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. selected publications Reyes-Prieto et al Annu Rev Genet 2007 Jun 28 Hackett et al Mol Biol Evol. 2007 Aug;24(8):1702-13 Reyes-Prieto et al Mol Biol Evol. 2007 Nov;24(11):2358-61 Reyes-Prieto et al Mol Phylogenet Evol. 2007 Oct;45(1):384-91. Nosenko et al J. Phycol. 43,763–767 (2007) Zhang et al J. Mol. Evol. 2007 Oct;65(4):463-74. Haugen et al BMC Evol. Bio. 2007 Sep 8;7:159. Tyra et al Genome Biol. 2007 Oct 5;8(10):R212 Nosenko et al BMC Evol. Bio. 2007 Sep 25;7(1):173 Patterson et al. Microbiology Today 2007 Feb:44-45 Reeb et al. JME 2007 Feb 8; Reyes-Prieto et al. Curr. Biol. 2006 Dec 5;16(23):2320-5 Yoon et al JAppPhycol (2006) 18: 475-481 Parfrey et al PLoS 2006, Dec 22;2(12):e220 Yoon et al BMC Evolutionary Biology 2006, 6:78 Yoon et al Curr Biol 2006 Sept; Vol 16 No 17, R670-R672 Lin et al AEM 2006 Aug;72(8):5626-30. Nosenko et al MBE AdvAccess 2006 Jul 28 Yoon et al J Phycol 2006 J. Phycol. 42,482-492 Reyes-Prieto et al Algae 2006 Vol 21(1): 1-10 Li et al MBE 2006 Mar;23(3):663-74 Hackett et al BMC Genomics 2005 May 29;6(1):80 Simon et al Curr Genet 2005 Jun;47(6):389-99 Simon et al JME 2005 Apr;60(4):434-46 Genome Workshop JEU 2005 Mar-Apr;52(2):170-2 Yoon et al MBE 2005 May;22(5):1299-308 Zhang et al J Phycol 2005 41, 411-420 Haugen et al TIGS 2005 Feb;21(2):111-9 Hagopian et al JME 2004 Oct;59(4):464-77 Hackett et al AJB 2004 91(10): 1523-1534 Williford et al EvoDevo 2004 Mar-Apr;6(2):67-77 Ciniglia et al Mol Ecol 2004 Jul;13(7):1827-38 Yoon et al MBE 2004 May;21(5):809-18 Haugen et al RNA 2004 Jul;10(7):1084-96 Haugen et al NAR 2004 Apr 6;32(6):2049-57 Haugen et al MBE 2004 Jan;21(1):129-40 Hackett et al Curr Bio 2004 Feb 3;14(3):213-8 Bhattacharya et al BioEssays 2004 Jan;26(1):50-60

Home > Major Projects > Origin of Introns > Year 1 activities

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

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 used the broken-stick model (MacArthur, R.H., PNAS 43:293 [1957])to test the distribution of rRNA introns on the primary structure. In this model, the introns “break” the rRNA (“stick”) into discrete pieces. The pieces are ordered by size and the resulting distribution can be analyzed to test for departure from the null expectation of a uniform distribution. Co-PI Jian Huang wrote a computer program to conduct this analysis, calculate different test statistics (see Goss, P.J.E., Lewontin, R.C., Genetics 142:589 [1996]), and run simulations to test the significance of our findings. The intron distribution analysis included all known group I, group II, and spliceosomal introns in both nuclear and organellar rRNA. Co-PI Robin Gutell amassed the data set, incorporating our unpublished introns. We also did comparative analyses of flanking sequences at 49 different spliceosomal intron sites show that the G—intron—G motif is the conserved flanking sequence at sites of intron insertion. Analysis using information theory shows that these rRNA introns contain relatively less information in comparison to yeast introns, but in contrast, the flanking exons are information-rich. Purine-content analysis shows that the proximal 5’ flanking sequence of these introns is enriched in purines, in particular runs of Gs. This work was published in BMC Evol Biol (2003, 3:7)

	The exon context and distribution of Euascomycetes rRNA spliceosomal introns Debashish Bhattacharya1*, Dawn Simon1, Jian Huang2, Jamie J. Cannone3, Robin R. Gutell3
	1 Department of Biological Sciences and Center for Comparative Genomics, University of Iowa, Iowa City IA USA. 2Department of Statistics and Actuarial Science, University of Iowa, Iowa City IA USA. 3Institute for Cellular and Molecular Biology, University of Texas, Austin TX USA. *Corresponding author
	Background We have studied spliceosomal introns in the ribosomal (r)RNA of fungi to discover the forces that guide their insertion and fixation.
	Results Comparative analyses of flanking sequences at 49 different spliceosomal intron sites showed that the G—intron—G motif is the conserved flanking sequence at sites of intron insertion (Fig. 1). Information analysis showed that these rRNA introns contain significant information in the flanking exons. Analysis of all rDNA introns in the three phylogenetic domains and two organelles showed that group I introns are usually located after the most conserved sites in rRNA, whereas spliceosomal introns occur at less conserved positions. The distribution of spliceosomal and group I introns in the primary structure of small and large subunit rRNAs was tested with simulations using the broken-stick model as the null hypothesis. This analysis suggested that the spliceosomal and group I intron distributions were not produced by a random process. Sequence upstream of rRNA spliceosomal introns was significantly enriched in G nucleotides (Fig. 2). We speculate that these G-rich regions may function as exonic splicing enhancers that guide the spliceosome and facilitate splicing. Conclusions Our results begin to define some of the rules that guide the distribution of rRNA spliceosomal introns and suggest that the exon context is of fundamental importance in intron fixation.

We have generated a set of interesting and potentially powerful hypotheses for explaining the extant rRNA intron distribution. To test these analyses, we have enlisted the aid of chemist/biochemist Dr. Gloria Culver (Iowa State University, Ames) who has a complete set of recombinant E. coli ribosomal (r-) proteins which can be used to study in detail the role of each protein in 30S maturation (Culver, G.M., Noller, H.F. Methods Enzymol 318:446 [2000]). Post-doc Peik Haugen has made several constructs to test the role of intron folding and splicing on SSU rRNA maturation. The autocatalytic Anabaena PCC7120 tRNA(Leu) group I intron has been cloned into 4 different E. coli SSU rRNA regions that are predicted to be favorable (S789) or unfavorable (S1040, S1363, S1445) for splicing in the SSU rRNA. Criteria for identifying these sites were the extant intron distribution and knowledge about putatively hidden/exposed rRNA sites based on the 30S crystal structure (e.g., S789 is intron-rich and should be favorable for splicing, whereas S1445 is presumably exposed in the SSU ribosome but one never finds an intron there in nature). In vitro splicing assays in the absence of r-proteins show that these introns splice at the heterologous sites (e.g., Fig. 3).

Fig. 3. In vitro splicing of Anabaena tRNA(Leu) intron in the wild-type position (wt) and at positions S789 and S1445 (exper.) in the E. coli SSU rRNA. The negative control (neg.) was the SSU RNA without the intron. The assay favored forward splicing and products at time 0 min and 60 min are shown. Note the accumulation of the linear intron and intron circles over time. Lad. is the RNA ladder.

 

 

Gloria will use the clones to address the role of intron splicing on rRNA maturation in two ways: 1) the SSU rRNA containing the Anabeana introns will be over-expressed in bacteria to test the effects of intron expression on in vivo ribosome maturation. E. coli growth rates will be monitored to identify intron sites that have a negative effect on bacterial growth. Ribosome assembly will be monitored in these strains using standard polysome profile analysis. 2) intron-containing SSU rRNA transcripts that result in interesting phenotypes or altered polysome profiles will be further characterized. The ability of these transcripts to support in vitro 30S subunit reconstitution will be assessed and changes in specific assembly events will be monitored. This work, which will be done in Gloria’s lab, promises to answer an important question in group I intron evolution/biochemistry. How does intron splicing, constrained by the position of the intron in the rRNA, interact with the complex process of ribosome biogenesis? This process is of particular interest with lichens which may contain up to 10 introns in their rRNA. We are very excited about the prospects this research provides and will, with a relatively small investment from my lab, profit from the well-established protocols and expertise in Gloria’s group in RNA biochemistry.

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.

The emerging data set: We are continuing our strategy of intron discovery by screening Euascomycetes fungi. The screen within the large subunit rRNA of the Physciaceae, conducted by Dawn and REU-supported undergraduate Cora Hummel, is nearly complete and has turned up 10 novel spliceosomal intron sites and 67 group I introns of which 3 are at novel rRNA sites. A paper describing the phylogeny of the Physciaceae group I introns, done with collaborator Thomas Friedl (University of Göttingen), is in press at J Mol Evol. We feel that that we have now exhausted this valuable source of introns and have now turned our search to the relatively poorly studied but intron-rich Leotiomycetes (also called Helotiales or inoperculate discomycetes [20 cultures]) and the Chaetothyriales (10 cultures). This work is being done in collaboration with co-PI François Lutzoni. We are, however, already determining the phylogeny of our existing, predominantly unpublished data set (mostly from François’ lab) of over 200 lichen group I introns to address broader issues in intron evolution. One 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. To this end, we have enlisted the aid of Dr. Mark Pagel (University of Reading, England) who is an expert on applying maximum likelihood methods to understanding character evolution in phylogenies. In short, we are calculating group I intron presence/loss probabilities on different nodes in the host tree and then testing the predicted intron relationships (given a model of strict vertical inheritance) with phylogenetic analysis. The results of these analyses are being prepared in a manuscript which should be of general relevance to investigators studying mobile elements. Ultimately, we plan to sample all the lichen fungal lineages so that we can accurately estimate intron presence/absence on a robust host tree (e.g., Fig. 2 in Lutzoni, F., Pagel M., Reeb, V. Nature 411:937 [2001]). Dawn, Cora, and Valérie Reeb (Ph.D. student in François’ lab) will conduct this intron screen. In addition, Valérie is currently spending a 3 week research stay with us (funded by the Mellon Foundation) and is completing the sequencing of the group I introns in another large group of lichen fungi, the Acarosporaceae. The rRNA gene sequencing was supported by grant DEB-9615542 to François and a DDIG (DEB-0105194) to Valérie. She has discovered over 120 introns in this group and we are currently completing (with funds from this grant) the intron sequences and analyzing this remarkable data set. Most interesting is Valérie’s finding of intron open reading frames in 5 European isolates of Pleopsidium chlorophanum. These ORFs encode apparently functional His-Cys box endonucleases which are absent in 8 North American isolates of P. chlorophanum. We are currently reconstructing the phylogeny of the host genome (using ITS sequences), of the small subunit rRNA (S)943 intron in which the ORFS are found, and of the ORFs themselves to elucidate the evolutionary history of these 3 genetic elements (i.e., nucleus, intron, ORF) and of the populations that contain them. Valérie is being trained in the analysis of group I introns (alignment, secondary structure prediction) and is also learning how to test their self-splicing ability (from Peik) and whether the ORFs are expressed. Interestingly, the ORFs are encoded on the non-coding strand in loop P8 as are the ORFs found in a S943 intron in a green alga and in the S956 intron in the myxomycete, Didymium iridis. We are intrigued by these findings and hope to use the Pleopsidium system to understand better the role of endonucleases in promoting nuclear group I intron mobility. We expect a number of collaborative publications to emerge from this research.

Intron phylogenetic analyses: Partial support for the RS model regarding group I intron spread has been borne out in our intron phylogenetic analyses that show introns of heterologous origin often form well-supported monophyletic groups, consistent with a common origin. Inspection of the exon flanking

Fig. 4. Secondary structure models of helices P1 and P10 in lichen-fungal SSU rDNA group I introns. The P1 and P10 (boxed) helices of the Rinodina cacuminum 114 intron in its native site and in the heterologous 303 site are shown. The arrows indicate the 5' and 3' splice sites in these introns, with exon sequences shown in lower case and intron sequences in upper case. The intron sequences between P1 and P10 are represented with a solid line.

 

 

upstream sequence at the heterologous sites, consistent with the RS model of intron spread. These findings, with regard to the Physciaceae lichen fungi, are being published in the JME paper. Additional examples of reverse-splicing spread of group I introns in lichens will be described in the upcoming paper that involves François and Mark Pagel’s groups. Corroborative evidence for the RS model is the ability of the mobile introns to build stable secondary structures at the native and heterologous rDNA sites. This prediction is met by all the mobile introns that we have identified (e.g., S114-S303 introns, Fig. 4). To test this idea, Master’s student Joe Runge is first testing that the mobile fungal introns can self-splice and then will make constructs in which these introns are inserted in the native and heterologous fungal rDNA sites; for example, the S114 intron will be inserted into the S303 site, and vice versa. In vitro self-splicing assays will be used to determine, whether consistent with the RS model, the mobile lichen group I introns can splice efficiently at the heterologous sites. These introns will then be expressed in E. coli and RT-PCR will be used to identify sites of reverse-splicing in vivo. Knowing the sequence requirements for the P1 pairing in each mobile intron allows us to predict all the potential insertion sites in bacterial rRNA. Comparing the potential sites to the actual sites of insertion will help us understand the power of the RS model in predicting the specificity of lichen intron reverse-splicing. These analyses are under the supervision of Peik who is an expert in intron expression. REU-supported summer student, Courtney Siebrecht, will be primarily responsible for conducting the splicing experiments. The work is relatively inexpensive and provides a functional test of our phylogenetic predictions. We expect, during the course of the grant, to identify additional examples of group I intron reverse-splicing using phylogenetic and other comparative methods. The reverse-splicing assay described here will complement this work and test the power of our theoretical predictions.

Another example of this research approach is our nearly 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. An abstract describing this project is shown below. David will present this material at a workshop on the Molecular Biology of Cyanobacteria in Sweden in June.

Universal Retention of the tRNA-Leu (UAA) Intron in the Chloroplasts of Land Plants is Coupled with the Near Pervasive Loss of the Intron in all Other Plastids David Fewer, Dawn Simon, Thomas Friedl, and Debashish Bhattacharya

The intron interrupting the leucine transfer RNA gene in cyanobacteria and plastids is widely held to be the most ancient group I intron. The tRNA Leu (UAA) intron is ubiquitous in chloroplasts of the land plant lineage and widely used in micro-evolutionary studies. However, high levels of sequence identity and the paralogy of introns interrupting the tRNA-Leu genes in cyanobacteria have led to suggestions that the intron may have a more recent origin. To readdress the evolutionary history of this intron, a systematic survey was undertaken to determine the phylogenetic distribution of the intron in cyanobacteria and plastids. It is shown here that the distribution of the intron is much more sporadic than previously suspected. However, no instances of horizontal transfer received support in phylogenetic analyses. The present-day distribution of the intron is consistent with an evolutionary history characterized by vertical transmission, with no losses in land plants, widespread loss in cyanobacteria, several losses among green algae and their secondary derivatives, and near pervasive loss amongst red algae and their secondary derivatives. The long-standing hypothesis that the plastid introns have lost their ability to catalyze their own excision is confirmed.

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 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. In summary, 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., role of introns in ribosome folding [with Gloria Culver], use of information content to predict spliceosomal intron positions [with Jian Huang]). These new initiatives could potentially provide a much fuller understanding of the intron biology in rRNAs and generally in all nuclear genes. We anticipate a number of publications will come from these efforts as will a much more robust basis for thinking about intron evolution.

Origin of Introns: Year one activities Year two activities Year three activities