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Testing the Reverse-Splicing Model of Intron Spread with Ribosomal DNA (MCB 01-10252)
 

ANNUAL REPORT – YEAR 2 (2003)
  
PROJECT ACTIVITIES AND FINDINGS: The main objective of our grant is to isolate and analyze group I and spliceosomal introns interrupting the ribosomal (r)DNA of Euascomycetes fungi to understand generally how introns spread in nuclear genes. Our previous results suggest that most introns in nuclear rDNA originate from existing introns through a reversal of the forward splicing reaction (reverse-splicing [RS]). The RS model makes 4 major predictions about intron distribution that are being tested with our research. These predictions are listed below, as are the results of our ongoing analyses.

  
Prediction 1:
Introns are non-randomly distributed, with most of them clustering in regions that are not hidden by rRNA tertiary structure.  
Prediction 2:
Spliceosomal introns are inserted in target exon sequences that have a high affinity for splicing factors.  

We have recently published a paper in BMC Evolutionary Biology (http://www.biomedcentral.com/content/pdf/1471-2148-3-7.pdf) that addresses these issues. This work significantly advances our understanding of the exon context of rDNA spliceosomal introns. A number of novel applications to understanding intron distribution and evolution were used in this research (e.g., information analysis of exons, broken-stick model application to intron distribution, measurement of the association of intron and G-rich regions and estimation of the probability through simulations).

 
Prediction 3:
Group I introns reverse-splice into rRNA regions which contain a short (4-6 nt) 5’ exon flanking sequence that builds a helix with the intron internal guide sequence required for forward- and reverse-splicing.  

One of the major concerns about the reverse-splicing model in explaining rRNA intron movement is the unknown contribution of homing endonuclease genes (HEGs) to promoting group I intron mobility. We have recently completed an exhaustive study of all known nuclear HEGs and have in press at Molecular Biology and Evolution a detailed study of these sequences. We find strong evidence that HEGs promote nuclear intron mobility but more interesting is the highly mobile nature of the HEGs themselves that often move independent of the group I introns and transpose to sites in heterologous introns in neighboring sites (see below).		  

  The evolution of homing endonuclease genes and group I introns in nuclear rDNA
Peik Haugen1, Valérie Reeb2, François Lutzoni2 and Debashish Bhattacharya*
  
 
1Department of Biological Sciences and Center for Comparative Genomics, University of Iowa, 210 Biology Building, Iowa City, IA 52242-1324, USA. 2Department of Biology, Duke University, Durham, NC 27708-0338, USA
*Corresponding author
  
 
Abstract: Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intron-less allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse-splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the “switching” of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA (Fig. 2). These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system (Fig. 3). The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.
 

Figure 2


Figure 2 – Secondary structure of the ericoid mycorrhizal PSIV S943 group I intron. Paired elements (P1-P10) and numbering of every tenth nucleotide positions in the intron are marked on the structure. Shaded sequence positions were used to infer S943 intron phylogeny (not shown). HEG insertions located in the P8 structural element of S943 introns of different species are boxed. Arrows indicate the orientation of the HEG. Four of the HEGs are encoded by the same strand as the ribozyme (sense), whereas four are found on the strand complementary to that of the ribozyme, denoted as the anti-sense strand.

 




Figure 3

Comparison of phylogenies of HEG-containing nuclear group I introns and the corresponding homing ENases.

Figure 3 – Comparison of phylogenies of HEG-containing nuclear group I introns and the corresponding homing ENases. The ENase tree is based on 119 aa and was built using protein maximum likelihood ([proml] JTT+Γ model). The bootstrap values above the internal nodes on the left of the slash marks were inferred with the proml method, whereas the bootstrap values on the right of the slash marks were inferred with a minimum evolution analysis (Poisson model). The bootstrap values under the branches were inferred with an unweighted maximum-parsimony analysis. The thick branches denote >95% posterior probability for groups to the right resulting from a Bayesian inference (WAG + Γ model). The intron tree was built using DNA maximum likelihood (K80 [K2P]+Γ model). Bootstrap values above the internal nodes were inferred with minimum evolution (ME) analysis (K80 [K2P]+Γ model), whereas the bootstrap values under the nodes were inferred with ME using LogDet distances. Only bootstrap values >60% are shown. The thick branches denote >95% posterior probability for groups to the right resulting from a Bayesian inference (GTR+I+Γ model). The deep separation of introns belonging to the IC1 or IE subgroups is shown on the tree (large IC1 and IE text). Shaded areas highlight groups of introns from the same intron insertion site (shown in large gray text) and regions where the two tree topologies show strong similarity. Cases of clear tree incongruence are boxed and shown in bold text.


The emerging data set: We are continuing our strategy of intron discovery by screening Euascomycetes fungi. The screen within the large subunit rRNA of 74 Physciaceae (53 species in 13 genera), conducted by Dawn and REU-supported undergraduate Cora Hummel, is complete and has turned up 9 novel spliceosomal introns from 6 positions and 77 group I introns ofrom 12 positions. We will prepare a paper describing these results in Summer 2003. Cora will continue her work in our lab with a 1/2-time position in Summer and Fall 2003. After reviewing 6 candidates, we have completed our search for the second REU position and have appointed Biology undergraduate Jessica Moline. Both Cora and Jessica will finish the intron discovery project in other fungal groups (e.g., Leotiomycetes, also called Helotiales or inoperculate discomycetes [20 cultures]), and the Chaetothyriales [10 cultures]). Co-PI François Lutzoni has provided us with these cultures or DNAs. We expect to be finished with this component of the grant by the end of 2003.
DB traveled to Duke on Dec. 8-10, 2002 with Peik Haugen to confer with François and collaborator Mark Pagel (University of Reading, England) on our intron mapping project. This project has been finalized and we are in the process of putting together the final data set. Gert Helms (Ph.D. student in Thomas Friedl’s lab in Goettingen, Germany) was supported from this grant to spend a month in François’ lab to finish up the intron sequencing. The goal of this work is to develop a maximum likelihood method for calculating the probability of intron gain/loss on fungal host trees (provided by François). This will allow us to assess with greater confidence the probability that introns at particular sites are ancient or of a more recent origin. Mark is an expert on applying maximum likelihood methods to understanding character evolution in phylogenies.
Valérie Reeb (a Ph.D. student in François’ lab) has received additional funding with an A. W. Mellon training grant to spend a month in DB’s lab in Fall 2003 to work on the Acarosporaceae group I intron data set (100 sequences). By this time, Valérie will have completed the Acarosporaceae nuclear tree so we are set to understand intron evolution using this host tree as a comparative tool. During her stay, she will be further trained in the analysis of group I introns (alignment, secondary structure prediction) and will learn how to test their self-splicing ability (from Peik).
Another example of our approach to studying group I intron evolution is shown in the completed collaborative project with Ph.D. student David Fewer from Thomas Friedl’s lab. Dawn and David have studied the evolution of the tRNA(Leu) group I intron in cyanobacteria and plastids using phylogenetic analyses and splicing assays. This paper is in press at the Journal of Molecular Evolution (see below).
  

  Phylogeny and Self-Splicing Ability of the tRNA-Leu Group I Intron
Dawn Simon1, David Fewer2,3, Thomas Friedl2, Debashish Bhattacharya1
  
 
1Department of Biological Sciences and Center for Comparative Genomics, University of Iowa, 210 Biology Building, Iowa City, IA 52242-1324, USA. 2Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Experimentelle Phykologie und Sammlung von Algenkulturen, Universität Göttingen, 37073 Göttingen, Germany. 3Present address: Department of Applied Chemistry and Microbiology, Viikki Biocenter, PO Box 56, Viikinkaari 9, 00014 University of Helsinki, Finland.
*Corresponding author
  
 
Abstract: Group I introns are mobile RNA enzymes (ribozymes) that encode conserved primary and secondary structures required for autocatalysis. The group I intron that interrupts the tRNA-Leu gene in cyanobacteria and plastids is remarkable because it is the oldest known intervening sequence, and may have been present in the common ancestor of the cyanobacteria (i.e., 2.7 – 3.5 billion years old). This intron entered the eukaryotic domain through primary plastid endosymbiosis. We reconstructed the phylogeny of the tRNA-Leu intron and tested the in vitro self-splicing ability of a diverse collection of these ribozymes to address the relationship between intron stability and autocatalysis. Our results suggest that the present day intron distribution in plastids is best explained by strict vertical transmission, with no intron losses in land plants, and pervasive loss among green algae, as well as in the red algae and their secondary plastid derivatives. Interestingly, all tested land plant introns could not self-splice in vitro and presumably have become dependent on a host factor to facilitate in vivo excision. The host-dependence likely evolved once in the common ancestor of land plants. However, in all other plastid lineages there have been multiple putative intron losses and these ribozymes could either self-splice (all cyanobacteria) or complete only the first step of autocatalysis in vitro (green algae, glaucophytes, and Stramenopiles). Our data support a possible negative correlation between long-term vertical ancestry of the tRNA-Leu group I intron and self-splicing ability (Fig. 4).
  

Figure 4

Figure 4 – The distribution of self-splicing ability mapped on the phylogeny of the tRNA-Leu group I intron. A. Schematic representation of the phylogeny of the tRNA-Leu group I intron. The taxa that were tested for in vitro self-splicing ability are shown, as is their ability to complete the splicing reaction (+), to complete only the first step (no mark), and to have no apparent self-splicing ability (-). B. Examples of typical results in our experiments for tRNA-Leu introns that were self-splicing in vitro (i.e., the cyanobacteria Arthrospira PCC 8005 and Nodularia sphaerocarpa SAG 50.79) and those that could only complete the first step of splicing (the glaucophyte Cyanoptyche gloeocystis SAG 4.97 and the stramenopile Bodanella lauterborni SAG 123.79). The pre-RNA is shown as is the first step (intron + 3’exon), free intron, and ligated exons. The expected size of the ligated exons in our intron constructs was 78 nt.

Prediction 4:
 Group I introns insert through reverse-splicing into neighboring sites in rRNA primary, secondary, and tertiary structure.

This prediction will be tested once we have completed the majority of the sequencing this summer and can then compare the phylogenetic relationships of many lichen group I introns to their physical positions in the SSU and LSU rRNAs. The preliminary data provide some support for this hypothesis (e.g., monophyly of S114-S303 introns) but it is too early to reach any conclusions.

Supplementary Research on Group I Intron Evolution

An important aspect of group I intron evolution is the interplay between primary, secondary, and tertiary structure evolution and splicing and intron maintenance in the rDNA. There are, however, few models to study the long-term evolution of group I introns that offers variation in all of these aspects of intron biology. We have identified such a model, the 788 group I intron that is widely distributed in fungal SSU rRNA and is vertically inherited in these taxa. We have characterized the phylogeny, secondary structure, and in vitro splicing of all available (>20 sequences) S788 introns (see Fig. 5A) and are preparing these data for publication. This work is led by Peik Haugen and is the major component of the MS thesis research of Joe Runge. We observe an interesting trend in the S788 introns: the early-diverging forms in the intron tree (not shown) are large introns that have complete P5 domains (e.g., Geosmithia viridis), whereas the later diverging, more derived forms have incomplete P5 domains. This RNA helical region is critical for proper intron folding and catalysis, and loss of sequence normally results in the requirement for host-mediated splicing (e.g., Mohr et al., Nature 370, 147-150 [1994]). We find this trend, predicted from analysis of a few model introns, to be the case in the “natural” S788intron family.

  


Figure 5 A and B

Figure 5 – (see above) A. Secondary structure models of fungal S788 group I introns - the variation in P5 is shown. The species with the extended P5 region (i.e., P5abc) are earlier-diverging in the intron phylogeny. B. In vitro splicing analysis of the Barrnaelia S788 intron. The intron construct was digested with different restriction enzymes (MluI, HincII) in the 3’ exon to detect a size shift in the gel and verify splicing. The pre-RNA, free intron, and ligated exons of two different sizes are marked in the gel. The excised intron remains the same size, whereas the ligated exon differs in size based on the restriction digest. This is a PAGE gel that was stained with EtBr to visualize the RNA fragments.

Introns with more extensive P5 domains (e.g., Barrmaelia oxyacanthae [Fig. 5B], Penicillium oblatum) show catalytic activity, whereas the “short” introns in the derived Physiacae (e.g., Lecanora dispersa, Physcia stellaris) show no in vitro activity. We are presently completing the in vitro splicing assays and will try to rescue splicing activity in two ways, 1) over-expression in E. coli to test whether a bacterial protein can facilitate splicing, and 2) addition of Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18), that has been shown to rescue splicing in P5abc-deficient group I introns (Mohr et al., Cell 69, 483-494 [1992]). The Lambowitz group has provided us with an aliquot of CYT18. This work is in review at the journal RNA.
In a second project, Dawn Simon is studying fungal intron population biology. Basically, Dawn wishes to understand the natural history of spliceosomal introns. Although we have known of the existence of spliceosomal introns for over 25 years, little is understood about how they insert into genes and their rates of gain and loss in natural populations. Understanding these population-level phenomena is critical to understanding the rate at which introns invade, establish, and are lost from species. To address this important issue in spliceosomal intron evolution, Dawn has sampled multiple populations of lichens (Physcia aipolia and P. stellaris) that have been shown from past work to be intron-rich. Straightforward PCR and sequence analyses provide the raw data about intron presence/absence in different populations at different genic sites and the level of allelic variation in the different individuals. Dawn will build host trees primarily using ITS regions (Fig. 6) of ribosomal DNA and then use phylogenetic (e.g., Bayesian and maximum likelihood character analysis) and population genetics tools to understand the evolutionary history of the different introns and their effect on the flanking exon sequences (e.g., HKA – test). Presently, Dawn has collected 70 Physcia thalli from 9 sites in North Carolina and 2 sites in Iowa. From the North Carolina sites, multiple single spore isolates were started (and are still viable at Duke). DNA was extracted from 20 of the 70 thalli and preliminary PCR screening of the entire LSU from 6 of the thalli revealed the presence of 10 group I intron and 6 spliceosomal intron insertion sites. Of these 16 total sites, 10 are variable (7 group I sites, 3 spliceosomal intron sites) with regard to intron presence or absence. A 600 bp region of the LSU rDNA, replete with introns was identified in all 20 thalli and were sequenced (or are currently being sequenced). In addition, ITS 1, 5.8S, and ITS2 sequences were obtained and a host tree was constructed (see Fig. 6). We expect to complete the practical portion of this study by the end of Summer 2003 and prepare the manuscript by the end of the year.

  
Figure 6

 

Figure 6 – Population allelogram based on rDNA (ITS 1, 5.8S, ITS2, and ca. 500 bp of the LSU) sequence comparisons. The host tree was made with the ME method using Kimura2p distances. The bootstrap values result from 2000 pseudosamples. The LSU rRNA introns at 636, 779, and 1092 are putatively novel positions. The presence (+) or absence (-) of these introns is shown for each individual. The taxa with “A” in their names are Physcia aipolia, whereas those with “S” are P. stellaris. The outgroup is the closely related Physconia perisidiosa (PHYPER). Note the novel spliceosomal intron insertion at 1054 in individual 6Ah and the loss of the ancestral (i.e., present in P. perisidiosa) 1094 group I intron in the common ancestor of the aipolia group defined by 2Ac, 1Aa, and 6Ah.

 

And finally, we are continuing our work with Gloria Culver (Iowa State University) on testing the effects of group I intron splicing on in vitro 30S ribosome reconstitution. Peik and REU researcher Courtney Siebrecht have prepared all of the necessary intron constructs and we plan to do the experiments in Summer 2003. This project is described in detail in the 2002 Annual Report and focuses on the effect of rRNA intron insertion site on efficient autocatalysis.  

Summary
In the second year of this grant, we have made significant advances in gathering data that is already providing interesting insights into rRNA intron evolution. The important contributions of the Co-PIs have made this rapid progress possible. Most gratifying to us is that the grant, though focusing on phylogenetic and bioinformatic analyses, has opened new doors and allowed us to enter new arenas of research (e.g., interplay of intron secondary structure evolution and splicing, intron population biology, role of introns in ribosome folding [with Gloria Culver], use of information content to study spliceosomal introns [with Jian Huang]). These new initiatives will provide a much fuller understanding of the intron biology in rRNAs and generally in all nuclear genes. We anticipate a number of additional publications will come from these efforts as will a much more robust basis for thinking about intron evolution.
We have further trained grant-supported post-doc Peik Haugen and Ph.D. student Dawn Simon in intron molecular evolution, splicing, and bioinformatic methods. Undergraduate Cora Hummel was trained in Summer and Fall, 2002 and will continue her work in Summer and Fall, 2003 through generous REU support. Cora has matured tremendously during her time in the lab and has developed into an independent researcher with the self-confidence to tackle challenging problems on her own. The second REU student, Courtney Siebrecht, was a wonderful and effective lab worker who created most of the intron constructs to be used in the upcoming in vitro reconstitution experiments. We are truly sorry to lose her to a biotech company in Washington, D.C. We hope to have similar success with newly appointed REU researcher Jessica Moline. We are, therefore, fully committed and engaged in recruiting and training both undergraduate and graduate students. This process will hopefully be helped by the addition of two new faculty members to our Center for Comparative Genomics this year and by the offering of an undergraduate course in Molecular Phylogenetics that D. Bhattacharya will teach for the first time in Fall 2003.