RESUMEN
High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenous NOTCH2 and MAP1B mRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation of NOTCH2 mRNA at the endoplasmic reticulum, and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.
RESUMEN
CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Bases de Datos Genéticas , Endodesoxirribonucleasas , Sistemas CRISPR-Cas/genética , Filogenia , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/clasificación , Proteínas Asociadas a CRISPR/genética , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/clasificación , Endodesoxirribonucleasas/genética , Enciclopedias como AsuntoRESUMEN
Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans-cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90-99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.
Asunto(s)
Sistemas CRISPR-Cas , ARN , Animales , Humanos , Sistemas CRISPR-Cas/genética , Complejos Multiproteicos , Mamíferos/genéticaRESUMEN
CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.
Asunto(s)
Sistemas CRISPR-Cas , Cromatina , Humanos , Cromatina/genética , Edición Génica , Reparación del ADN por Recombinación , Recombinación Homóloga , N-Metiltransferasa de Histona-LisinaRESUMEN
Depletion of architectural factors globally alters chromatin structure but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Cohesin loss disrupts the Xi superstructure, unveiling superloops between escapee genes with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure, compromises spreading of Xist RNA-Polycomb complexes, and attenuates Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss creates an Xi superloop, and cohesin retention creates Xi megadomains on the active X chromosome. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X inactivation and inter-chromosomal interactions across the genome.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/metabolismo , Silenciador del Gen , Proteínas del Grupo Polycomb/metabolismo , ARN Largo no Codificante/metabolismo , Inactivación del Cromosoma X , Cromosoma X , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Ratones , Conformación de Ácido Nucleico , Proteínas del Grupo Polycomb/genética , Conformación Proteica , Proteínas/genética , Proteínas/metabolismo , ARN Largo no Codificante/genética , Relación Estructura-Actividad , CohesinasRESUMEN
X chromosome inactivation (XCI) is a global silencing mechanism by which XX and XY mammals equalize X-linked gene dosages. XCI begins with an establishment phase during which Xist RNA spreads and induces de novo heterochromatinization across a female X chromosome and is followed by a maintenance phase when multiple epigenetic pathways lock down the inactive X (Xi) state. Involvement of Polycomb repressive complexes 1 and 2 in XCI has been intensively studied but with conflicting conclusions regarding their recruitment and role in Xi silencing. Here, we reveal that establishment of XCI has two phases and reconcile the roles that Xist repeats A and B play in gene silencing and Polycomb recruitment. Repeat A initiates both processes, whereas repeat B bolsters or stabilizes them thereafter. Once established, XCI no longer requires repeat A during maintenance. These findings integrate disparate studies and present a unified view of Xist's role in Polycomb-mediated silencing.
Asunto(s)
Proteínas del Grupo Polycomb/metabolismo , ARN Largo no Codificante/metabolismo , Inactivación del Cromosoma X , Animales , Células Cultivadas , Ratones , Proteínas del Grupo Polycomb/genética , ARN Largo no Codificante/química , ARN Largo no Codificante/genética , Cromosoma X/genéticaRESUMEN
X-chromosome inactivation triggers fusion of A/B compartments to inactive X (Xi)-specific structures known as S1 and S2 compartments. SMCHD1 then merges S1/S2s to form the Xi super-structure. Here, we ask how S1/S2 compartments form and reveal that Xist RNA drives their formation via recruitment of Polycomb repressive complex 1 (PRC1). Ablating Smchd1 in post-XCI cells unveils S1/S2 structures. Loss of SMCHD1 leads to trapping Xist in the S1 compartment, impairing RNA spreading into S2. On the other hand, depleting Xist, PRC1, or HNRNPK precludes re-emergence of S1/S2 structures, and loss of S1/S2 compartments paradoxically strengthens the partition between Xi megadomains. Finally, Xi-reactivation in post-XCI cells can be enhanced by depleting both SMCHD1 and DNA methylation. We conclude that Xist, PRC1, and SMCHD1 collaborate in an obligatory, sequential manner to partition, fuse, and direct self-association of Xi compartments required for proper spreading of Xist RNA.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/genética , Complejo Represivo Polycomb 1/metabolismo , ARN Largo no Codificante/metabolismo , Cromosoma X/química , Cromosoma X/genética , Animales , Metilación de ADN/genética , Histonas/metabolismo , Lisina/metabolismo , Ratones , Modelos Genéticos , Inactivación del Cromosoma X/genéticaRESUMEN
During X-inactivation, Xist RNA spreads along an entire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct binding partner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topological Xi reconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/Polycomb spreading, silencing, and changes in chromosome architecture.
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Fibroblastos/metabolismo , Eliminación de Gen , Silenciador del Gen , Células Madre Embrionarias de Ratones/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 2/genética , ARN Largo no Codificante/genética , Inactivación del Cromosoma X , Cromosoma X/genética , Animales , Línea Celular Transformada , Femenino , Regulación del Desarrollo de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Masculino , Ratones , Motivos de Nucleótidos , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , ARN Largo no Codificante/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Cromosoma X/metabolismoRESUMEN
The noncoding RNA Xist recruits silencing factors to the inactive X chromosome (Xi) and facilitates re-organization of Xi structure. Here, we examine the mouse epigenomic landscape of Xi and assess how Xist alters chromatin accessibility. Xist deletion triggers a gain of accessibility of select chromatin regions that is regulated by BRG1, an ATPase subunit of the SWI/SNF chromatin-remodeling complex. In vitro, RNA binding inhibits nucleosome-remodeling and ATPase activities of BRG1, while in cell culture Xist directly interacts with BRG1 and expels BRG1 from the Xi. Xist ablation leads to a selective return of BRG1 in cis, starting from pre-existing BRG1 sites that are free of Xist. BRG1 re-association correlates with cohesin binding and restoration of topologically associated domains (TADs) and results in the formation of de novo Xi 'superloops'. Thus, Xist binding inhibits BRG1's nucleosome-remodeling activity and results in expulsion of the SWI/SNF complex from the Xi.
Asunto(s)
Cromatina/metabolismo , ARN Largo no Codificante/metabolismo , Cromosoma X/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética/genética , Epigénesis Genética/fisiología , Femenino , Ratones , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Largo no Codificante/genética , ARN no Traducido/genética , ARN no Traducido/metabolismo , Cromosoma X/genéticaRESUMEN
X chromosome inactivation is an epigenetic dosage compensation mechanism in female mammals driven by the long noncoding RNA, Xist. Although recent genomic and proteomic approaches have provided a more global view of Xist's function, how Xist RNA localizes to the inactive X chromosome (Xi) and spreads in cis remains unclear. Here, we report that the CDKN1-interacting zinc finger protein CIZ1 is critical for localization of Xist RNA to the Xi chromosome territory. Stochastic optical reconstruction microscopy (STORM) shows a tight association of CIZ1 with Xist RNA at the single-molecule level. CIZ1 interacts with a specific region within Xist exon 7-namely, the highly repetitive Repeat E motif. Using genetic analysis, we show that loss of CIZ1 or deletion of Repeat E in female cells phenocopies one another in causing Xist RNA to delocalize from the Xi and disperse into the nucleoplasm. Interestingly, this interaction is exquisitely sensitive to CIZ1 levels, as overexpression of CIZ1 likewise results in Xist delocalization. As a consequence, this delocalization is accompanied by a decrease in H3K27me3 on the Xi. Our data reveal that CIZ1 plays a major role in ensuring stable association of Xist RNA within the Xi territory.
Asunto(s)
Cromosomas de los Mamíferos , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares , ARN Largo no Codificante , Secuencias Repetitivas de Ácidos Nucleicos , Cromosoma X , Animales , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Ratones , Células Madre Embrionarias de Ratones/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Motivos de Nucleótidos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromosoma X/genética , Cromosoma X/metabolismoRESUMEN
In mammals, monoallelic gene expression can result from X-chromosome inactivation, genomic imprinting, and random monoallelic expression (RMAE). Epigenetic regulation of RMAE is not fully understood. Here we analyze allelic imbalance in chromatin state of autosomal genes using ChIP-seq in a clonal cell line. We identify approximately 3.7% of autosomal genes that show significant differences between chromatin states of two alleles. Allelic regulation is represented among several functional gene categories including histones, chromatin modifiers, and multiple early developmental regulators. Most cases of allelic skew are produced by quantitative differences between two allelic chromatic states that belong to the same gross type (active, silent, or bivalent). Combinations of allelic states of different types are possible but less frequent. When different chromatin marks are skewed on the same gene, their skew is coordinated as a result of quantitative relationships between these marks on each individual allele. Finally, combination of allele-specific densities of chromatin marks is a quantitative predictor of allelic skew in gene expression.
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Desequilibrio Alélico , Cromatina/genética , Alelos , Animales , Línea Celular , Epigénesis Genética , Femenino , Fibroblastos/metabolismo , Expresión Génica , Genoma , Impresión Genómica , Masculino , Ratones , Ratones de la Cepa 129RESUMEN
The inactive X chromosome (Xi) serves as a model to understand gene silencing on a global scale. Here, we perform "identification of direct RNA interacting proteins" (iDRiP) to isolate a comprehensive protein interactome for Xist, an RNA required for Xi silencing. We discover multiple classes of interactors-including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers, and modifiers-that synergistically repress Xi transcription. Inhibiting two or three interactors destabilizes silencing. Although Xist attracts some interactors, it repels architectural factors. Xist evicts cohesins from the Xi and directs an Xi-specific chromosome conformation. Upon deleting Xist, the Xi acquires the cohesin-binding and chromosomal architecture of the active X. Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ARN Largo no Codificante/metabolismo , Inactivación del Cromosoma X , Cromosoma X/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Células Cultivadas , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Ratones , Complejos Multiproteicos/metabolismo , Conformación de Ácido Nucleico , Proteómica , ARN Helicasas/metabolismo , Cromosoma X/química , Cromosoma X/genética , CohesinasRESUMEN
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.
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Pintura Cromosómica/métodos , Cromosomas/genética , Haplotipos , Hibridación Fluorescente in Situ/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Línea Celular , Drosophila , Biblioteca de Genes , Sondas de Oligonucleótidos/metabolismo , Coloración y EtiquetadoRESUMEN
In mammals, several classes of monoallelic genes have been identified, including those subject to X-chromosome inactivation (XCI), genomic imprinting, and random monoallelic expression (RMAE). However, the extent to which these epigenetic phenomena are influenced by underlying genetic variation is unknown. Here we perform a systematic classification of allelic imbalance in mouse hybrids derived from reciprocal crosses of divergent strains. We observe that deviation from balanced biallelic expression is common, occurring in â¼20% of the mouse transcriptome in a given tissue. Allelic imbalance attributed to genotypic variation is by far the most prevalent class and typically is tissue-specific. However, some genotype-based imbalance is maintained across tissues and is associated with greater genetic variation, especially in 5' and 3' termini of transcripts. We further identify novel random monoallelic and imprinted genes and find that genotype can modify penetrance of parental origin even in the setting of large imprinted regions. Examination of nascent transcripts in single cells from inbred parental strains reveals that genes showing genotype-based imbalance in hybrids can also exhibit monoallelic expression in isogenic backgrounds. This surprising observation may suggest a competition between alleles and/or reflect the combined impact of cis- and trans-acting variation on expression of a given gene. Our findings provide novel insights into gene regulation and may be relevant to human genetic variation and disease.
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Desequilibrio Alélico , Transcriptoma , Alelos , Animales , Análisis por Conglomerados , Cruzamientos Genéticos , Perfilación de la Expresión Génica , Variación Genética , Impresión Genómica , Genotipo , Ratones , Especificidad de Órganos/genéticaRESUMEN
CTCF is a master regulator that plays important roles in genome architecture and gene expression. How CTCF is recruited in a locus-specific manner is not fully understood. Evidence from epigenetic processes, such as X chromosome inactivation (XCI), indicates that CTCF associates functionally with RNA. Using genome-wide approaches to investigate the relationship between its RNA interactome and epigenomic landscape, here we report that CTCF binds thousands of transcripts in mouse embryonic stem cells, many in close proximity to CTCF's genomic binding sites. CTCF is a specific and high-affinity RNA-binding protein (Kd < 1 nM). During XCI, CTCF differentially binds the active and inactive X chromosomes and interacts directly with Tsix, Xite, and Xist RNAs. Tsix and Xite RNAs target CTCF to the X inactivation center, thereby inducing homologous X chromosome pairing. Our work elucidates one mechanism by which CTCF is recruited in a locus-specific manner and implicates CTCF-RNA interactions in long-range chromosomal interactions.
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ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Cromosoma X/genética , Animales , Factor de Unión a CCCTC , Células Cultivadas , Emparejamiento Cromosómico , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Sitios Genéticos , Ratones , Unión ProteicaRESUMEN
Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years as a potentially new and crucial layer of biological regulation. lncRNAs of all kinds have been implicated in a range of developmental processes and diseases, but knowledge of the mechanisms by which they act is still surprisingly limited, and claims that almost the entirety of the mammalian genome is transcribed into functional noncoding transcripts remain controversial. At the same time, a small number of well-studied lncRNAs have given us important clues about the biology of these molecules, and a few key functional and mechanistic themes have begun to emerge, although the robustness of these models and classification schemes remains to be seen. Here, we review the current state of knowledge of the lncRNA field, discussing what is known about the genomic contexts, biological functions, and mechanisms of action of lncRNAs. We also reflect on how the recent interest in lncRNAs is deeply rooted in biology's longstanding concern with the evolution and function of genomes.
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Epigénesis Genética , Procesamiento Postranscripcional del ARN , ARN Largo no Codificante/genética , Animales , Evolución Molecular , Humanos , ARN Largo no Codificante/metabolismo , Transcripción GenéticaRESUMEN
The multiprotein exon junction complex (EJC) that is deposited upstream of spliced junctions orchestrates downstream events in the life of a metazoan mRNA, including its surveillance via the nonsense-mediated decay (NMD) pathway. However, the mechanism by which the spliceosome mediates EJC formation is not well understood. We show that human eIF4G-like spliceosomal protein (h)CWC22 directly interacts with the core EJC component eIF4AIII in vitro and in vivo; mutations at the predicted hCWC22/eIF4AIII interface disrupt association. In vivo depletion of hCWC22, as for yeast Cwc22p, causes a splicing defect, resulting in decreased levels of mature cellular mRNAs. Nonetheless, hCWC22 depletion yields increased levels of spliced RNA from the unusual nonsense codon-containing U22 host gene, which is a natural substrate of NMD. To test whether hCWC22 acts in NMD through coupling splicing to EJC deposition, we searched for mutations in hCWC22 that affect eIF4AIII deposition without affecting splicing. Addition of hCWC22(G168Y) with a mutation at the putative hCWC22/eIF4AIII interface exacerbates the defect in splicing-dependent deposition of eIF4AIII(T334V) with a mutation reported to be in direct contact with mRNA, linking hCWC22 to the process of EJC deposition in vitro. Importantly, the addition of hCWC22(G168Y) affects deposition of eIF4AIII(T334V) without inhibiting splicing or the efficiency of deposition of the endogenous eF4AIII(WT) in the same reaction, demonstrating hCWC22's specific role in eIF4AIII deposition in addition to its role in splicing. The essential splicing factor CWC22 has, therefore, acquired functions in EJC assembly and NMD during evolution from single-celled to complex eukaryotes.
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Proteínas Portadoras/metabolismo , Exones/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , Empalme del ARN/genética , Empalmosomas/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Isomerasa de Peptidilprolil , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Despite the lack of an exon junction complex (EJC), Saccharomyces cerevisiae contains Fal1p, a DEAD-box helicase highly homologous to eIF4AIII. We show that yeast Fal1p is functionally orthologous to human eIF4AIII, since expression of human eIF4AIII complements both the lethal phenotype and the 18S rRNA biogenesis defect of fal1Δ(null) yeast. We further show that yeast Fal1p interacts genetically with an eIF4G-like protein, Sgd1p: One allele of sgd1 acts as a dominant extragenic suppressor of a mutation in a predicted RNA-binding residue of Fal1p, whereas another synthetically exacerbates the growth defect of this fal1 mutation. Both sgd1 mutations map to a single, short, evolutionarily conserved patch that matches key eIF4A-interacting residues of eIF4G when superimposed on the X-ray structure of the eIF4A/eIF4G complex. We demonstrate direct physical interactions between yeast Sgd1p and Fal1p, and between their human orthologs (NOM1 and eIF4AIII) in vitro and in vivo, identifying human NOM1 as a missing eIF4G-like interacting partner of eIF4AIII. Knockdown of eIF4AIII and NOM1 in human cells demonstrates that this novel conserved eIF4A/eIF4G-like complex acts in pre-rRNA processing, adding to the established functions of eIF4A/eIF4G in translation initiation and of eIF4AIII as the core component of the EJC.