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 This is an electronic version of an article published in Journal of Phycology ©2005, The Phycological Society of America. This is an electronic version of an article published in Journal of Phycology ©2006, The Phycological Society of America. This is an electronic version of an article published in Journal of Phycology ©2007, The Phycological Society of America. |
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| Home > Major Projects > Origin of Introns > Year 3 activities |
Testing the Reverse-Splicing
Model of Intron Spread with Ribosomal DNA (MCB 01-10252)
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ANNUAL
REPORT – YEAR 3 (2004) |
| 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. |
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Prediction 1: |
Introns are non-randomly distributed, with most of them clustering in regions that are not hidden by rRNA tertiary structure. | |
We (and others) established last year that rDNA introns are non-randomly distributed ( Bhattacharya, D., D. Simon, J. Huang, J.J. Cannone, and R.R. Gutell. 2003. The exon context and distribution of rRNA introns. BMC Evol. Biol. 3:7. ) and have now taken a functional approach to this important question. In collaboration with Gloria Culver at Iowa State University , we are studying the effect of group I intron splicing on 30S ribosome assembly. We have inserted the self-splicing Anabana tRNA Leu group I intron into different E. coli 16S rRNA sites to test the idea that particular rRNA regions are more or less amenable to group I intron splicing (and by extension, retention). Below is the abstract of this work for the upcoming RNA meeting. This work is in preparation for submission to the journal RNA in Summer 2004. |
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| Testing the assembly of the ribosomal small subunit using group I intron-containing 16S rRNA Peik Haugen1 , Jennifer Maki 2 , Gloria Culver 2 and Debashish Bhattacharya1 |
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| 1 Department of Biological Sciences and Center for Comparative Genomics, University of Iowa, 210 Biology Building, Iowa City, IA 52242, USA 2 Iowa State University, Department of Biochemistry, Biophysics, and Molecular Biology, Ames, Iowa 50011, USA. |
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| More than one thousand group I introns have been found in the rRNA genes of protists and fungi. The introns are non-randomly distributed on the rRNA primary, secondary, and 3-D structure. In 3-D, they appear remarkably clustered on the interface surfaces of the SSU and LSU ribosomal subunits. Several hypotheses have been proposed to explain the clustered distribution, but the evolutionary constraints underlying this pattern remain unclear. We have inserted the self-splicing Anabaena tRNA-leu group I intron into different sites in the E. coli 16S rRNA gene (see Fig. 1). These positions reflect different predictions about the positional effects on intron splicing. In E. coli 16S rRNA is a component of SSU that sediments at 30S. Intron-containing 16S rRNAs are being used in the reconstitution of 30S particles by adding a mix of purified "Total Protein derived from 30S subunits" (TP30). Intron splicing and activity of the reconstituted 30S particle are then monitored. Preliminary results show that the Anabaena intron, when inserted at position 789 that is neighboring a site (788) frequently populated by introns in nature, efficiently self-splices from the naked 16S rRNA when subjected to conditions that favor both autocatalysis and 30S reconstitution. Interestingly, the 16S rRNA containing a null mutant of this intron, when mixed with TP30, reconstitutes into a particle that sediments at approximately 32S (Fig. 2). We are characterizing these 32S particles to determine if they are capable of performing normal SSU function, as well as testing the structural and functional implications of three other intron insertion sites into 16S rRNA. These additional sites, that never contain introns in nature are either buried in the SSU structure as a result of RNA-RNA (1040)- or RNA-protein (1364) interactions, or are exposed on the SSU surface (1445). The results of these analyses will potentially allow us to gain novel insights into why group I introns are tolerated in some regions of the SSU rRNA, and are completely absent from others. |
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Prediction 2: |
Spliceosomal introns are inserted in target exon sequences that have a high affinity for splicing factors. | |
| This issue was addressed last year in the BMC Evol. Biol. paper and Ph.D. student Dawn Simon is working on a follow-up paper that analyzes intron flanking sequences in different model taxa that will be submitted for publication in Fall 2004. | ||
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| 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 published two papers that comprehensively address the role of HEGs in group I intron mobility. |
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| Haugen, P., V. Reeb, F. Lutzoni, and D. Bhattacharya. 2004. The evolution of homing endonuclease genes and group I introns in nuclear rDNA. Mol. Biol. Evol. 21(1):105-116. |
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| Haugen, P., and D. Bhattacharya. 2004, The spread of LAGLIDADG homing endonuclease genes in rDNA. Nucleic Acids Res. 32:2049-2057. (see below for details) |
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Abstract: Group I introns that encode homing endonuclease genes (HEGs) are highly invasive genetic elements. Their movement into a homologous position in an intron-less allele is termed homing. Although the mechanism of homing is well understood, the evolutionary relationship between HEGs and their intron partners remains unclear. Here we have focused on the largest family of HEGs (encoding the protein motif, LAGLIDADG) to understand how HEGs and introns move in rDNA. Our analysis shows the phylogenetic clustering of HEGs that encode a single copy of the LAGLIDADG motif in neighboring, but often evolutionarily distantly related group I introns. These endonucleases appear to have inserted into existing introns independent of ribozymes. In contrast, our data support a common evolutionary history for a large family of heterologous introns that encode HEGs with a duplicated LAGLIDADG motif. This finding suggests that intron/double-motif HEG elements can move into heterologous sites as a unit. Our data also suggest that a subset of the double-motif HEGs in rDNA originated from the duplication and fusion of a single-motif HEG encoded by present-day ribozymes in LSU rDNA (see Fig. 3). |
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Prediction 4: |
Group I introns insert through reverse-splicing into neighboring sites in rRNA primary, secondary, and tertiary structure. | |
| This prediction is being tested using phylogenetic methods and a comprehensive data set of fungal group I introns. One paper, regarding the mobility of large subunit rDNA group I introns in lichen fungi has been submitted to J. Mol. Evol. and a second paper on the small subunit rDNA introns in these taxa is being prepared for submission in Summer 2004. |
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Simon, D., J. Moline, G. Helms, T. Friedl, and D. Bhattacharya. Submitted. Divergent histories of rDNA group I introns in the lichen fungi. J. Mol. Evol. |
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| Abstract: The wide but sporadic distribution of group I introns in protists, plants and fungi, as well as in eubacteria likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene. |
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Bhattacharya, D., V. Reeb, D. Simon, and F. Lutzoni. In preparation. Symbiotaphrina and group I intron spread in fungal rDNA |
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| Abstract: Group I introns are mobile genetic elements that can catalyze their own excision from pre-RNA (i.e., they are self-splicing). Understanding how these RNA-enzymes (ribozymes) have spread into about 170 different sites in nuclear ribosomal DNA (rDNA) remains an active area of research. Two models are invoked to explain group I intron movement. The first, intron homing, is mediated by an intron-encoded homing endonuclease that can efficiently spread the ribozyme into intron-minus alleles. Recent analyses suggest that homing may have played an important role in the establishment of group I introns in nuclear rDNA and that they can mobilize these sequences through endonuclease mobility. The second pathway is reverse-splicing, whereby group I introns recognize their target sequence through complementary base-pairing with a short (4-6 nt) internal guide sequence. Because of the limited sequence requirement, reverse-splicing potentially provides a greater possibility of intron movement into heterologous sites than does homing that relies on a 15-45 nt recognition sequence. The role of reverse-splicing in group I intron spread in rDNA, however, remains unclear because of its reliance on chance integration, reverse-transcription, and recombination to promote spread. To address reverse-splicing intron spread, we used phylogenetic methods to identify 9 putatively ancient group I introns in the rDNA of the yeast-like symbiont Symbiotaphrina (Euascomycetes). These introns appear to be vertically inherited in later-diverging lichen fungi in which they have spread, putatively through reverse-splicing, into 5 heterologous rDNA sites. Together, these 14 introns explain one-half of the known diversity of Euascomycetes rDNA group I introns. We suggest that the widespread and often confusing distribution of fungal group I introns is not solely the result of homing of laterally transferred introns followed by sporadic loss but rather also by the movement of existing introns into novel rDNA sites. |
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. This work is now complete and will appear in the July issue of RNA. |
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Abstract: More than 1000 group I introns have been identified in fungal rDNA. Little is known, however, of the splicing and secondary structure evolution of these ribozymes . Here we use a combination of comparative and biochemical methods to address the evolution and splicing of a vertically inherited group I intron found at position 788 in the fungal small subunit (S) rRNA. The ancestral state of the S788 intron contains a highly conserved core and an extended P5 domain typical of IC1 introns (Fig. 5). In contrast, the more derived introns have lost most of P5 and have an accelerated divergence rate within the core region with three functionally important substitutions that unambiguously separate them from the ancestral pool. Of fourteen S788 group I introns that were tested for splicing, five, all of the ancestral type, were able to self-splice and produced intron RNA circles in vitro. The more derived S788 introns did not self-splice and potentially rely on fungal-specific factors to facilitate splicing (Fig. 6). In summary, we demonstrate one possible fate of vertically inherited group I introns: the loss of secondary structure elements, lessened selective constraints in the intron core, and ultimately, dependence on host-mediated splicing. |
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| In a second project, Dawn Simon has been 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 25 populations of lichens ( Physcia aipolia and P. stellaris ) in the midwestern United States and North Carolina that have been shown from past work to be intron-rich. PCR, sequence, and slot-blot analyses have provided the raw data about intron presence/absence in different populations at different genic sites and the level of allelic variation in the different individuals. DNA from 40 individual thalli was extracted and the ITS regions was sequenced. A preliminary phylogeny has been constructed (see Fig. 7). Whereas the tree lacks resolution at the population level, several important conclusions can still be made. First, P. stellaris and P. aipolia form clearly separated monophyletic lineages. Second, there appears to be gene flow between the midwest and North Carolina populations (details not shown). For the intron portion of the study, Dawn chose to focus on the first half of the large subunit (LSU) rDNA gene. Preliminary work was first done to determine the positions of introns in this region. This was accomplished by sequencing the 562/1245 (numbering is based on the homologous positions in E.coli ) region of the LSU rDNA for a total of 17 thalli from a range of midwest and North Carolina sites. Based on this sequencing, introns were found at 7 sites (4 group I and 3 spliceosomal). To efficiently screen the remaining thalli, intron specific probes were hybridized to slot blots containing genomic DNA. Surprisingly, it was found that each sample on the blot hybridized with all 5 intron probes tested until now (using both randomly-primed and specific oligonucleotide probes). This means that in some samples, sequencing showed the absence of an intron, whereas the slot blot hybridization suggested presence. Coupled with the inherent bias in PCR for preferential amplification of smaller fragments, these results likely indicate either heterogeneity between rDNA copies within one individual, or the presence of multiple strains of fungi within one lichen. Differentiating between these two possibilities is the primary goal of Dawn's summer research. If the heterogeneity is determined to occur between rDNA repeats, then the number of intron-containing copies will be quantified. This work is to completed by the end of Summer 2004 and is slated to be submitted for publication in the Fall. |
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