ADV PHOTO EDITING & DSGN
ADV PHOTO EDITING & DSGN J 395
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SE Gene regulation by microRNAs Richard W Carthew The role of small RNAs as key regulators of mRNA turnover and translation has been well established Recent advances indicate that the small RNAs termed microRNAs play important roles in animal development and physiology Cellular activities such as proliferation morphogenesis apoptosis and differentiation are regulated by microRNAs The expression of various genes are regulated by microRNAs and several microRNAs act in reciprocal negative feedback loops with protein factors to control cell fate decisions that are triggered by signal transduction activity These observations implicate small RNAs as important mediators of gene regulation in response to cell cell signaling The mechanism by which microRNAs silence gene expression is post transcriptional possibly influencing the stability compartmentalization and translation of mRNAs This mechanism is an efficient means to regulate production of a diverse range of proteins Addresses Department of Biochemistry Molecular Biology and Cell Biology 2205 Tech Drive Northwestern University Evanston IL 60208 USA Corresponding author Carthew Richard W r carthewnorthwesternedu Current Opinion in Genetics amp Development 2006 16203 208 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Susan Parkhurst and Toshio Tsukiyama Available online 28th February 2006 0959437X see front matter 2005 Elsevier Ltd All rights reserved DOI 101016jgde200602012 Introduction Although the role that proteins play in gene regulation is well understood it has become clear that RNAs are also important gene regulatory factors Small RNAs including microRNAs miRNAs and short interfering RNAs siR NAs are components of an RNA based mechanism of gene regulation found in eukaryotes siRNAs are used throughout the Eukaryota to inhibit viruses and transpo sable elements They also play a role in chromosome organization and in silencing the expression of protein coding genes The miRNA branch of RNA based gene regulation is less widespread miRNAs are found in plants and animals but are apparently absent in fungi This review focuses upon recent advances in our understand ing of miRNAs and their manifest functions in animal development and physiology Full text provided by wwwsciencedirectcom SCIENCEltampDIFIECT MicroRNA biogenesis and mechanism The number of miRNA genes found in sequenced animal species corresponds to approximately 05 15 of the total number of genes in their genomes httpwww sangeracukSoftwareRfammirnaindexshtml The fruit y Drosop id med ogdsz er and the nematode Cde or de elites eegems have at the latest count 78 and 114 miRNA genes respectively Humans have at least 326 miRNA genes MicroRNAs are transcribed by RNA polymerase II and the primary transcripts contain hairpin loop domains that fold back into duplex like structures 12 The hairpin loop region is cleaved in the nucleus by the RNase III enzyme Drosha in complex with a double strand RNA binding protein termed DGCRS 3 6 The resulting hairpin loop fragment is then exported from the nucleus to the cytoplasm where it is cleaved by the RNase III enzyme Dicer in partnership with another RNAbinding protein called TRBP imm activation responsive RNA binding protein 7 9 The fully mature miRNA accumulates as a single stranded species comprising one arm of each miRNA hairpin precursor and it incorporates into a ribonucleoprotein complex that carries out its function of silencing gene expression Although the exact silencing mechanism is not known it is clear that miRNAs use a mode of silencing related to that employed by siRNAs which cleave mRNA tran scripts In a similar fashion to siRNAs miRNAs inhibit a target mRNA by base pairing to complementary sequences within the message However an animal miRNA typically makes imperfect base pair contacts with its target mRNA Contacts usually occur within 3 untranslated regions UTRs The historical mechanistic view has been that miRNA bound target mRNAs are not cleaved but instead are unable to generate encoded proteins due to an effect on the elongation phase of protein synthesis 10 Recently this view has come into question with a bevy of discoveries suggesting that miR NAs might inhibit gene expression by other mechanisms Human let7 miRNA inhibits translation of reporter mRNAs through a mRNA 5 cap dependent mechanism that affects initiation a similar effect was seen with an arti cial miRNA 1112 This discrepancy is further exacerbated by independent ndings that implicate miR NAs as destabilizers of imperfectly base paired mRNAs Human miRNAs miR 1 and miR 124 cause a reduction in the abundance of target transcripts 13 Moreover miR 16 is complementary to AU rich elements found in the 3 UTRs of some short lived transcripts and miR 16 is essential for the rapid turnover of these mRNAs 14 Finally both lie4 and let7 miRNAs in C eegems wwwsciencedirectcom Current Opinion in Genetics amp Development 2006 16203 208 204 Chromosomes and expression mechanisms destabilize target mRNAs even though they are imper fectly basepaired 15 It is not yet clear whether these observed reductions in mRNA levels are a result of cleavage by a siRNAlike mechanism or whether the bound miRNAs enlist other degradation machineries The data are further compounded by recent observations that miRNAs might repress gene expression by seques tering targeted mRNAs into PIGWbodies processing bodies 1617 These are cytoplasmic foci that contain nontranslated mRNAs and exclude the translation machinery Such target localization could potentially embody part or all of the underlying cause of repression One simple model is that the interaction between miRNA and target results in transport of mRNAs to P GWbodies where they are unavailable to the protein synthetic machinery but are subject to decapping and degradation by resident nucleases It is also conceivable that miRNAs act through multiple cooperative mechan isms to repress their targets Consistent with this notion other speci c translation repression mechanisms have been observed to feed into PIGWbodies What are the targets of miRNA regulation A major question about miRNAs concerns the extent of their regulation of animal genomes Computational meth ods have been helpful in estimating this value When the 3 UTRs from sequenced mammalian genomes were aligned sequences capable of forming 6 8 bp perfect duplexes with the 5 ends of many human miRNAs were identi ed 18 Given the frequency of 3 UTR motifs with complementary miRNAs found it was estimated that approximately 20 to 30 of all human genes are targets of miRNA regulation and there is an average of 200 targets per miRNA 1819 Most binding sites are dominated by complementarity to the miRNA seed with little or no support from pairing to the 3 end 20 By contrast other binding sites contain imperfect seed pair ing and are compensated with strong 3 pairing 2021 These 3 compensatory sites are estimated to constitute 20 of all miRNAbinding sites Bioinformatic studies have suggested that the extent of miRNAbased gene regulation has remained relatively stable throughout ani mal evolution with 15 to 30 of animal genomes under direct regulation A formidable task remains in testing these modeling predictions Although smallscale experimental validation studies have con rmed the ef cacy of these computa tional models it has been more dif cult to determine the genomewide extent of miRNA control Two approaches have been adopted with some success One approach sought targets by transfecting tissuespeci c miRNAs into HeLa cells and identifying which mRNAs were consequently reduced in abundance 13 Of the tran scripts that were repressed by each miRNA a negative correlation was observed between their expression and the expression of each effector miRNA in various tissues This suggests that the transcripts that are downregulated in HeLa cells are biological targets of the same miRNAs expressed in human tissues For each miRNA approxi mately 100 transcripts were downregulated suggesting a large miRNAtarget ratio in the same order as that pre dicted by computational methods A second approach to nding genomewide targets by experimental analysis used proteomics 22 Unfertilized oocytes were harvested from mutant Dmmp ia females missing the ierI gene and thus depleted of properly processed miRNAs The proteome from such oocytes was compared to the normal oocyte proteome and 4 of the detected proteins were found to be downregulated when miRNAs are normally processed Of the proteins identi ed by mass spectrometry virtually all had predicted miRNAbinding sites within their 3 UTRs The two genomewide approaches have provided some what contradictory conclusions The range and breadth of targets is noticeably restricted in the Dromp id oocyte in comparison with that in HeLa cells A much smaller proportion of the genome appears to be repressed by y miRNAs compared with the proportion in sam le human miRNAs tested Moreover most of the Dromp id targets are involved in some aspect of global protein metabolism whereas the human targets have more diverse functions The Dmmp ia targets contain primar ily 3 compensatory binding sites and most target tran scripts are not reduced in abundance These differences between Dromp id and human results are not yet fully explainable It might reflect an intrinsic difference in miRNA mechanisms between the two species but more probably it reflects the unusual features of latestage oocytes in general Oocytes shut off all RNA synthesis and RNA metabolism is highly regulated as is protein metabolism Intriguingly under certain stress conditions mammalian cells use miRNAs to regulate global protein metabolism 23 Biological regulation by miRNAs global approaches If 30 of the coding genome is repressed by miRNAs then the breadth of regulated biological processes might be enormous To address this issue several groups have i i the A of eliminarin miRNA maturation by examining the phenotypes of Dir mutant animals Surprisingly they have revealed rather focused de cits that suggest more speci c roles for miRNAs Knockout of mouse Dir results in embryonic lethality and conditional removal ofDiier from the embryonic limb causes extensive apoptosis 24 However patterning and differentiation of the mutant limb remains mostly normal This strong dependence on Dir for cell survival was also observed in the Tcell lineage of the hematopoietic system 25 Loss of Dir in zebra sh results in abnormal Current Opinion in Genetics amp Development 2006 16203 208 wwwscien cedirectcom during 39 i i and somitogenesis but again overall patterning of the body axis is normal 26 Such tissuespeci c roles for micro RNAs are supported by the documented expression patterns of zebra sh miRNAs most mi N s are expressed in highly tissuespeci c patterns during seg mentation and later stages A different role for miRNAs has been revealed in stem cells One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environ ment where most of the cells are quiescent A crucial question in stem cell biology has been how stem cells escape division stop signals It appears that miRNAs are required for division of germ line stem cells GSCs in Dromp id 28 If GSCs are mutant for Biker1 then the frequency of GSC division drops by 80 and many fewer eggs and sperm are produced This cessation is not caused by a defect in GSC differentiation but rather by a defect in cell cycle control mutant GSCs are blocked at the G1 8 checkpoint as a result of upregulation of the cyclin dependant kinaseinhibitor Dacapo Binding sites for several miRNAs are located in the 3 UTR of Dacapo suggesting that miRNAs are required for GSCs to bypass the normal Gl S checkpoint by downregulating this inhibitor The miRNA pathway might be part ofa general mechanism that promotes stem cell proliferation because Diier mutant embryonic stem cells also lose their prolif erative capacity in vivo and in vim Z9 A flip side to these studies concerns whether in adult cells miRNAs can stimulate proliferation that leads to a cancerous state Indeed more than half of the known human miRNA genes are located near chromosomal breakpoints associated with cancer and in some docu mented cases the miRNA genes are ampli ed leading to overexpression 30 For one miRNA cluster overexpres sion promotes tumor progression in an animal lymphoma model 31 Thus the proliferative potential inherent in miRNAs is a causative agent for cancer Table 1 Gene regulation by microRNAs Carthew 205 Biological regulation by miRNAs singlegene approaches Another method to determine the functions of miRNAs is to inactivate each miRNA individually Table 1 Due to the small size of each gene mutagenesis has been tech nically challenging but recent progress has been forth coming As one would expect speci c miRNA genes have been found to be important for cell growth morphogen esis and apoptosis The largest Drosop id miRNA gene amily the miRZ family is required for the suppression of apoptosis during embryogenesis 32 This observa tion was made in an elegant study that used antisense RNAs rather than traditional mutagenesis to system atically inactivate miRNAs Human miRl7 5p and miR ZOa appear to be regulators ofproliferation they bind and repress EZFl mRNA in response to stimulation by cMyc 33 This might allow for tight control of the proliferative signal And an evolutionarily conserved function for miR 1 in muscle proliferation and morphogenesis was recently described Musclespeci c expression of miR 1 is acti vated by the promyogenic transcription factor MefZ in both Dromp id and mouse 3435 Murine miR l sup presses the proliferation of ventricular cardiomyocytes by downregulating the Handl transcription factor 34 By contrast loss of Dromp id miRl results in abnormal growth and morphogenesis of muscles 35 The paradigm for this singlegene approach has been C elegdm in which the rst miRNAs in4 and let7 were discovered The miRNA let7 is expressed late in the nematode life cycle and it promotes the larva L4 to adult transition in diverse tissues Several targets of let7 have been identi ed in four of these tissues and all were found to be transcription factors consistent with the idea that let7 regulates the timing of differentiation 36 On the basis of sequence similarity several other miRNAs are highly related to let7 Three of these miRNAs function together to control the timing of the LZtoL3 transition 3738 Consistent with these observations mutants in the C elegdm Diier gene exhibit similar Summary of selected miRNAs and their biological activities miRNA Species Target genes Function References I t 7 Nematode def12 phad hbl 1 L4 adult transition 36 miR48 miR24l miR84 Nematode hbl 1 L2 L3 transition 3738 IsyB miR273 Nematode cog1 die1 ASE fate decision 42 miR84 Nematode let60 Secondary vulva cell fate 45 miRBl Nematode vav1 Secondary vulva cell fate 46quot miR2 miRB miRll miR308 Fruit fly hid grim rpr 5k A o tosis 3239 miRl Fruit fly Unknown Muscle growth 35 miR3l Fruit fly Unknown mbryo segmentation 3239 miR7 Fruit fly an Photoreceptor fate 44quot iRl ouse Hand1 Myocyte proliferation 34 miRl 96a M ouse Hoxb8 4i miRl75p miR20 Human 2F1 Proliferation 33 wwwsciencedirectcom Current Opinion in Genetics amp Development 2006 16203 208 206 Chromosomes and expression mechanisms developmental timing defects 3940 Thus miRNAs play an important role in controlling the timing of devel opmental decisions A few miRNAs have been found to regulate cell differ entiation Axial patterning of the embryo is aided by miR 31 in Dmmp ia and by miRl96a in the mouse 3Z 4l Although miR 3l in uences segmentation by affecting unidenti ed downstream genes a target ofmiR196a was identi ed to be Hoxb8 which is consequently restricted from the posterior of the mouse embryo 41 A more detailed View of the roles played by miRNAs in cell fate determination has come from the study of neuronal development In C elegdm two gustatory neurons adopt either an ASEL or ASER fate from a bipotential ASE precursor The fate decision is controlled by two miR NAs 531 and miR Z73 which mutually repress each other s expression 42 They do so by inhibiting tran scription factors that activate 531 and MIR273 transcrip tion Figure 1a This fourcomponent feedback loop is doublenegative in character and it forms a bistable system Bistable systems exist almost exclusively in one of two possible states that are stabilized by feedback loops 43 In the case of the ASE decision the two states are the ASEL and ASER fates The bistable nature of the feedback mechanism ensures that the adoption of one state is maintained or stabilized For example if a weak signal to differentiate is received by an ASE cell the istable switch could amplify the signal into a strong uniform response Thus it would ensure that signal variation between different cells has less impact on their ability to uniformly respond Likewise if a signal to differentiate is transient the bistable switch could trans late the transient signal into a longlasting response The relationship between bistable feedback loops and miRN s is more pervasive than this one example Expression of the Dromp id miRNA miR7 is turned on in cells as they begin to differentiate into photore ceptors 44quot This is dependent on EGF receptor EGFRsignaling which triggers ERK extracellular sig 1 TrAl A rAtquot 1 1quot and degradation of the transcription factor Yan In nonsti mulated cells stabilized Yan represses miR 7 transcrip tion In turn miR7 miRNA represses Yan protein expression in photoreceptors directly by binding to complementary sequences within its mRNA 3 UTR This reciprocal negative feedback between Yan and miR7 ensures mutually exclusive expression with Yan in progenitor cells and miR7 in photoreceptor cells Figure lb Expression is switched when EGFRsignal ing transiently triggers Yan degradation The longterm depletion of Yan from differentiating cells is crucial because it inhibits transcription of multiple cellspeci c genes This mechanism involving miR7 explains how signal transduction activity can robustly generate a stable change in gene expression patterns in the Drom id eye Figure 1 a rsys dim A s 599719 1 mil273 3 8 lt ASEL cell fate ASEFl cell fate b yan RNA Vb m Target genes Target genes EG FR C 2 P7p Cun39enl Opinion in Genetics amp Developmem Cell fate determination and the roles of miRNAs a ASE cell fate determination in the C elegans nervous system Two ASE cells undergo fate decisions the cell on the left side of the animal becomes an ASEL and the cell on the right becomes an ASER Adoption of the ASEL fate is determined by miRNA Isy6 whereas the ASER fate is determined by miRNA miR273 These RNAs mutually inhibit each other s expression by repressing the transcription factors cogl and die1 b Photoreceptor cell fate determination in the Drosophila eye In the absence of an EGF receptor signal the transcription factor Yan represses expression of the miR7 gene When the EGF receptor is activated Yan protein is degraded making it incapable of repressing miR7 The synthesized miR7 miRNA binds to the 3 UTR of yan transcripts thereby repressing Yan expression This reciprocal negative feedback loop between miR7 and Yan exerts itself upon the differentiation state of cells through the action of Yan upon downstream genes It is also possible that miR7 regulates other downstream genes outside of the feedback loop c Vulva cell development in C elegans The let23 EGF receptor is activated in the P6pVPC which induces its primary cell fate and secondarily leads to secretion of the DSL protein DSL interacts with its cognate Notch receptor LIN12 on the surface of the neighboring P5p and P7p cells Signal transduction through the let23 pathway is attenuated in these cells by the action of miR84 on LET60 Ras expression Activation of LIN12 leads to expression of miRBT which represses Vavi Given that the Vavi protein normally inhibits LIN12 its 39 ence a positive feedback loop to be engaged This helps to switch these cells to a secondary cell fate Current Opinion in Genetics amp Development 2006 16203 208 wwwscien cedirectcom Other examples of interactions between miRNAs and signal transduction networks are emerging Figure 1c Development of the C elegdm vulva requires cell cell interactions to specify vulval precursor cells VPCs into primary secondary and tertiary fates An inductive signal from the gonad activates the EGFR pathway in a VPC causing it to differentiate as a primary cell The EGFR pathway is suppressed by miR 84 which is expressed in early secondary cells and acts directly upon an EGFR pathway component Ras Hence miR 84 acts to attenuate signaling activity that promotes primary cell ate determination Once determined the primary cell produces a lateral signal that is received by the Notch receptor on the surface of neighboring VPCs When activated Notch is cleaved and the intracellular fragment localizes to the VPC nucleus and stimulates transcription of downstream genes One of these genes encodes miRNA miR61 46quot In turn miR6l binds to the mRNA encoding Vav1 the U elegdm ortholog of the Vav oncogene product and downregulates its expression Because Vav1 represses Notch the downregulation of Vav1 by miR 6l augments Notch signaling activity and promotes the VPC to adopt a secondary fate Thus a reciprocal negative feedback loop between a miRNA and protein in this case Vav plays an important role in a cell fate decision triggered by a cell cell signal the mechanism bears a striking resemblance in certain respects to the interaction between miR7 and Yan Conclusions It is becoming clear that miRNAs play diverse regulatory roles in animal cells They might use several mechanisms to repress gene expression although it is still uncertain if these are related to each other Cellular activities such as proliferation morphogenesis apoptosis and differentia tion are regulated by mi s and in some cases upstream and downstream genes have been linked to the miRNAs Several miRNAs have been found to act in reciprocal negative feedback loops with protein factors to control cell fate decisions that are triggered by signal transduction activity It remains to be seen how generally miRNAs will be involved in this type of mechanism But the potential of rapidly evolving miRNA regulation could be important for evolving new regulatory circuits and ultimately new patterns within body plans Acknowledgements This Work has been supported by the National Institutes of Health References and recommended reading Papers of particular interest published within the annual period of review have been highlighted as o of special interest so of outstanding interest 1 Cai X Hagedom CH Cullen BR Human microRNAs are processed from capped polyadenylated transcripts that can also function as mRNAs RNA 2004 1019571966 9 F Squot 57 l 9 0 0 N 9 P 0 S N 9 Gene regulation by microRNAs Carthew Lee Y IQm M Han J Yeom KH Lee S Baek SH Kim VN MicroRNA genes are transcribed by RNA polymerase II EMBO J 2004 2340514060 Landthaler M Yalcin A Tuschl T The human DiGeor e syndrome critical region gene 8 and its D melanogaster homolog are required for miRNA biogenesis Curr Biol 2004 1421622167 Denli AM Tops BB Plasterk RH Ketting RF Hannon GJ Processing of primary microRNAs by the Microprocessor complex Nature 2004 432231235 Han J Lee Y Yeom KH Kim YK Jin H Kim VN The Drosha DGCR8 complex in primary microRNA processing Genes Dev 2004 1830163027 Gregory RI Yan KP Amuthan G Chendrimada T Doratotaj B ooch N Shiekhattar R The Microprocessor complex mediates the genesis of microRNAs Nature 2004 4322 5240 Jiang F Ye X Liu X Fincher L McKearin D Liu Q Dicer1 and R3D1L catalyze microRNA maturation in Drosophila Genes Dev 2005 191674 1679 Chendrimada TP Gregory RI Kumaraswamy E Norman J Cooch N Nishikura K Shiekhattar R TRBP recruits the Dicer complex to go2 for microRNA processing an ene silencing Nature 2005 436740744 Saito K Ishizuka A Siomi H Siomi MC Processing of premicroRNAs by the Dicer1Loquacious complex in Drosophila cells PLoS Biol 2005 3e235 Olsen PH Ambros V The in4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking L N14 protein synthesis e on of translation Dev Biol 1999 216671680 Pillai RS Bhattacharyya SN Artus CG Zoller T Cougot N E Bertrand E Filipowicz W Inhibition of translational atlon by Let7 MicroRNA in human cells Science 2005 30915731576 Humphreys DT Westman BJ Martin DI Preiss T MicroRNAs control translation initiation by in 39 karyotic initiation factor 4Ecap and polyA tail fun Natl Acad Sci USA 2005 1021696116966 Lim LP Lau NC GarrettEngele P Grimson A Schelter JM CastIeJ Bartel DP Linsley PS Johnson JM Microarray analysis shows t at some micro downregulate large numbers of target mRNAs Nature 2005 433769773 Jing Q Huang S Guth S Zarubin T Motoyama A Chen J Di Padova F Lin SC Gram H Han J nvolvement of microRNA in AUrich elementmediated mRNA instability Cell 2005 1206 3634 Ing eu n Proc Bagga S Bracht J Hunter S Massirer K Holtz J Eachus R Pasquinelli AE Regulation by let7 and lin4 miRNAs results in target mRNA degradation Cell 2005 122553563 Sen GL BIau HM Argonaute 2RISC resides in sites of mammalian mRN ecay known s cytoplasmic bodies Nat Cell Biol 2005 7633636 Liu J ValenciaSanchez MA Hannon GJ Parker R MicroRNA dependent localization of targeted m NAs to mammalian Pbodies Nat Cell Biol 2005 7719723 Lewis BP Burge CB Bartel DP Conserved seed pairing often flanked by adenosines indicates that thousands of human enes are microRNA targets Cell 2005 1201520 Krek A Grun D Poy MN Wolf R Rosenberg L Epstein EJ MacMenamin P da Piedade I Gunsalus KC Stoffel M et al Combinatorial microRNA target predictions Nat Genet 2005 37495500 Brennecke J Stark A Russell RB Cohen SM Prin p es of microRNAtarget recognition PLoS Biol 2005 3e85 John B Enright AJ Aravin A Tuschl T Sander 0 Marks DS Human MicroRNA targets PLoS Biol 2004 2e363 wwwsciencedirectcom Current Opinion in Genetics amp Development 2006 16203 208 Review ELS TRENDS in Genetics Vol2 l No2 February 2005 Full text provided by wwwsciencedirectcom SCIENCEltdDIRECT39 The natural history of group I introns Peik Haugen Dawn M Simon and Debashish Bhattacharya Department of Biological Sciences and Roy J Carver Center for Comparative Genomics University of Iowa 312 Biology Building Iowa City IA 522421324 USA There are four major classes of introns selfsplicing group I and group II introns tRNA andor archaeal introns and spliceosomal introns in nuclear premRNA Group I introns are widely distributed in protists bac teria and bacteriophages Group II introns are found in fungal and land plant mitochondria algal plastids bacteria and Archaea Group II and spliceosomal introns share a common splicing pathway and might be related to each other The tRNA andor archaeal introns are found in the nuclear tRNA of eukaryotes and in archaeal tRNA rRNA and mRNA The mechanisms underlying the selfsplicing and mobility of a few model group I introns are well understood By contrast the role of these highly distinct processes in the evolution of the 1500 group I introns found thus far in nature eg in algae and fungi has only recently been clarified The explosion of new sequence data has facilitated the use of comparative methods to understand group I intron evolution in a broader context and to generate hypotheses about intron insertion splicing and spread that can be tested experimentally Introduction Mobile genetic elements have had a major impact on eukaryotic genomes 1 Group I introns are small RNAs they range in size from 250 500nt and comprise one of the four distinct types of introns Box 1 These elements are found in a wide variety of organisms eg in fungi algae and in many other unicellular eukaryotes genes ie protein rRNA and tRNA coding genes and genomes throughout the tree of life Group I introns spread ef ciently at the DNA level into intronless cognate sites by a process termed homing see Glossary The success of group I introns in genomes is also a result of their ability to selfsplice from RNA transcripts which potentially renders them neutral to the host The structure folding and autocatalysis of a few model group I introns have been rigorously studied and we now have a detailed under standing of how these elements act as catalytic RNAs ribozymes during splicing of precursor RNA 2 4 Box 2 The mechanism of group I intron spread in DNA has also been studied in detail Homing endonuclease genes HEGs that invade noncritical regions ie terminal loops of group I introns promote intron mobility by encoding highly sitespeci c homing endonucleases HEs HEGs are themselves sel sh genetic elements and the vast majority of group I introns in nature do not contain a Corresponding author Bhattacharya D dbhattacblueweeguiowaedu Available online 21 December 2004 HEG see the following section for intron life cycle HEs and putative HEs encoded by HEGlike sequences are divided into four families based on the presence of conserved amino acid motifs these are the HisCys box LAGLIDADG GIYYIG and HNH families Differences in the endonuclease sequences suggest independent origins of the four families of HE and putative HE proteins 5 Despite extensive knowledge about group I intron bio chemistry understanding the complex and often unpre dictable evolutionary history of group I introns and their associated HEGs has proven to be a formidable challenge The explosive growth of DNA sequence data in GenBank httpwwwncbinlmnihgovGenBankGenBankstats html has however turned the tide enabling a more Box 1 Types of introns Four major classes of introns are recognized based on their splicing mechanism These are autocatalytic group I and group II introns spliceosomal introns tRNA andor archaeal introns Group I introns are widespread in protist nuclear rDNA genes fungal mitochondria bacteria and bacteriophages These pre RNA insertions self splice by a well characterized and distinctive two step pathway that relies on an external guanosine nucleotide as the cofactor Group II introns are found in bacterial and organellar genomes and self splice through a pathway that is different from group I introns but is similar to the mechanism of spliceosomal intron removal In these introns rather than using an external guanosine the 2 OH of an adenine residue within the intron is the nucleophile The shared splicing mechanism suggests that the small nuclear snRNA components of the spliceo some are derived from a group II intron Spliceosomal introns are the most common insertions found in nuclear pre mRNA genes The tRNA introns are found in eukaryotic nuclei and in Archaea and are enzymatically removed by a cut and rejoin mechanism that requires ATP and an endonuclease This pathway is completely different from that of spliceosomal introns Glossary Homing the process by which an intron or intein spreads into the homo logous position in an allele that lacks the intron Homing endonuclease a highly specific endonuclease protein that is typically encoded by selfsplicing introns They promote intron mobility through homing lntrons intervening sequences in genes that are removed from precursor RNA in a process termed splicing Monophyletic a group of organisms or molecular sequences which includes the most recent common ancestor of all of its members and all of the descen dants of that most recent common ancestor Monophyletic groups are also called clades Phylogeny the evolutionary relationships among organisms or genes Reverse splicing the reversal of the forward selfsplicing reaction in which a free intron integrates into another RNA molecule This term is sometimes used to describe the process by which a group I intron becomes stably integrated into the genome following the reverse splicing reaction and subsequent reverse transcription and reintegration into the genome Ribozymes catalytic RNAs RNA enzymes wwwsciencedirectcom 01689525 see front matter 2004 Elsevier Ltd All rights reserved doi101016jtig200412007 TRENDS in Genetics Vol21 Box 2 Group I intron secondary structure and splicing quot39L 39 39 structureofagroupl39 39 roxi mately ten paired elements P1 P10 plus the optional P11 P17 Figure la that are organized into three domains at the tertiary structure level 4 The intron recognizes the 5 exon sequence by a 4 6nt base pairing the internal guide sequence lGSl interaction Approximately 100 nucleotides constitute the central catalytic core of the intron RNA the shaded regions in Figure la Group introns Figure lbl Many introns can self splice in vitro as naked RNAs ie no proteins are required for intron splicinglwhereas others need protein factors to facilitate correct folding of the ribozyme co re A prerequisite forsplicingis h h39 d39 g f g 39 f to a pocket in the catalytic core ofthe intron called the G binding site During the first step of splicing the cofactor attacks the 5 splice site 88 and attaches to the intron resulting in the release ofthe upstream exon The exoG leaves the G binding site and is replaced by the last nucleotide of the intron which is always a G called L0G The second step is initiated by an attack by the 3 end of the released exon on the 3 88 which results in ligation of the exons and release of the intron RNA Successful catalysis is dependent on the correct folding of the intron 3 P91 P13 P13 b A i rst step r 5 lIOH exoG mGl3 A l r second step r Ligated exons 5 I 3 Free intron exoG mG TRENDS In GenetCs Figure I Secondary structure and selfrsplicing of group l introns www5ciencedirectcom No2 Februaw 2005 detailed picture to be drawn of intron distribution and spread through populations and species In this article we present an overview of recent advances in the under standing of group I intron distribution and evolution and describe the driving forces that bring introns to their genomic destinations Group I intron distribution in nature The distribution of group I introns on the tree of life is shown in Figure 1 derived from httpwwwrnaicmb utexasedu 6 We accounted for intron redundancy by A 39 with single entries sets of sequences from taxa with identical names that represent population samples eg 59 introns at the same site in different isolates of the green alga Closterium ehrenbergii or different cultivated strains As of June 8 2004 a total of 1400 group I introns were reported in eukaryotic genomes Of these 800 are in the nucleus at 47 different sites in small subunit SSU ribosomal rRNA and 44 sites in large subunit LSU rRNA 220 are in mitochondrial genes and 370 are in plastid DNA Nuclear group Iintrons are limited to rDNA genes whereas in the organelles they are found in rRNA tRNA and protein coding regions Group I introns have a sporadic and highly biased dis tribution in nature and many microbial eukaryotes are rich in these sequences Figure 1 However group I introns appear to be absent in animals except for three cases Two are found in the mitochondrial genome of the sea anemone Metridium senile and one is in the mito chondrial gene cytochrome c oxidase subunit I of the coral Acropora tenuis Group I introns have not yet been found in Archaea Lineages in the tree of life that are particularly intron rich are fungi plants and the red and green algae These taxa contain 90 of all group I introns that have been identi ed to date However DNA sequencing efforts in particular for organelle genomes have been biased towards particular groups of genes and organisms eg in animals and plants of the 623 sequenced or partially se uenced mitochondrial genomes 555 are from animals 9 nrbi nlm nib quot oh m and it is possible that larger pools of introns remain to be discovered eg in excavates a group of potentially anciently diverged agellates that bear a unique cluster of agellae httpmicroscopembledu as the taxonomic breadth of sequencing programs increase In contrast to the situation in eukaryotes group I introns are rare in bacteria Only fty introns representing nine different insertion sites occur in this prokaryotic lineage ie four in tRNA genes four in rRNA genes and one in the recA gene 6 8 Group I introns also occur infrequently in viruses 9 and phages 1011 and are not shown in gure 1 Group I intron mobility The evolutionary fates of group I introns The sporadic distribution of group I introns suggests that many of these mobile elements have undergone horizontal transfer through homing or reverse splicing into differ ent species and genes The potential evolutionary fates of laterally transferred group I introns are loss from the lineage vertical inheritance andor movement within the TRENDS in Genetics Vol21 No2 Februaw 2005 113 l i P 39 Rhizavia N I 39 5 VSO a BS 9 MN m 5 00 as N 0 39n a 652 Y 9 g Y c WP on r G a a a v9 8 E s If E PM M71799 0 7 gods s 0 0710 m PD 6wa stramenopiies m M gt WM Oomyceles Algae e 39 Hat 38 r DmsltN eucaphyte alga La Brow e Opalinjds bylvmhunds 0 13936 NP NM 1 C bar lt M gt r 036 am Yplophytes Cy DICSYOSI r 39 06985 S I s 41 slime mom Cay dlt HaDtoph 96 N P 765mm 0 yfss N Pramquot c A Ore 613508 4176 V sW 1175 quot70de Amoeuozoa e m 13 WWW N Ne 6 40 307 Usage a D a an 98 05086 Q 0 b s a s z 0 7r 0w Y a an a g co a 0 Us N 3 Q s m 9 1 a M 0 LE 3 9 Zv 6 one s g 3 lt2 as N 3 1939 l m Opes Imkoms Evra lam Discicristates 7 Archaea Eubaclerla lt B Figure 1 Tne distribution of group l introns on lhelree of We Tne presence of introns in nuclear N plas d P mhochondrlal M or bacterial B genomes ls Indicated by quot UI39 I ive39 IS I I I I h gh 139 l e mu39wme we we euoe of 1725 26750517100 1017250 or 5159roup l introns is indicated in largenexx of land plantsThevee is a consensus of molecular and ultraslruclural data for more details see Ref 43 ons in the nuclear DNA andme broken Hnes represennhe possible posmons onne root me oroksryono Hneages on me consensus phylogeny of eukaryoles Adapted w39nn permission from Ref 43 lineage with partial loss or xation ie stable inheritance with no losses and no apparent mobility within the lineage An additional level of complexity is that many group I introns appear to be mosaic genetic elements Unlike their counterparts in group II introns HEGs in group I introns are less likely to share the same evolutionary history as the catalytic RNA 12 In fact a group I intronencoded HEG from one intron eg intron one can invade another group I intron at the homologous same or heterologous other position independent of the group I ribozyme encoded by intron one see the following section The intron lifecycle and homing In 1999 Goddard and Burt published a model of intron evolution that involved cyclical gain and loss Figure 2 13 Analysis of the yeast omega group I ribozyme showed that a group I intron with a fulllength HEG invades an intronminus population and spreads into all homologous same sites through homing see Ref 5 for mechanistic details of homing Once the intron becomes xed in the population the HEG no longer has a biological function ie the HEG function is to confer intron mobility mutations and is 39 nr lost Unable to spread the intron is also destined to be lost over 1 i i www5ciencedirectcom time and will not reappear until the same intron with the fulllength HEG is reintroduced through gene ow To evaluate if the omega lifecycle applies to other HEGassociated group I introns it is necessary to examine many different introns from natural populations If multiple populations of an organism that carries a HEGcontaining intron are sampled then one would expect to nd introns with fulllength HEGs degenerate HEGs introns lacking HEGs in addition to examples of intron absence This pattern ts well with what has been reported previously 61114 20 although every step in the intron cycle is not always found in each case probably as a result of insuf cient sampling An extensive study of SSU rDNA 8516 and 81506 the numbering re ects the homologous position in Escherichia coli rDNA group I introns from geographically distinct populations of red algae identi ed individuals lacking introns those contain ing introns without a HEG and those containing introns with various deletions andor mutations in the HEG 17 In summary the Goddard and Burt intron lifecycle model which was established with the yeast omega intron is supported by the intron and HEG distribution in red algae and is consistent with the intron distribution in other naturally occurring populations This suggests that recurrent gain and loss is likely to be a general TRENDS in Genetics Vol21 No2 Februaw 2005 Intron with non 3 functional HEG No intron Precursor RNA Intron invasion Precise loss and xation Intron loss Homing Precursor RNA Functional mobile intron H EG degeneration Loss of H EG function Precursor RNA TRENDS in G enetics Figure 2Th39is model was proposed byGoddard and Bun 13 and 39is consistent with otheranalyses of 5quot lintrons lnih39i r hama hquot 39 witha functional HEG invades an intronrless popuiation and becomes fixed in aii nomoiogous same insertion sites tnrougn noming Because tne HEG no longer can promote intron spread aii sites are occupied tne HEG becomes redundant and eventuaiiy iost Tne intron lifercycle is compieted pytne precise ioss oitne intronmodei iortne recurrent gain and loss oftne omega group i intron feature of HEGassociated group I introns Introns do how ever manage to escape from the cycle by inserting into a new genic position discussed in the following section Introns in the same insertion site are evolutionarily related Reconstruction of group I intron phylogeny is made pos sible by the existence of the conserved RNA secondary structures Pl P9 that can be used to guide the align ment of these sequences The alignments are used as input to computer programs eg PAUP PHYLIP MrBayes and MEGA to infer evolutionary trees The core region used in these analyses is short in length 100 250 nt but as shown in Figure 3 it still encodes suf cient phylo genetic signal to resolve many nodes in the intron trees An important insight into group I intron evolution that has come from phylogenetic analyses is that sequences from homologous genic sites are closely related mono phyletic even though they can reside in distantly related organisms eg eukaryotes and prokaryotes 21 It is unlikely that this result is explained by a single origin of each intron before the eukaryotic radiation or the split of bacteria and eukaryotes followed by widespread loss in different lineages Introns are highly divergent sequences and it is therefore unlikely that they remain conserved across the millions or billions of years that separate taxa such as prokaryotes and eukaryotes A more parsimonious scenario is that group I introns are laterally transferred across the tree of life resulting in their sporadic and highly biased distribution If the second idea is correct then the observation that intron movement nearly always occurs to the homologous site in different organisms suggests that strong restrictions exist to guide introns to their target ie high sequence speci city in homing Intron movement through reverse splicin Group I introns do however occasionally escape to hetero logous genic sites but the mechanisms responsible for www5ciericedirectcom these invasions have proven elusive Because of the limited sequence requirement 4 6nt for group I ribo zymes to locate their target region reverse splicing of free intron RNAs into target RNAs provides a more plausible model for intron spread into ectopic sites than homing which relies on a 15 45nt recognition sequence for the intronencoded homing endonuclease Whether this path way has an important role in group I intron spread however is unknown because of the lack of any direct evidence of reversesplicing mediated intron movement from genetic crosses Furthermore in contrast to homing that has been shown to be highly ef cient it is likely that reverse splicing with its reliance on chance integration followed by two additional steps ie reversetranscription and then recombination would be less effective in promoting intron movement Rare reverse splicing events are therefore most likely to be recognized in the context of a broadly sampled intron phylogeny A nal consideration is that rDNA exists as a multicopy gene family necessi tating that alleles containing transferred introns must rise to high frequency presumably through concerted evolution or less parsimoniously repeated reverse splicing events in individuals and in populations to ensure survival and xation In spite of these constraints conclusive experimental evidence exists that group I introns can ful ll one crucial step in the reverse splicing mobility pathway full inte gration into foreign RNA in viva 2223 In one analysis 19 variants of the Tetrahymena intron all with different internal guide sequences were expressed in E coli 23 Partial reverse splicing at 69 sites and complete inte gration at one novel site in the 238 rRNA were found with this approach Phylogenetic analyses provide support for another prediction of the reverse splicing pathway statistical support for the close relationship of introns at ectopic rDNA sites eg 81046 81052 8289 81199 18 In these cases sequence similarity is limited to the proxi mal 5 anking exon in each pair of related introns This w TRENDS in Genetics Vol21 No2 Februaw 2005 01 substitutions per site 02 substitutions per site TRENDS in Genetics Figure 3 The phyiogeny of nuclear rDNA group i introns A summary ofthe phyiog 39 L in o htra 39 39 H J a A a Rat 21 aiiului and the size of the triangies represents the number of sequences in each ciade nding is consistent with the 4 6nt sequence requirement in the 5 exon that is predicted for the RNAbased reverse splicing and integration pathway In addition HEGs have not been found within the introns at these insertion sites making it less likely that endonucleases mediated the invasions of new sites at the DNA level Although we still lack conclusive proof of reverse splicingmediated intron mobility accumulating experimental and comparative data are in favor of this mechanism having contributed to the spread of group I introns Intronindependent HEG mobility It has been known for some time that sequences encoding HEGs insert into the peripheral regions of ribozymes and mobilize them through homing We now have a more detailed picture of the intronHEG relationship and the evolutionary history of the two partners turns out to be more complicated than previously thought The most direct approach to identify intronindependent HEG move ment is through the comparison of intron and HEG trees Unfortunately this approach is stymied by the rare occurrence of intact HEGs in nature ie they decay rapidly following intron xation 20 HEG mobility can however leave clear evolutionary footprints Figure 4 For example www3cierlcedirectcom iiy enetic reiationships a between introns in aigae and fungi and b between introns in man 260ui 39 39 posterior 39 in am Both 39 p orbtueJukesrCantorrnodei Formore an Raf i i Branch 39 39 39 39 tutiie riumuer He HEGs that are closely related to the HisCys box family are inserted into different peripheral loops or encoded on different strands of homologous group I introns eg introns inserted after positions 516 943 and 1506 in the gene that encodes the nuclear SSU rRNA 20 Movement to different peripheral loopregions within the intron provides evidence for HEG mobility within a single ribozyme lineage Furthermore closely related HEGs are found in phylogenetically distantly related group Iintrons that are located at adjacent rDNA sites sites L1923 L1925 and L1926 suggesting that local HEG mobility into heterologous introns occurs 20 A study of rDNA introns that encode HEGs of the LAGLIDADG family also identi ed related HEGs in adjacent introns in some cases resulting from HEG mobility alone or in other cases as a result of the mobility of the intron HEG element as a unit 24 Did a gene duplication event trigger the spread of LAGLIDADG HEGs Homing endonucleases that contain the conserved LAGLIDADG motif represent the largest family of HEGs and are widespread in nature occurring in several differ ent types of mobile elements ie group I and II introns inteins and archaeal introns and as freestanding open TRENDS in Genetics Vol21 No2 Februaw 2005 HEG i Allele quotAquot HEG Allele quotAquot a Intron homing b HEG invades homologous H EG intron or intron in adjacent site Allele IIAII HEG Allele quotAquot Both alleles are protected against ENase activity Allele quotaquot DSBR HEG Allele quotaquot ma Allele quotaquot n 7 Allele quotaquot DSB in intron l egltlmate I recombination A HEG Em Allele quotaquot of l l l l WAllelequotaquot 1 l HEG or HEG l l l l Allele quotaquot or T Allele quotaquot H EG TRENDS ln Genetcs Figure 4Amodelforthe movemenl of 4 l Homing 4 l l 39 lo move wllhlhe groupllntron as alsunlloy homlng orlb lndependent onhe noozyme Homing results in thetran ferofthe lnlronhEG elemenl plusflanklng exon sequences conconverslon lnlolhe homologous genlo ooslllonThe HF quot 39 39n red D39h 39 an ell new lo result lnlhelnsenlon onhe HEG lnlo homologous HEGrmlnus lntrons both shown in orange ol 39 l 39 39ln A hl 39h respect lo the donorlntron HEGs can lnsen lnlo 39 lhelnlron 39 39 439 39 39 region f h 39 39 39 by the different locations ofthe HEG on the schematic representation ofthe intron reading frames ORFs 25 Comparative studies suggest that the successful spread of LAGLIDADG HEGs was driven by a gene duplication and fusion event followed by the rapid evolution of the DNAbinding domain 242627 Approximately 40 group I intronassociated LAGLIDADG HEGs contain one copy of the motif ie LAGLIDADG motif whereas the remainder contain two copies gt200 cases in total 524 Interestingly the singlemotif HEGs are restricted to introns located at six insertion sites in rDNA ve out of six of these are clustered between L1917 and L1951 By contrast the doublemotifHEGs are more widely distributed in rDNA introns and in introns in other types of genes The difference in the success of these HEGs in spreading into novel introns probably re ects how they recognize target sequences The singlemotif HEs function wwwsciencedirectcom as homodimers and therefore need a high degree of symmetry in the DNA target sequence whereas the doublemotif HEs function as monomers e N an Cterminal halves of the monomeric HEs ie each half corresponds to one protein in the dimers of singlemotif HEs have accumulated many amino acid substitutions and might have a less stringent requirement for DNA target symmetry 26 Most doublemotif LAGLIDADG HEGs in rDNA can be traced to one of two lineages 24 One of these designated Clade 1 in Ref 24 appears to be derived from a duplication and fusion event involving a singlemotif HEG in an intron located between the rDNA positions L1917 L1951 whereas the origin of the other group of doublemotif HEGs Clade 2 is unclear In summary evolution has found an effective solution to target site restriction in LAGLIDADG HEGs Duplication w and divergence of the ancestral singlemotif HEGs to form a twomotif unit appears to have freed them to explore a larger set of host introns and genic sites The role of proteins in group I intron splicing HEGs can avoid loss by gaining maturase activity A fascinating aspect of the group I intronHEG relation ship is that some endonucleases also function as matu rases ie they assist in intron folding 28 The existence of these bifunctional proteins suggests that the gain of maturase activity has provided the HEs andor their asso ciated introns with an evolutionary advantage So what advantage could maturase activity oifer One hypothesis is that the gain of a HEGencoded DNA typically 1kb in size in the group I intron might lower the ef ciency of self splicing The maturase activity conferred by the HEG could therefore be selected for because it rescues or su ciently improves splicing ability to compensate for the presence of the ORF 28 Another intriguing possibility is that maturase activity evolves or is retained because it gives the HEG an opportunity to escape inactivation or loss 29 ie endonuclease function will be lost if the intron is xed in the population whereas maturase activity might be retained to ensure correct intron excision This scenario appears to have played out recently in Saccharomyces cerevisiae in which two nonadjacent amino acid changes activate endonuclease activity in an intronencoded LAGLIDADG maturase 3031 Other important insights have come from structural and biochemical studies of the IAniI HE protein 293233 In this case the RNA and DNAbinding domains of the protein are nonoverlapping ie they are located at different places in the protein TRENDS in Genetics Vol21 No2 Februaw 2005 structure It is therefore likely that the maturase activity evolved as a secondary adaptation In summary there appears to be a dynamic relationship between ribozyme HE and maturase activities in HEGassociated group I introns The relative evolutionary importance of these processes re ects primarily the potential for intron spread in populations or species and the extent of dependence on the maturase for intron splicing The longterm evolution of selfsplicing ability Insights into the evolution of RNA structure and auto catalysis can be gained by comparing group I introns that have been vertically inherited for millions of years These sequences provi e potential examples of intronhost coevolution and are candidates for introns that might have evolved a function that is bene cial to the host Two outstanding examples of longterm vertical inheritance are the ancient group I intron inserted in the tRNALeu gene of cyanobacteria and plastids 34 36 and the S788 intron in the ascomycete fungi 37 The tRNALeu intron has probably been vertically inherited for more than a billion years in plastids 38 and twotothree billion years in the cyanobacterial ancestors of these organelles 34 whereas the S788 intron was probably acquired in ascom ycetes 400 600 million years ago 37 Although there are some obvious diiferences in the evolutionary history of these two group I introns there are also striking simi larities Most importantly when selfsplicing activity is mapped on the intron phylogeny which generally recapi tulates the host tree as a result of intron vertical inheritance in both cases the early diverging ribozymes selfsplice ef ciently as naked RNAs By contrast the later a Euphyiiophytlna Coleocnaetales Chara e5 Zygnernatales Euphyllophytina Charophyta Streptophyta Glaucophyta 1st Cyanobacteria b r P5abcd Streptophyta Charophytes P5abcd Figure 5 Analysis ofthe pnylogenyand in vitro splicing oftwo enican a into thetR A Leu gene in plastids and in cyanopactena undergothefirst step y and b after position 733 in of splicing 15 or show no activity in vitro are indicated on the tree The boxed ciadogram in a shows the host relationships oftaxa containing i intron y 39 Jtnein vitro splicing apility ofintronsinserted the ssu rRNA of ascomycete fungi are shown introns that splice efficiently H s uiiiiiiai thetRNAsLeu intron quot39 i P5abcd and a later diverging ciade that lacks this domain 7P5abcd The trees are modified with permission from Ref 36 and Ref 37 www5ciencedirectcom tie illtrullan allce tral t H i ii 11B TRENDS in Genetics Vol21 No2 Februaw 2005 diverging introns can either only complete the rst step of splicing ie attack of the 5 splice site by the exogenous guanosine which becomes covalently attached to the intron the 5 exon is freed or lack autocatalytic activity entirely gure 5 It is tempting to speculate that the partial or complete loss of activity in both ribozymes is due to their dependence over evolutionary time on particular host proteins to facilitate splicing Furthermore the xa tion of the tRNALeu intron in all plants combined with their loss of selfsplicing ability suggests that a reciprocal dependence between the intron and the host might have evolved in the common ancestor of this highly successful lineage Although unproven this idea is sup orted by a study that shows chloroplast ribonucleoproteins chNPs associate with unspliced tRNALeu transcripts in N icotiana tabacum 39 The chNP tRNA complexes confer stab ility and ribonuclease resistance to the RNAs and poten tially act as a scaffold for hostmediated splicing of the introncontaining tRNAs 39 The S788 group Iintron is also of great interest because derived members of this ribozyme lineage have undergone major structural changes with many nucleotide substi tutions in the central catalytic core some of which are crucial for intron folding and the loss of the larger peri pheral P5abcd domain Figure 5 Two distinct clades are identi ed in phylogenetic analyses of the S788 intron an ancestral pool that contains the P5 extensions P5abcd Figure 5 and a later diverging clade that lacks this domain P5abcd 37 The loss of P5abcd is correlated with the inability to selfsplice in vitro This example therefore pro vides a direct link between group I intron structure evolu tion and autocatalysis in a vertically inherited intron Concluding remarks The detailed 39 m intron and mobil ity have been established through rigorous biochemical analyses Intron evolution is being clari ed through the analysis of intron phylogeny distribution secondary structure and splicing in a diverse group of microbial eukaryotes Bringing together these two disparate pools of information is essential to understand the natural history of group I introns Recent work has successfully integrated structural andfunctional insights regarding group I introns into a broader evolutionary context Examples of important contributions to our understanding of intron biology and evolution are the compilation of introns into a compre hensive database 6 development of the cyclical model for the gain and loss of HEGassociated introns 13 novel insights into the dynamic intronH relationship 24262729 and the recognition that group I introns can be vertically inherited over long periods of evolutionary time and their interaction with the host cell can provide important insights into intron biology 34 3739 It will be important to investigate the role of host factors in intron splicing across the tree of life eg mitochondrial tyrosyltRNA synthetase Cyt18 31 leucyl tRNA synthetase LeuRS 40 and mitochondrial RNA splicing 1 Mrs1 41 and the contribution of these factors to intron xation and mobility It is likely that host proteins gene rally have a role in the splicing of many group I introns however their broader impact on intron evolution is 1 1 1 www5ciencedirectcom currently unknown Another unanswered question is how group I introns are transferred between species Viruses have often been suggested as possible vectors but evi dence is still lacking nally the true biological roles for group I intron RNAs remains to be clari ed for more details see Ref 42 Acknowledgements This work was generously supported by grants awarded to DB from the National Science Foundation MCB 0110252 DEB 0107754 agrant from The Norwegian Research Council to RH and Avis E Cone and Stanley fellowships from the University of Iowa to D S We thank H Joseph Runge Iowa for helpful discussions References Hurst GD and Werren JH 2001 The role of sel sh genetic elements in eukar otic evolution Nat Rev Ge 2 59 606 Cech TR 2002 Ribozymes the rst 20 years Biachem Sac Trans 30 1162 1166 Westhof E 2002 Group Iintrons and RNA folding Biachem Sac Trans 30 1149 1152 Adams PL et al 2004 Crystal structure of a selfsplicing group I intron with both exons Nature 430 45 50 Chevalier BS and Stoddard BL 2001 Homing endonucleases structural and functional insight into the catalysts of intronintein mobility Nucleic Acids Res 29 3757 3774 Cannone JJ et al 2002 The comparative RNA web CRW site an online database of comparative sequence and structure information for ribosomal intron and o er RNAs B C Biainfarmatics 3 2 oi 1011861471210532 httpJwwwbiomedcentralcom1471210532 Ko M et al 2002 Group I selfsplicing intron in the recA gene of Bacillus anthracis J Bacterial 184 3917 3922 N L and Doolittle WF 2003 Active selfsplicing group I introns in 23S rRNA genes of hyperthermophilic bacteria derived S H m w as m m 4 m from introns in eukaryotic organelles Prac Natl Acad Sci U 100 10806 10811 9 Nishida K et al 1998 Group I introns found in Chlarella Viruses biological implications Wralagy 242 319 326 E gell DR et al 0 Barriers to intron promiscuity in bacteria J Bacterial 182 5281 5289 Sandegren L and Sj39oberg BM 2004 Distribution sequence homolo and homing of group I introns among Tevenlike bacterio phages evidence for recent transfer of old introns J Biol Chem 279 22218 22227 Toor N et al 2001 Coevolution of group II intron RNA structures with their intronencoded reverse transcriptases RNA 7 1142 1152 Goddard MR and Burt A 1999 current invasion and extinction ofa sel sh g ne Prac Natl Acad Sci U S A 96 13880 13885 Cho Y et al 1998 Explosive invasion of plant mitochondria by a group I intron Prac Natl Acad Sci U S A 95 14244 14249 Haugen P et al 1999 Complex groupI introns in nuclear SSU rDNA ofred and green algae evidence ofhomingendonuclease pseudogenes in the Bangiophyceae Curr Genet 36 345 353 Foley S et al 2000 Widespread distribution ofa group Iintron and its three deletion derivatives in the 1 sin gene of Streptococcus thermaphilus bacteriophages J Wral 74 611 618 17 Muller KM et al 2001 A structural and phylogenetic analysis of the group IC1 introns in the order Bangiales Rhodophyta Mal Bial Eval 18 1654 1 67 18 Bhattacharya D et al 2002 Vertical evolution and intragenic spread gal group I introns J Mal v 5 74 84 H o H H H m H w H as H m H m of lichenfun 19 Nozaki H et al 2002 Evolution of rbcL group IA introns and intron open reading frames within the colonial Volvocales Chlorophyceae Mal Phylagenet Eval 23 326 338 20 Haugen P et al 2004 The evolution of homing endonuclease genes an group Iintrons in nuclear rDNA Mal Bial Eval 21 129 140 21 Nikoh N and Fukatsu T 2001 Evolutionary dynamics of multiple group Iintrons in nuclear ribosomal RNA genes of endoparasitic fungi ofthe genus Cardyceps Mal Bial Eval 18 1631 1642
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