RESUMEN
Self-splicing proteins, called inteins, are present in many human pathogens, including the emerging fungal threats Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga), the causative agents of cryptococcosis. Inhibition of protein splicing in Cryptococcus sp. interferes with activity of the only intein-containing protein, Prp8, an essential intron splicing factor. Here, we screened a small-molecule library to find addititonal, potent inhibitors of the Cne Prp8 intein using a split-GFP splicing assay. This revealed the compound 6G-318S, with IC50 values in the low micromolar range in the split-GFP assay and in a complementary split-luciferase system. A fluoride derivative of the compound 6G-318S displayed improved cytotoxicity in human lung carcinoma cells, although there was a slight reduction in the inhibition of splicing. 6G-318S and its derivative inhibited splicing of the Cne Prp8 intein in vivo in Escherichia coli and in C. neoformans Moreover, the compounds repressed growth of WT C. neoformans and C. gattii In contrast, the inhibitors were less potent at inhibiting growth of the inteinless Candida albicans Drug resistance was observed when the Prp8 intein was overexpressed in C. neoformans, indicating specificity of this molecule toward the target. No off-target activity was observed, such as inhibition of serine/cysteine proteases. The inhibitors bound covalently to the Prp8 intein and binding was reduced when the active-site residue Cys1 was mutated. 6G-318S showed a synergistic effect with amphotericin B and additive to indifferent effects with a few other clinically used antimycotics. Overall, the identification of these small-molecule intein-splicing inhibitors opens up prospects for a new class of antifungals.
Asunto(s)
Empalme de Proteína/fisiología , Proteínas de Unión al ARN/genética , Antifúngicos/farmacología , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidad , Proteínas Fúngicas/metabolismo , Humanos , Inteínas/genética , Intrones/genética , Empalme de Proteína/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia/métodosRESUMEN
Inteins (or protein introns) autocatalytically excise themselves through protein splicing. We challenge the long-considered notion that inteins are merely molecular parasites and posit that some inteins evolved to regulate host protein function. Here we show substrate-induced and DNA damage-induced splicing, in which an archaeal recombinase RadA intein splices dramatically faster and more accurately when provided with ssDNA. This unprecedented example of intein splicing stimulation by the substrate of the invaded host protein provides compelling support in favor of inteins acting as pause buttons to arrest protein function until needed; then, an immediate activity switch is triggered, representing a new form of post-translational control.
Asunto(s)
Proteínas Arqueales/genética , Daño del ADN/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Inteínas/genética , Empalme de Proteína/genética , Proteínas Arqueales/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Regulación de la Expresión Génica/genética , Modelos Biológicos , Mutación , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.
Asunto(s)
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Empalmosomas/metabolismo , Asparagina/química , Asparagina/metabolismo , Sitios de Unión , Clonación Molecular , Cobre/química , Cobre/metabolismo , Cryptococcus neoformans/metabolismo , Cristalografía por Rayos X , Cisteína/química , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inteínas , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Empalmosomas/ultraestructura , Homología Estructural de Proteína , Zinc/química , Zinc/metabolismoRESUMEN
Group II introns are self-splicing ribozymes and mobile genetic elements. Splicing is required for both expression of the interrupted host gene and intron retromobility. For the pRS01 plasmid-encoded Lactococcus lactis group II intron, Ll.LtrB, splicing enables expression of the intron's host relaxase protein. Relaxase, in turn, initiates horizontal transfer of the conjugative pRS01 plasmid and stimulates retrotransposition of the intron. Little is known about how splicing of bacterial group II introns is influenced by environmental conditions. Here, we show that low temperatures can inhibit Ll.LtrB intron splicing. Whereas autocatalysis is abolished in the cold, splicing is partially restored by the intron-encoded protein (IEP). Structure profiling reveals cold-induced disruptions of key tertiary interactions, suggesting that a kinetic trap prevents the intron RNA from assuming its native state. Interestingly, while reduced levels of transcription and splicing lead to a paucity of excised intron in the cold, levels of relaxase mRNA are maintained, partially due to diminished intron-mediated mRNA targeting, allowing intron spread by conjugal transfer. Taken together, this study demonstrates not only the intrinsic cold sensitivity of group II intron splicing and the role of the IEP for cold-stress adaptation, but also maintenance of horizontal plasmid and intron transfer under cold-shock.
Asunto(s)
Frío , Conjugación Genética , Intrones/genética , Lactococcus lactis/genética , Empalme del ARN , Secuencia de Bases , Respuesta al Choque por Frío , ADN Nucleotidiltransferasas/metabolismo , Evolución Molecular , Transferencia de Gen Horizontal , Plásmidos/genética , Plásmidos/metabolismo , ARN Catalítico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , RetroelementosRESUMEN
Group II introns are the putative progenitors of nuclear spliceosomal introns and use the same two-step splicing pathway. In the cell, the intron RNA forms a ribonucleoprotein (RNP) complex with the intron-encoded protein (IEP), which is essential for splicing. Although structures of spliced group II intron RNAs and RNP complexes have been characterized, structural insights into the splicing process remain enigmatic due to lack of pre-catalytic structural models. Here, we report two cryo-EM structures of endogenously produced group II intron RNPs trapped in their pre-catalytic state. Comparison of the catalytically activated precursor RNP to its previously reported spliced counterpart allowed identification of key structural rearrangements accompanying splicing, including a remodeled active site and engagement of the exons. Importantly, altered RNA-protein interactions were observed upon splicing among the RNP complexes. Furthermore, analysis of the catalytically inert precursor RNP demonstrated the structural impact of the formation of the active site on RNP architecture. Taken together, our results not only fill a gap in understanding the structural basis of IEP-assisted group II intron splicing, but also provide parallels to evolutionarily related spliceosomal splicing.
Asunto(s)
Intrones , Empalme del ARN , ARN/metabolismo , Ribonucleoproteínas/química , Exones , Lactococcus lactis/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Empalmosomas/metabolismoRESUMEN
Group II (gII) introns are mobile retroelements that can spread to new DNA sites through retrotransposition, which can be influenced by a variety of host factors. To determine if these host factors bear any relationship to the genomic location of gII introns, we developed a bioinformatic pipeline wherein we focused on the genomic neighborhoods of bacterial gII introns within their native contexts and sought to determine global relationships between introns and their surrounding genes. We found that, although gII introns inhabit diverse regions, these neighborhoods are often functionally enriched for genes that could promote gII intron retention or proliferation. On one hand, we observe that gII introns are frequently found hiding in mobile elements or after transcription terminators. On the other hand, gII introns are enriched in locations in which they could hijack host functions for their movement, potentially timing expression of the intron with genes that produce favorable conditions for retrotransposition. Thus, we propose that gII intron distributions have been shaped by relationships with their surrounding genomic neighbors.
Asunto(s)
Genoma Bacteriano , Intrones , Secuencias Repetitivas Esparcidas , ReplicónRESUMEN
The duality of group II introns, capable of carrying out both self-splicing and retromobility reactions, is hypothesized to have played a profound role in the evolution of eukaryotes. These introns likely provided the framework for the emergence of eukaryotic retroelements, spliceosomal introns and other key components of the spliceosome. Group II introns are found in all three domains of life and are therefore considered to be exceptionally successful mobile genetic elements. Initially identified in organellar genomes, group II introns are found in bacteria, chloroplasts, and mitochondria of plants and fungi, but not in nuclear genomes. Although there is no doubt that prokaryotic and organellar group II introns are evolutionary related, there are remarkable differences in survival strategies between them. Furthermore, an evolutionary relationship of group II introns to eukaryotic retroelements, including telomeres, and spliceosomes is unmistakable.
Asunto(s)
Intrones , Bacterias/genética , Células Eucariotas , Secuencias Repetitivas Esparcidas , ARN Catalítico/genética , EmpalmosomasRESUMEN
Group II introns, the putative progenitors of spliceosomal introns and retrotransposons, are ribozymes that are capable of self-splicing and DNA invasion. In the cell, group II introns form ribonucleoprotein (RNP) complexes with an intron-encoded protein, which is essential to folding, splicing and retromobility of the intron. To understand the structural accommodations underlying splicing, in preparation for retromobility, we probed the endogenously expressed Lactococcus lactis Ll.LtrB group II intron RNP using SHAPE. The results, which are consistent in vivo and in vitro, provide insights into the dynamics of the intron RNP as well as RNA-RNA and RNA-protein interactions. By comparing the excised intron RNP with mutant RNPs in the precursor state, confined SHAPE profile differences were observed, indicative of rearrangements at the active site as well as disengagement at the functional RNA-protein interface in transition between the two states. The exon-binding sequences in the intron RNA, which interact with the 5' exon and the target DNA, show increased flexibility after splicing. In contrast, stability of major tertiary and protein interactions maintains the scaffold of the RNA through the splicing transition, while the active site is realigned in preparation for retromobility.
Asunto(s)
Proteínas Bacterianas/genética , Intrones/genética , Lactococcus lactis/genética , Empalme del ARN , Ribonucleoproteínas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Exones/genética , Lactococcus lactis/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Unión Proteica , ARN Catalítico/química , ARN Catalítico/genética , ARN Catalítico/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
Inteins, or intervening proteins, are mobile genetic elements translated within host polypeptides and removed through protein splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional protein splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading protein function. The RadA recombinase from the archaeon Pyrococcus horikoshii represents an intriguing example of CPS, whereby protein splicing is inhibited by interactions between the intein and host protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the splicing rate and accuracy. Here, we investigate the mechanism by which ssDNA exhibits this influence and find that ssDNA strongly promotes a specific step of the splicing reaction, cyclization of the terminal asparagine of the intein. Interestingly, inhibitory interactions between the host protein and intein that block splicing localize to this asparagine, suggesting that ssDNA binding alleviates this inhibition to promote splicing. We also find that ssDNA directly influences the position of catalytic nucleophiles required for protein splicing, implying that ssDNA promotes assembly of the intein active site. This work advances our understanding of how ssDNA accelerates RadA splicing, providing important insights into this intriguing example of CPS.
Asunto(s)
ADN de Cadena Simple/genética , Inteínas/genética , Empalme del ARN , Recombinasas/química , Pyrococcus horikoshii/enzimologíaRESUMEN
We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.
Asunto(s)
Proteínas Bacterianas/química , Inteínas , Mycobacterium tuberculosis/química , Rec A Recombinasas/química , Tiazoles/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Mycobacterium tuberculosis/genética , Conformación Proteica , Empalme de Proteína , Rec A Recombinasas/genética , Tuberculosis/microbiologíaRESUMEN
Inteins are mobile genetic elements that self-splice at the protein level. Mycobacteria have inteins inserted into several important genes, including those corresponding to the iron-sulfur cluster assembly protein SufB. Curiously, the SufB inteins are found primarily in mycobacterial species that are potential human pathogens. Here we discovered an exceptional sensitivity of Mycobacterium tuberculosis SufB intein splicing to oxidative and nitrosative stresses when expressed in Escherichia coli. This effect results from predisposition of the intein's catalytic cysteine residues to oxidative and nitrosative modifications. Experiments with a fluorescent reporter system revealed that reactive oxygen species and reactive nitrogen species inhibit SufB extein ligation by forcing either precursor accumulation or N-terminal cleavage. We propose that splicing inhibition is an immediate, posttranslational regulatory response that can be either reversible, by inducing precursor accumulation, or irreversible, by inducing N-terminal cleavage, which may potentially channel mycobacteria into dormancy under extreme oxidative and nitrosative stresses.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Escherichia coli/genética , Inteínas , Mycobacterium tuberculosis/genética , Empalme de Proteína , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Catálisis , Simulación por Computador , Cisteína/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Mycobacterium tuberculosis/metabolismo , Nitrógeno/química , Estrés Oxidativo , Oxígeno/química , Plásmidos/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Group II introns are self-splicing RNAs found in eubacteria, archaea, and eukaryotic organelles. They are mechanistically similar to the metazoan nuclear spliceosomal introns; therefore, group II introns have been invoked as the progenitors of the eukaryotic pre-mRNA introns. However, the ability of group II introns to function outside of the bacteria-derived organelles is debatable, since they are not found in the nuclear genomes of eukaryotes. Here, we show that the Lactococcus lactis Ll.LtrB group II intron splices accurately and efficiently from different pre-mRNAs in a eukaryote, Saccharomyces cerevisiae. However, a pre-mRNA harboring a group II intron is spliced predominantly in the cytoplasm and is subject to nonsense-mediated mRNA decay (NMD), and the mature mRNA from which the group II intron is spliced is poorly translated. In contrast, a pre-mRNA bearing the Tetrahymena group I intron or the yeast spliceosomal ACT1 intron at the same location is not subject to NMD, and the mature mRNA is translated efficiently. Thus, a group II intron can splice from a nuclear transcript, but RNA instability and translation defects would have favored intron loss or evolution into protein-dependent spliceosomal introns, consistent with the bacterial group II intron ancestry hypothesis.
Asunto(s)
Proteínas Bacterianas/genética , Núcleo Celular/metabolismo , Regulación Bacteriana de la Expresión Génica , Intrones/genética , Lactococcus lactis/genética , Empalme del ARN/genética , Citoplasma/metabolismo , Exones/genética , Estabilidad del ARN , Saccharomyces cerevisiae/genética , Empalmosomas/fisiologíaRESUMEN
Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis Using conserved catalytic nucleophiles at their N- and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH3)2Cl2], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N- and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents.
Asunto(s)
Antiinfecciosos/farmacología , Proteínas Bacterianas/metabolismo , Cisplatino/farmacología , Mycobacterium tuberculosis/metabolismo , Rec A Recombinasas/metabolismo , Proteínas Bacterianas/genética , Humanos , Mycobacterium tuberculosis/genética , Dominios Proteicos , Rec A Recombinasas/genéticaRESUMEN
Inteins, also called protein introns, are self-splicing mobile elements found in all domains of life. A bioinformatic survey of genomic data highlights a biased distribution of inteins among functional categories of proteins in both bacteria and archaea, with a strong preference for a single network of functions containing replisome proteins. Many nonorthologous, functionally equivalent replicative proteins in bacteria and archaea carry inteins, suggesting a selective retention of inteins in proteins of particular functions across domains of life. Inteins cluster not only in proteins with related roles but also in specific functional units of those proteins, like ATPase domains. This peculiar bias does not fully fit the models describing inteins exclusively as parasitic elements. In such models, evolutionary dynamics of inteins is viewed primarily through their mobility with the intein homing endonuclease (HEN) as the major factor of intein acquisition and loss. Although the HEN is essential for intein invasion and spread in populations, HEN dynamics does not explain the observed biased distribution of inteins among proteins in specific functional categories. We propose that the protein splicing domain of the intein can act as an environmental sensor that adapts to a particular niche and could increase the chance of the intein becoming fixed in a population. We argue that selective retention of some inteins might be beneficial under certain environmental stresses, to act as panic buttons that reversibly inhibit specific networks, consistent with the observed intein distribution.
Asunto(s)
Archaea/genética , Bacterias/genética , Análisis por Conglomerados , Eucariontes/genética , Evolución Molecular , Inteínas/genética , Animales , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , AdnB Helicasas/química , AdnB Helicasas/genética , AdnB Helicasas/metabolismo , Genoma , Genómica/métodos , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Modelos Moleculares , Filogenia , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de ProteínasRESUMEN
Group II introns are hypothesized to share common ancestry with both nuclear spliceosomal introns and retrotransposons, which collectively occupy the majority of genome space in higher eukaryotes. These phylogenetically diverse introns are mobile retroelements that move through an RNA intermediate. Disruption of Escherichia coli genes encoding enzymes that catalyze synthesis of global regulators cAMP and ppGpp inhibits group II intron retromobility. These small molecules program genetic transitions between nutrient excess and starvation. Accordingly, we demonstrated that glucose depletion of wild-type cells and cAMP supplementation of mutants stimulated retromobility. Likewise, amino acid starvation, which induces the alarmone ppGpp, activated retromobility. In both cases, retrotransposition to ectopic sites was favored over retrohoming. Interestingly, these stimulatory effects are mediated at the level of the DNA target, rather than of expression of the retroelement. Thereby, during metabolic stress, cAMP and ppGpp control group II intron movement in concert with the cell's global genetic circuitry, stimulating genetic diversity.
Asunto(s)
AMP Cíclico/fisiología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Guanosina Tetrafosfato/fisiología , Intrones/genética , Retroelementos/fisiología , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Aminoácidos/metabolismo , Cromosomas Bacterianos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Plásmidos/genética , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , ARN Bacteriano/metabolismo , Retroelementos/genéticaRESUMEN
Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.
Asunto(s)
Proteínas Arqueales/química , Proteínas de Unión al ADN/química , Exteínas , Inteínas , Empalme de Proteína , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/química , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Mutación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pyrococcus horikoshii/genética , Rec A Recombinasas/química , TemperaturaRESUMEN
Mobile genetic elements either encode their own mobilization machineries or hijack them from other mobile elements. Multiple classes of mobile elements often coexist within genomes and it is unclear whether they have the capacity to functionally interact and even collaborate. We investigate the possibility that molecular machineries of disparate mobile elements may functionally interact, using the example of a retrotransposon, in the form of a mobile group II intron, found on a conjugative plasmid pRS01 in Lactococcus lactis. This intron resides within the pRS01 ltrB gene encoding relaxase, the enzyme required for nicking the transfer origin (oriT) for conjugal transmission of the plasmid into a recipient cell. Here, we show that relaxase stimulates both the frequency and diversity of retrotransposition events using a retromobility indicator gene (RIG), and by developing a high-throughput genomic retrotransposition detection system called RIG-Seq. We demonstrate that LtrB relaxase not only nicks ssDNA of its cognate oriT in a sequence- and strand-specific manner, but also possesses weak off-target activity. Together, the data support a model in which the two different mobile elements, one using an RNA-based mechanism, the other using DNA-based transfer, do functionally interact. Intron splicing facilitates relaxase expression required for conjugation, whereas relaxase introduces spurious nicks in recipient DNA that stimulate both the frequency of intron mobility and the density of events. We hypothesize that this functional interaction between the mobile elements would promote horizontal conjugal gene transfer while stimulating intron dissemination in the donor and recipient cells.
Asunto(s)
Conjugación Genética/fisiología , Transferencia de Gen Horizontal , Lactococcus lactis/genética , Retroelementos/fisiología , Secuencia de Bases , ADN de Cadena Simple/metabolismo , Endorribonucleasas/fisiología , Epistasis Genética , Intrones/genética , Organismos Modificados Genéticamente , Empalme del ARN/genéticaRESUMEN
Group II introns are commonly believed to be the progenitors of spliceosomal introns, but they are notably absent from nuclear genomes. Barriers to group II intron function in nuclear genomes therefore beg examination. A previous study showed that nuclear expression of a group II intron in yeast results in nonsense-mediated decay and translational repression of mRNA, and that these roadblocks to expression are group II intron-specific. To determine the molecular basis for repression of gene expression, we investigated cellular dynamics of processed group II intron RNAs, from transcription to cellular localization. Our data show pre-mRNA mislocalization to the cytoplasm, where the RNAs are targeted to foci. Furthermore, tenacious mRNA-pre-mRNA interactions, based on intron-exon binding sequences, result in reduced abundance of spliced mRNAs. Nuclear retention of pre-mRNA prevents this interaction and relieves these expression blocks. In addition to providing a mechanistic rationale for group II intron-specific repression, our data support the hypothesis that RNA silencing of the host gene contributed to expulsion of group II introns from nuclear genomes and drove the evolution of spliceosomal introns.
Asunto(s)
Núcleo Celular/genética , Núcleo Celular/metabolismo , Intrones , Precursores del ARN/genética , Precursores del ARN/metabolismo , Emparejamiento Base , Secuencia de Bases , Citoplasma/genética , Citoplasma/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Evolución Molecular , Exones , Expresión Génica , Silenciador del Gen , Humanos , Conformación de Ácido Nucleico , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Precursores del ARN/química , Empalme del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
Group II introns are large catalytic RNAs that form a ribonucleoprotein (RNP) complex by binding to an intron-encoded protein (IEP). The IEP, which facilitates both RNA splicing and intron mobility, has multiple activities including reverse transcriptase. Recent structures of a group II intron RNP complex and of IEPs from diverse bacteria fuel arguments that group II introns are ancestrally related to eukaryotic spliceosomes as well as to telomerase and viruses. Furthermore, recent structural studies of various functional states of the spliceosome allow us to draw parallels between the group II intron RNP and the spliceosome. Here we present an overview of these studies, with an emphasis on the structure of the IEPs in their isolated and RNA-bound states and on their evolutionary relatedness. In addition, we address the conundrum of the free, albeit truncated IEPs forming dimers, whereas the IEP bound to the intron ribozyme is a monomer in the mature RNP. Future studies needed to resolve some of the outstanding issues related to group II intron RNP function and dynamics are also discussed.