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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 Testing the
Reverse Splicing Model of Intron Spread |
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| PI: | Debashish Bhattacharya | |
| Co-PIs: | François Lutzoni | |
| Jian Huang | ||
| Robin Gutell |
| PROJECT SUMMARY: The existence of intervening sequences of non-coding DNA interrupting the coding region of nuclear genes, only to be later removed from transcribed nuclear pre-mRNAs by spliceosomes, poses fundamental questions: What is the evolutionary origin and significance of introns? And, how do they spread in genes? We have found a wealth of spliceosomal introns in the nuclear ribosomal DNA (rDNA) genes of Euascomycetes fungi, making these organisms ideal for the study of a recent intron invasion into a family of genes that is otherwise universally free of spliceosomal introns. We propose to use the Euascomycetes to determine how introns spread in rRNA genes. Our analysis of the Euascomycetes suggests that a reversal of splicing, mediated by the spliceosome, may facilitate the spread of existing introns into new rRNA sites, with these introns being preferentially inserted into exposed ribosomal regions that are susceptible to a reverse-splicing "attack" (the RS-model). Following insertion, the intron-containing RNA may become fixed in the genome through reverse-transcription and homologous recombination. The RS-model also offers an explanation for the spread of group I introns, another widespread class of intervening sequences in fungal and protist nuclear rDNA. Group I introns, however, are autonomous mobile elements that can insert into RNA through autocatalytic splicing reversal. Despite this difference in the mechanism of insertion, the distribution of spliceosomal and group I introns is expected to reflect constraints on reverse-splicing due to rRNA primary, secondary, and tertiary structure. These introns offer, therefore, parallel systems with which to test the RS-model. We will address, through a strategy of intron discovery, four major predictions of the RS-model regarding the expected distribution of rDNA introns. To do this, novel introns will be identified with PCR analysis of the small and large subunit rDNA in 150 taxa of the intron-rich Euascomycetes ("Lecanoromycetes"). Intron and exon flanking sequences will then be sequenced in the rDNA coding regions that contain insertions. Prediction 1: Introns are non-randomly distributed, with most of them clustering in regions that are not hidden by rRNA tertiary structure. To test this prediction we will determine spliceosomal and group I intron positions within primary, secondary, and tertiary structures of rRNAs to determine if the introns are randomly distributed. Existing RNA cross-linking, short-range reagent-labeling, and rRNA tertiary structure data will be used to test whether introns are positioned on the surface of ribosomes. Prediction 2: Spliceosomal introns are inserted in target exon sequences ("proto-splice" sites) that have a high affinity for splicing factors. We will test whether a proto-splice site exists in rRNA genes. Exon flanking sequences of Euascomycetes spliceosomal introns will be studied to determine if conserved nucleotide motifs are present. Comparative analyses that have already been completed of flanking sequences at 18 different spliceosomal intron sites suggest that the AG-intron-G motif is the minimal requirement for an Euascomycetes rDNA proto-splice site. 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. We will determine whether 5’ exon sequences flanking mobile group I introns are conserved. Mobile introns will be identified with phylogenetic analyses. Reverse-splicing of group I introns will be inferred when the 5’ exon sequence is conserved between homologous introns that are found in heterologous rRNA sites. Prediction 4: Group I introns insert through reverse-splicing into neighboring sites ("intron sliding") in rRNA primary, secondary, and tertiary structure. We will determine whether group I introns slide into sites that are in close proximity in the rRNA tertiary structure to test if there is a statistically significant relationship between the position of group I introns in phylogenetic trees and their insertion sites in folded rRNAs. This will be done by studying the correlation between matrices representing intron sequence divergence and the physical distance between introns in the ribosomes. The proposed research will help us understand how introns arise in all nuclear coding regions, and approach the long-term goal of understanding the significance of introns to gene evolution. |
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