RESUMEN
Mutations in RNA/DNA-binding proteins cause amyotrophic lateral sclerosis (ALS), but the underlying disease mechanisms remain unclear. Here, we report that a set of ALS-associated proteins, namely FUS, EWSR1, TAF15, and MATR3, impact the expression of genes encoding the major histocompatibility complex II (MHC II) antigen presentation pathway. Both subunits of the MHC II heterodimer, HLA-DR, are down-regulated in ALS gene knockouts/knockdown in HeLa and human microglial cells, due to loss of the MHC II transcription factor CIITA. Importantly, hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells bearing the FUSR495X mutation and HPCs derived from C9ORF72 ALS patient induced pluripotent stem cells also exhibit disrupted MHC II expression. Given that HPCs give rise to numerous immune cells, our data raise the possibility that loss of the MHC II pathway results in global failure of the immune system to protect motor neurons from damage that leads to ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/genética , Presentación de Antígeno/genética , Genes MHC Clase II , Complejo Mayor de Histocompatibilidad , Neuronas Motoras , Proteínas de Unión al ARN/genética , Proteínas Asociadas a Matriz NuclearRESUMEN
To ensure efficient and accurate gene expression, pre-mRNA processing and mRNA export need to be balanced. However, how this balance is ensured remains largely unclear. Here, we found that SF3b, a component of U2 snRNP that participates in splicing and 3' processing of pre-mRNAs, interacts with the key mRNA export adaptor THO in vivo and in vitro. Depletion of SF3b reduces THO binding with the mRNA and causes nuclear mRNA retention. Consistently, introducing SF3b binding sites into the mRNA enhances THO recruitment and nuclear export in a dose-dependent manner. These data demonstrate a role of SF3b in promoting mRNA export. In support of this role, SF3b binds with mature mRNAs in the cells. Intriguingly, disruption of U2 snRNP by using a U2 antisense morpholino oligonucleotide does not inhibit, but promotes, the role of SF3b in mRNA export as a result of enhanced SF3b-THO interaction and THO recruitment to the mRNA. Together, our study uncovers a U2-snRNP-independent role of SF3b in mRNA export and suggests that SF3b contributes to balancing pre-mRNA processing and mRNA export.
Asunto(s)
Fosfoproteínas , Precursores del ARN , Factores de Empalme de ARN , ARN Mensajero , Ribonucleoproteína Nuclear Pequeña U2 , Sitios de Unión/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN/genética , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismoRESUMEN
The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.
Asunto(s)
Exosomas/genética , Exosomas/metabolismo , Proteínas Nucleares/fisiología , ARN Helicasas/metabolismo , Animales , Núcleo Celular/metabolismo , Células Madre Embrionarias , Células HEK293 , Células HeLa , Humanos , Ratones , Proteínas Nucleares/genética , Unión Proteica , Dominios Proteicos , Transporte de Proteínas/genética , Estabilidad del ARN/genéticaRESUMEN
Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Atrofia Muscular Espinal/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteína EWS de Unión a ARN/genética , Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ARN/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Sistemas CRISPR-Cas , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Edición Génica , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patología , Proteínas Asociadas a Matriz Nuclear/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteína EWS de Unión a ARN/deficiencia , Proteína FUS de Unión a ARN/deficiencia , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Empalmosomas/química , Empalmosomas/metabolismo , Factores Asociados con la Proteína de Unión a TATA/deficiencia , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismoRESUMEN
A significant fraction of mRNAs are degraded by the nuclear exosome in normal cells. Here, we studied where and when these exosome target mRNAs are sorted away from properly exported ones in the cells. We show that upon exosome inactivation, polyA RNAs are apparently accumulated in nuclear foci that are distinct from nuclear speckles (NSs), and provide several lines of evidence supporting that these polyA RNAs mainly correspond to accumulating exosome target mRNAs. These results suggest that exosomal mRNA degradation mostly occurs outside of NSs. In support of this possibility, targeting exosome target mRNAs to NSs stabilizes them by preventing exosomal degradation. Furthermore, inhibiting mRNA release from NSs does not attenuate exosomal degradation in normal cells, and results in polyA RNA accumulation both inside and outside of NSs in exosome inactivated cells, suggesting that passage through NSs is not required for sorting mRNAs for degradation or export. Indeed, exosome target mRNAs that normally do not enter NSs are exported upon exosome inactivation. Together, our data suggest that exosome target mRNAs are mainly degraded in the nucleoplasm before entering NSs and rapid removal of these mRNAs is important for preventing their nuclear export.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/metabolismo , Exosomas/metabolismo , Estabilidad del ARN/fisiología , Transporte de ARN/fisiología , ARN Mensajero/metabolismo , Núcleo Celular/ultraestructura , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/fisiología , Complejo Multienzimático de Ribonucleasas del Exosoma/fisiología , Células HeLa , Humanos , Poli A/genética , ARN Helicasas/fisiología , Proteínas de Unión al ARN/fisiologíaRESUMEN
Mutations in multiple RNA/DNA binding proteins cause Amyotrophic Lateral Sclerosis (ALS). Included among these are the three members of the FET family (FUS, EWSR1 and TAF15) and the structurally similar MATR3. Here, we characterized the interactomes of these four proteins, revealing that they largely have unique interactors, but share in common an association with U1 snRNP. The latter observation led us to analyze the interactome of the U1 snRNP machinery. Surprisingly, this analysis revealed the interactome contains ~220 components, and of these, >200 are shared with the RNA polymerase II (RNAP II) machinery. Among the shared components are multiple ALS and Spinal muscular Atrophy (SMA)-causative proteins and numerous discrete complexes, including the SMN complex, transcription factor complexes, and RNA processing complexes. Together, our data indicate that the RNAP II/U1 snRNP machinery functions in a wide variety of molecular pathways, and these pathways are candidates for playing roles in ALS/SMA pathogenesis.
Asunto(s)
Proteínas Asociadas a Matriz Nuclear/metabolismo , Mapas de Interacción de Proteínas , ARN Polimerasa II/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Células HeLa , HumanosRESUMEN
The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN Helicasas/metabolismo , Estabilidad del ARN , Transporte de ARN/genética , ARN Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/genética , Exosomas/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Unión Proteica , ARN Helicasas/genética , Estabilidad del ARN/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Factores de Transcripción/genéticaRESUMEN
Mutations in FUS cause amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to neurodegeneration remain obscure. We previously found that U1 snRNP is the most abundant FUS interactor. Here, we report that components of the U1 snRNP core particle (Sm proteins and U1 snRNA), but not the mature U1 snRNP-specific proteins (U1-70K, U1A and U1C), co-mislocalize with FUS to the cytoplasm in ALS patient fibroblasts harboring mutations in the FUS nuclear localization signal (NLS). Similar results were obtained in HeLa cells expressing the ALS-causing FUS R495X NLS mutation, and mislocalization of Sm proteins is RRM-dependent. Moreover, as observed with FUS, knockdown of any of the U1 snRNP-specific proteins results in a dramatic loss of SMN-containing Gems. Significantly, knockdown of U1 snRNP in zebrafish results in motor axon truncations, a phenotype also observed with FUS, SMN and TDP-43 knockdowns. Our observations linking U1 snRNP to ALS patient cells with FUS mutations, SMN-containing Gems, and motor neurons indicate that U1 snRNP is a component of a molecular pathway associated with motor neuron disease. Linking an essential canonical splicing factor (U1 snRNP) to this pathway provides strong new evidence that splicing defects may be involved in pathogenesis and that this pathway is a potential therapeutic target.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Señales de Localización Nuclear/genética , Proteína FUS de Unión a ARN/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Citoplasma/metabolismo , Gemini de los Cuerpos Enrollados/metabolismo , Gemini de los Cuerpos Enrollados/patología , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/antagonistas & inhibidores , Ribonucleoproteína Nuclear Pequeña U1/genética , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas Nucleares snRNP/genética , Proteínas Nucleares snRNP/metabolismoRESUMEN
Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Transporte Activo de Núcleo Celular , ADN Complementario/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Caperuzas de ARN/metabolismo , Transporte de ARN , Proteínas de Unión al ARN/fisiología , Ribonucleoproteínas/aislamiento & purificaciónRESUMEN
The mRNA export complex TREX (TREX) is known to contain Aly, UAP56, Tex1 and the THO complex, among which UAP56 is required for TREX assembly. Here, we systematically investigated the role of each human TREX component in TREX assembly and its association with the mRNA. We found that Tex1 is essentially a subunit of the THO complex. Aly, THO and UAP56 are all required for assembly of TREX, in which Aly directly interacts with THO subunits Thoc2 and Thoc5. Both Aly and THO function in linking UAP56 to the cap-binding protein CBP80. Interestingly, association of UAP56 with the spliced mRNA, but not with the pre-mRNA, requires Aly and THO. Unexpectedly, we found that Aly and THO require each other to associate with the spliced mRNA. Consistent with these biochemical results, similar to Aly and UAP56, THO plays critical roles in mRNA export. Together, we propose that Aly, THO and UAP56 form a highly integrated unit to associate with the spliced mRNA and function in mRNA export.