RESUMEN
LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two ORFs, of which L1-ORF2p nicks genomic DNA and reverse transcribes L1 mRNA using the nicked DNA as a primer which base-pairs with poly(A) tail of L1 mRNA. To better understand the importance of non-templated L1 3' ends' dynamics and the interplay between L1 3' and 5' ends, we investigated the effects of genomic knock-outs and temporal knock-downs of XRN1, DCP2, and other factors. We hypothesized that in the absence of XRN1, the major 5'â3' exoribonuclease, there would be more L1 mRNA and retrotransposition. Conversely, we observed that loss of XRN1 decreased L1 retrotransposition. This occurred despite slight stabilization of L1 mRNA, but with decreased L1 RNP formation. Similarly, loss of DCP2, the catalytic subunit of the decapping complex, lowered retrotransposition despite increased steady-state levels of L1 proteins. In both XRN1 and DCP2 depletions we observed shortening of L1 3' poly(A) tails and their increased uridylation by TUT4/7. We explain the observed reduction of L1 retrotransposition by the changed qualities of non-templated L1 mRNA 3' ends demonstrating the important role of L1 3' end dynamics in L1 biology.
Asunto(s)
Elementos de Nucleótido Esparcido Largo , ARN Mensajero , Humanos , Células HeLa , Retroelementos/genética , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Retrotransposons, including LINE-1, Alu, SVA, and endogenous retroviruses, are one of the major constituents of human genomic repetitive sequences. Through the process of retrotransposition, some of them occasionally insert into new genomic locations by a copy-paste mechanism involving RNA intermediates. Irrespective of de novo genomic insertions, retrotransposon expression can lead to DNA double-strand breaks and stimulate cellular innate immunity through endogenous patterns. As a result, retrotransposons are tightly regulated by multi-layered regulatory processes to prevent the dangerous effects of their expression. In recent years, significant progress was made in revealing how retrotransposon biology intertwines with general post-transcriptional RNA metabolism. Here, I summarize current knowledge on the involvement of post-transcriptional factors in the biology of retrotransposons, focusing on LINE-1. I emphasize general RNA metabolisms such as methylation of adenine (m6 A), RNA 3'-end polyadenylation and uridylation, RNA decay and translation regulation. I discuss the effects of retrotransposon RNP sequestration in cytoplasmic bodies and autophagy. Finally, I summarize how innate immunity restricts retrotransposons and how retrotransposons make use of cellular enzymes, including the DNA repair machinery, to complete their replication cycles.
Asunto(s)
Regulación de la Expresión Génica , Retroelementos , Humanos , Retroelementos/genética , Elementos de Nucleótido Esparcido Largo/genética , ARN/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
CRISPR-Cas are adaptable natural prokaryotic defense systems that act against invading viruses and plasmids. Among the six currently known major CRISPR-Cas types, the type VI CRISPR-Cas13 is the only one known to exclusively bind and cleave foreign RNA. Within the last couple of years, this system has been adapted to serve numerous, and sometimes not obvious, applications, including some that might be developed as effective molecular therapies. Indeed, Cas13 has been adapted to kill antibiotic-resistant bacteria. In a cell-free environment, Cas13 has been used in the development of highly specific, sensitive, multiplexing-capable, and field-adaptable detection tools. Importantly, Cas13 can be reprogrammed and applied to eukaryotes to either combat pathogenic RNA viruses or in the regulation of gene expression, facilitating the knockdown of mRNA, circular RNA, and noncoding RNA. Furthermore, Cas13 has been harnessed for in vivo RNA modifications including programmable regulation of alternative splicing, A-to-I and C to U editing, and m6A modifications. Finally, approaches allowing for the detection and characterization of RNA-interacting proteins have also been demonstrated. Here, we provide a comprehensive overview of the applications utilizing CRISPR-Cas13 that illustrate its versatility. We also discuss the most important limitations of the CRISPR-Cas13-based technologies, and controversies regarding them. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , ARN/genética , Edición de ARN , Procesamiento Postranscripcional del ARNRESUMEN
In eukaryotes, almost all RNA species are processed at their 3' ends and most mRNAs are polyadenylated in the nucleus by canonical poly(A) polymerases. In recent years, several terminal nucleotidyl transferases (TENTs) including non-canonical poly(A) polymerases (ncPAPs) and terminal uridyl transferases (TUTases) have been discovered. In contrast to canonical polymerases, TENTs' functions are more diverse; some, especially TUTases, induce RNA decay while others, such as cytoplasmic ncPAPs, activate translationally dormant deadenylated mRNAs. The mammalian genome encodes 11 different TENTs. This review summarizes the current knowledge about the functions and mechanisms of action of these enzymes.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.
Asunto(s)
Ratones/genética , Nucleotidiltransferasas/genética , ARN/metabolismo , Ratas/genética , Animales , Ratones/metabolismo , Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , Ratas/metabolismoRESUMEN
LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Uridina/metabolismo , Animales , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Ratones , Proteínas Nucleares/genética , Unión Proteica , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/genética , ARN Helicasas/metabolismo , Interferencia de ARN , ARN Nucleotidiltransferasas/antagonistas & inhibidores , ARN Nucleotidiltransferasas/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Retroelementos/genéticaRESUMEN
Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos , Proteínas/metabolismo , Recombinación Genética , Terminación de la Transcripción Genética , Clonación Molecular , ADN Nucleotidiltransferasas/genética , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Nucleofosmina , Regiones Promotoras Genéticas , Proteínas/genéticaRESUMEN
Nuclease and helicase activities play pivotal roles in various aspects of RNA processing and degradation. These two activities are often present in multi-subunit complexes from nucleic acid metabolism. In the mitochondrial exoribonuclease complex (mtEXO) both enzymatic activities are tightly coupled making it an excellent minimal system to study helicase-exoribonuclease coordination. mtEXO is composed of Dss1 3'-to-5' exoribonuclease and Suv3 helicase. It is the master regulator of mitochondrial gene expression in yeast. Here, we present the structure of mtEXO and a description of its mechanism of action. The crystal structure of Dss1 reveals domains that are responsible for interactions with Suv3. Importantly, these interactions are compatible with the conformational changes of Suv3 domains during the helicase cycle. We demonstrate that mtEXO is an intimate complex which forms an RNA-binding channel spanning its entire structure, with Suv3 helicase feeding the 3' end of the RNA toward the active site of Dss1.
Asunto(s)
Endorribonucleasas/metabolismo , Exorribonucleasas/metabolismo , Proteínas Mitocondriales/metabolismo , Complejos Multienzimáticos/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN Helicasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Candida glabrata/enzimología , Candida glabrata/genética , Candida glabrata/metabolismo , Cristalografía por Rayos X , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/química , Endorribonucleasas/genética , Exorribonucleasas/química , Exorribonucleasas/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Conformación de Ácido Nucleico , Polirribonucleótido Nucleotidiltransferasa/química , Polirribonucleótido Nucleotidiltransferasa/genética , Unión Proteica , Conformación Proteica , ARN/química , ARN/genética , ARN/metabolismo , ARN Helicasas/química , ARN Helicasas/genética , ARN Mitocondrial , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here, we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL_short RNA from the ferritin mRNA 5' UTR. Importantly, DIS3L2 contributes to surveillance of maturing snRNAs during their cytoplasmic processing. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exosome-associated DIS3. Using 3' RACE-Seq, we demonstrate that all novel DIS3L2 substrates are uridylated in vivo by TUT4/TUT7 poly(U) polymerases. Uridylation-dependent DIS3L2-mediated decay can be recapitulated in vitro, thus reinforcing the tight cooperation between DIS3L2 and TUTases. Together these results indicate that catalytically inactive DIS3L2, characteristic of Perlman syndrome, can lead to deregulation of its target RNAs to disturb transcriptome homeostasis.
Asunto(s)
Exorribonucleasas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Elementos Alu , Línea Celular , Macrosomía Fetal/genética , Macrosomía Fetal/metabolismo , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Unión Proteica , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato , Tumor de Wilms/genética , Tumor de Wilms/metabolismoRESUMEN
Structural rearrangement of the activated spliceosome (B(act)) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B(act) spliceosome. In the absence of Spp2, the B(act) spliceosome efficiently triggers Prp2's NTPase activity, but NTP hydrolysis is not coupled to ribonucleoprotein (RNP) rearrangements leading to catalytic activation of the spliceosome. Transformation of the B(act) to the B* spliceosome occurs only when Spp2 is present and is accompanied by dissociation of Prp2 and a reduction in its NTPase activity. In the absence of spliceosomes, Spp2 enhances Prp2's RNA-dependent ATPase activity without affecting its RNA affinity. Our data suggest that Spp2 plays a major role in coupling Prp2's ATPase activity to remodeling of the spliceosome into a catalytically active machine.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Catálisis , Coenzimas/metabolismo , Activación Enzimática , Hidrólisis , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.
Asunto(s)
Empalme del ARN , ARN de Hongos/metabolismo , Empalmosomas/metabolismo , Levaduras/genética , Catálisis , Dominio Catalítico , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Exones , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica , Sitios de Empalme de ARN , ARN de Hongos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espectrometría de Fluorescencia , Empalmosomas/genética , Levaduras/metabolismoRESUMEN
The spliceosome is a ribonucleoprotein machine that removes introns from pre-mRNA in a two-step reaction. To investigate the catalytic steps of splicing, we established an in vitro splicing complementation system. Spliceosomes stalled before step 1 of this process were purified to near-homogeneity from a temperature-sensitive mutant of the RNA helicase Prp2, compositionally defined, and shown to catalyze efficient step 1 when supplemented with recombinant Prp2, Spp2 and Cwc25, thereby demonstrating that Cwc25 has a previously unknown role in promoting step 1. Step 2 catalysis additionally required Prp16, Slu7, Prp18 and Prp22. Our data further suggest that Prp2 facilitates catalytic activation by remodeling the spliceosome, including destabilizing the SF3a and SF3b proteins, likely exposing the branch site before step 1. Remodeling by Prp2 was confirmed by negative stain EM and image processing. This system allows future mechanistic analyses of spliceosome activation and catalysis.
Asunto(s)
ARN de Hongos/aislamiento & purificación , ARN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , ARN Helicasas DEAD-box/aislamiento & purificación , ARN Helicasas DEAD-box/metabolismo , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica/métodos , Modelos Biológicos , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/aislamiento & purificación , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/aislamiento & purificación , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/aislamiento & purificación , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/ultraestructuraRESUMEN
RNA is now considered a key factor in the regulation of gene expression. There are several classes of small regulatory RNAs in plants, functioning in posttranscriptional gene silencing (PTGS) or epigenetic DNA modification. Trans-acting short interfering RNAs (tasiRNAs) form a class of small regulatory RNAs which has been distinguished only recently. To date, five genes encoding tasiRNAs have been identified in Arabidopsis thaliana. TasiRNAs derive from non-coding RNA precursors which are initially targeted for cleavage by a miRNA. Cleavage products are then converted into dsRNAs by a RNA dependent RNA polymerase and sequentially cleaved into 21-nt tasiRNAs. Like the majority of plant miRNAs, tasiRNAs regulate gene expression at the posttranscriptional level, guiding cleavage of ARF and PPR transcripts. Here, we briefly present tasiRNAs and speculate whether they form a homogeneous class of siRNAs.