a high level in mycelium and spores and is up-regulated during plant infection. We hypothesize that WEIRD is a functional non-coding RNA. We have also identified novel chimeras including a novel LINE retrotransposon Enigma and fungal Mg-SINE element. To conclude, we have shown that in M. grisea template switching during reverse transcription occurs at specific sites within the initial template RNA, and a consensus sequence for these chimerization sites is proposed.
DNA sequence analysis
Homology searches against GenBank were done using the BLAST Web-server at NCBI . Flanking regions of mammalian retroelements were investigated with the RepeatMasker program . For fungal genomes, flanking regions were aligned with known filamentous fungal transposable elements reported elsewhere . Direct repeats were detected by visual inspection of retroelement flanking sequences. Novel repeats were assigned to subfamilies according to the nomenclature proposed by Daboussi and Capy . For multiple alignments, BLAST pairwise search, Vector NTI and Clustal W programs  were used.
Fungal strains and growth conditions
Two Magnaporthe grisea strains – P1.2 and Guy11 – were used in this study. As all rice pathogenic Magnaporthe grisea isolates, these strains are also pathogenic on barley. Fungal strains were grown and stored as described .
Total RNAs were extracted from M. grisea mycelium, spores (strain P1.2) and infected barley leaves at 0 h, 8 h and 24 h (strain Guy11) using Rneasy Plus Mini Kit (Qiagen). cDNA synthesis was performed with 2 μg of total RNA using random primers and ThermoScript RT-PCR System (Invitrogen). The level of WEIRD and MGL transcription was assessed by real-time PCRs with genomic primers corresponding to the 5' and 3'-terminal parts of the elements using an ABI Prism 7700 Sequence Detection System (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems); primers Wfor, Wrev and MGLfor, MGLrev for WEIRD and MGL, respectively. Primer sequences for real-time PCR were chosen using Primer Express software (Applied Biosystems) and are presented in supplementary material (Additional file 4). All measurments were carried out in quadruplicate and expression levels were normalized to Ilv5 and Ef1 using the following formulae . All RT-PCR experiments were performed against the negative RT "-" controls (no reverse transcriptase added at the stage of the first strand cDNA synthesis) to control the DNA contamination. Only those samples displaying negative results on the RT "-" control experiments were further analyzed. To identify transcriptionally active MINEs a general RT-PCR was performed using primers designed to the 5' terminal part of WEIRD (in the sense orientation), and to the 3' end of MGL (in the reverse orientation) under the following conditions: 95°C – 2 min; 95°C – 25 s, 62°C – 25 s, 72°C – 2 min; 25 cycles. The products of RT-PCR were then diluted into 40 times and used as a template for the nested PCR (95°C – 2 min; 95°C – 25 s, 62°C – 25 s, 72°C – 2 min; 25 cycles). Nested PCR was performed to reduce the background originated due to the use of primers corresponding to repetitive sequences. The obtained products were sequenced by Genome Expressed (Meylan, France). Three transcriptionally active MINEs identified during this study were further analyzed by real-time RT-PCR using primers designed for the specific WEIRD-MGL junctions (q1 and q2 for MINE1; q3 and q4 for MINE2; q5 and q6 for MINE3).
The authors are extremely thankful to Professor Eugene D. Sverdlov for valuable discussion. Our efforts were supported by the following grants: Molecular and Cellular Biology Program of the Presidium of the Russian Academy of Sciences, grants 05-04-50770-a and 05-04-48682-a from the Russian Foundation for Basic Research, President of the Russian Federation grant МК-4227.2007.4, FEBS short-term fellowship, and CNRS/Bayer crop science funding. We express our gratitude to Tiffany Brunson and Isfahan Chambers for the critical reading of this manuscript.
- Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, et al: Initial sequencing and comparative analysis of the mouse genome. Nature. 2002, 420: 520-562. 10.1038/nature01262.PubMedView Article
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, et al: Initial sequencing and analysis of the human genome. Nature. 2001, 409: 860-921. 10.1038/35057062.PubMedView Article
- Buzdin AA: Retroelements and formation of chimeric retrogenes. Cell Mol Life Sci. 2004, 61: 2046-2059. 10.1007/s00018-004-4041-z.PubMedView Article
- Temin HM: Viruses, protoviruses, development, and evolution. J Cell Biochem. 1982, 19: 105-118. 10.1002/jcb.240190202.PubMedView Article
- Leib-Mosch C, Seifarth W: Evolution and biological significance of human retroelements. Virus Genes. 1995, 11: 133-145. 10.1007/BF01728654.PubMedView Article
- Matsutani S: Links between repeated sequences. J Biomed Biotechnol. 2006, 2006: 13569-PubMed CentralPubMedView Article
- Volff JN, Korting C, Schartl M: Multiple lineages of the non-LTR retrotransposon Rex1 with varying success in invading fish genomes. Mol Biol Evol. 2000, 17: 1673-1684.PubMedView Article
- Furano AV: The biological properties and evolutionary dynamics of mammalian LINE-1 retrotransposons. Prog Nucleic Acid Res Mol Biol. 2000, 64: 255-294.PubMedView Article
- Martin SL, Li WL, Furano AV, Boissinot S: The structures of mouse and human L1 elements reflect their insertion mechanism. Cytogenet Genome Res. 2005, 110: 223-228. 10.1159/000084956.PubMedView Article
- Kazazian HH: Mobile elements: drivers of genome evolution. Science. 2004, 303: 1626-1632. 10.1126/science.1089670.PubMedView Article
- Wei W, Gilbert N, Ooi SL, Lawler JF, Ostertag EM, Kazazian HH, Boeke JD, Moran JV: Human L1 retrotransposition: cis preference versus trans complementation. Mol Cell Biol. 2001, 21: 1429-1439. 10.1128/MCB.21.4.1429-1439.2001.PubMed CentralPubMedView Article
- Dewannieux M, Esnault C, Heidmann T: LINE-mediated retrotransposition of marked Alu sequences. Nat Genet. 2003, 35: 41-48. 10.1038/ng1223.PubMedView Article
- Buzdin A, Gogvadze E, Kovalskaya E, Volchkov P, Ustyugova S, Illarionova A, Fushan A, Vinogradova T, Sverdlov E: The human genome contains many types of chimeric retrogenes generated through in vivo RNA recombination. Nucleic Acids Res. 2003, 31: 4385-4390. 10.1093/nar/gkg496.PubMed CentralPubMedView Article
- Buzdin A, Vinogradova T, Lebedev Y, Sverdlov E: Genome-wide experimental identification and functional analysis of human specific retroelements. Cytogenet Genome Res. 2005, 110: 468-474. 10.1159/000084980.PubMedView Article
- Buzdin A, Ustyugova S, Gogvadze E, Vinogradova T, Lebedev Y, Sverdlov E: A new family of chimeric retrotranscripts formed by a full copy of U6 small nuclear RNA fused to the 3' terminus of l1. Genomics. 2002, 80: 402-406. 10.1006/geno.2002.6843.PubMedView Article
- Gogvadze EV, Buzdin AA, Sverdlov ED: Multiple template switches on LINE-directed reverse transcription: the most probable formation mechanism for the double and triple chimeric retroelements in mammals. Bioorg Khim. 2005, 31: 82-89.PubMed
- Fudal I, Bohnert HU, Tharreau D, Lebrun MH: Transposition of MINE, a composite retrotransposon, in the avirulence gene ACE1 of the rice blast fungus Magnaporthe grisea. Fungal Genet Biol. 2005, 42: 761-772. 10.1016/j.fgb.2005.05.001.PubMedView Article
- Gogvadze EV, Buzdin AA: New mechanism of retrogene formation in mammalian genomes: in vivo recombination during RNA reverse transcription. Mol Biol (Mosk). 2005, 39: 364-373.View Article
- Gilbert N, Lutz S, Morrish TA, Moran JV: Multiple fates of L1 retrotransposition intermediates in cultured human cells. Mol Cell Biol. 2005, 25: 7780-7795. 10.1128/MCB.25.17.7780-7795.2005.PubMed CentralPubMedView Article
- Babushok DV, Ostertag EM, Courtney CE, Choi JM, Kazazian HH: L1 integration in a transgenic mouse model. Genome Res. 2006, 16: 240-250. 10.1101/gr.4571606.PubMed CentralPubMedView Article
- Bibillo A, Eickbush TH: End-to-end template jumping by the reverse transcriptase encoded by the R2 retrotransposon. J Biol Chem. 2004, 279: 14945-14953. 10.1074/jbc.M310450200.PubMedView Article
- Nishihara H, Smit AF, Okada N: Functional noncoding sequences derived from SINEs in the mammalian genome. Genome Res. 2006, 16: 864-874. 10.1101/gr.5255506.PubMed CentralPubMedView Article
- Hayward BE, Zavanelli M, Furano AV: Recombination creates novel L1 (LINE-1) elements in Rattus norvegicus. Genetics. 1997, 146: 641-654.PubMed CentralPubMed
- Brosius J: Genomes were forged by massive bombardments with retroelements and retrosequences. Genetica. 1999, 107: 209-238. 10.1023/A:1004018519722.PubMedView Article
- Buzdin A, Gogvadze E, Lebrun MH: Chimeric retrogenes suggest a role for the nucleolus in LINE amplification. FEBS Lett. 2007, 581: 2877-2882. 10.1016/j.febslet.2007.05.034.PubMedView Article
- Bohnert HU, Fudal I, Dioh W, Tharreau D, Notteghem JL, Lebrun MH: A putative polyketide synthase/peptide synthetase from Magnaporthe grisea signals pathogen attack to resistant rice. Plant Cell. 2004, 16: 2499-2513. 10.1105/tpc.104.022715.PubMed CentralPubMedView Article
- Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H, Read ND, Lee YH, Carbone I, Brown D, Oh YY, Donofrio N, Jeong JS, Soanes DM, Djonovic S, Kolomiets E, Rehmeyer C, Li W, Harding M, Kim S, Lebrun MH, Bohnert H, Coughlan S, Butler J, Calvo S, Ma LJ, Nicol R, Purcell S, Nusbaum C, Galagan JE, Birren BW: The genome sequence of the rice blast fungus Magnaporthe grisea. Nature. 2005, 434: 980-986. 10.1038/nature03449.PubMedView Article
- Kachroo P, Leong SA, Chattoo BB: Mg-SINE: a short interspersed nuclear element from the rice blast fungus, Magnaporthe grisea. Proc Natl Acad Sci U S A. 1995, 92: 11125-11129. 10.1073/pnas.92.24.11125.PubMed CentralPubMedView Article
- Daboussi MJ, Capy P: Transposable elements in filamentous fungi. Annu Rev Microbiol. 2003, 57: 275-299. 10.1146/annurev.micro.57.030502.091029.PubMedView Article
- Nishimura M, Hayashi N, Jwa NS, Lau GW, Hamer JE, Hasebe A: Insertion of the LINE retrotransposon MGL causes a conidiophore pattern mutation in Magnaporthe grisea. Mol Plant Microbe Interact. 2000, 13: 892-894. 10.1094/MPMI.2000.13.8.892.PubMedView Article
- Piskareva O, Schmatchenko V: DNA polymerization by the reverse transcriptase of the human L1 retrotransposon on its own template in vitro. FEBS Lett. 2006, 580: 661-668. 10.1016/j.febslet.2005.12.077.PubMedView Article
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410.PubMedView Article
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680. 10.1093/nar/22.22.4673.PubMed CentralPubMedView Article
- Dioh W, Tharreau D, Notteghem JL, Orbach M, Lebrun MH: Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers. Mol Plant Microbe Interact. 2000, 13: 217-227. 10.1094/MPMI.2000.13.2.217.PubMedView Article
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.PubMedView Article