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1.
Proc Natl Acad Sci U S A ; 119(25): e2206046119, 2022 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-35704758

RESUMEN

Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.


Asunto(s)
Núcleo Celular , Motas Nucleares , Proteínas Quinasas , Empalme del ARN , Transporte Activo de Núcleo Celular , Núcleo Celular/enzimología , Células HeLa , Humanos , Proteínas Quinasas/metabolismo , ARN/metabolismo , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/genética
2.
PLoS Pathog ; 18(11): e1010930, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36318584

RESUMEN

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis.


Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , COVID-19/genética , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Citoplasma/metabolismo , Mamíferos
3.
RNA ; 25(5): 521-538, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30733326

RESUMEN

It has recently become clear that ribosomes are much more heterogeneous than previously thought, with diversity arising from rRNA sequence and modifications, ribosomal protein (RP) content and posttranslational modifications (PTMs), as well as bound nonribosomal proteins. In some cases, the existence of these diverse ribosome populations has been verified by biochemical or structural methods. Furthermore, knockout or knockdown of RPs can diversify ribosome populations, while also affecting the translation of some mRNAs (but not others) with biological consequences. However, the effects on translation arising from depletion of diverse proteins can be highly similar, suggesting that there may be a more general defect in ribosome function or stability, perhaps arising from reduced ribosome numbers. Consistently, overall reduced ribosome numbers can differentially affect subclasses of mRNAs, necessitating controls for specificity. Moreover, in order to study the functional consequences of ribosome diversity, perturbations including affinity tags and knockouts are introduced, which can also affect the outcome of the experiment. Here we review the available literature to carefully evaluate whether the published data support functional diversification, defined as diverse ribosome populations differentially affecting translation of distinct mRNA (classes). Based on these observations and the commonly observed cellular responses to perturbations in the system, we suggest a set of important controls to validate functional diversity, which should include gain-of-function assays and the demonstration of inducibility under physiological conditions.


Asunto(s)
Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN de Transferencia/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Animales , Archaea/genética , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Secuencia de Bases , Heterogeneidad Genética , Mamíferos/genética , Mamíferos/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/clasificación , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
4.
Cell Rep ; 42(3): 112273, 2023 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-36933216

RESUMEN

Chromatin regulation and alternative splicing are both critical mechanisms guiding gene expression. Studies have demonstrated that histone modifications can influence alternative splicing decisions, but less is known about how alternative splicing may impact chromatin. Here, we demonstrate that several genes encoding histone-modifying enzymes are alternatively spliced downstream of T cell signaling pathways, including HDAC7, a gene previously implicated in controlling gene expression and differentiation in T cells. Using CRISPR-Cas9 gene editing and cDNA expression, we show that differential inclusion of HDAC7 exon 9 controls the interaction of HDAC7 with protein chaperones, resulting in changes to histone modifications and gene expression. Notably, the long isoform, which is induced by the RNA-binding protein CELF2, promotes expression of several critical T cell surface proteins including CD3, CD28, and CD69. Thus, we demonstrate that alternative splicing of HDAC7 has a global impact on histone modification and gene expression that contributes to T cell development.


Asunto(s)
Código de Histonas , Histonas , Proteínas 14-3-3/genética , Empalme Alternativo/genética , Cromatina , Expresión Génica , Histona Desacetilasas/metabolismo
5.
Nat Struct Mol Biol ; 30(1): 31-37, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36536103

RESUMEN

To determine how different pioneer transcription factors form a targeted, accessible nucleosome within compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated nucleosome arrays in vitro with a central nucleosome containing binding sites for the hematopoietic E-Twenty Six (ETS) factor PU.1 and Basic Leucine Zipper (bZIP) factors C/EBPα and C/EBPß. Our long-read sequencing reveals that each factor can expose a targeted nucleosome on linker histone-compacted arrays, but with different nuclease sensitivity patterns. The DNA binding domain of PU.1 binds mononucleosomes, but requires an additional intrinsically disordered domain to bind and open compacted chromatin. The canonical mammalian SWI/SNF (cBAF) remodeler was unable to act upon two forms of locally open chromatin unless cBAF was enabled by a separate transactivation domain of PU.1. cBAF potentiates the PU.1 DNA binding domain to weakly open chromatin in the absence of the PU.1 disordered domain. Our findings reveal a hierarchy by which chromatin is opened and show that pioneer factors can provide specificity for action by nucleosome remodelers.


Asunto(s)
Cromatina , Nucleosomas , Animales , Factores de Transcripción/metabolismo , ADN , Adenosina Trifosfato/metabolismo , Ensamble y Desensamble de Cromatina , Mamíferos/genética
6.
Elife ; 112022 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-36264057

RESUMEN

Alternative splicing occurs in the vast majority of human genes, giving rise to distinct mRNA and protein isoforms. We, and others, have previously identified hundreds of genes that change their isoform expression upon T cell activation via alternative splicing; however, how these changes link activation input with functional output remains largely unknown. Here, we investigate how costimulation of T cells through the CD28 receptor impacts alternative splicing in T cells activated through the T cell receptor (TCR, CD3) and find that while CD28 signaling alone has minimal impact on splicing, it enhances the extent of change for up to 20% of TCR-induced alternative splicing events. Interestingly, a set of CD28-enhanced splicing events occur within genes encoding key components of the apoptotic signaling pathway; namely caspase-9, Bax, and Bim. Using both CRISPR-edited cells and antisense oligos to force expression of specific isoforms, we show for all three of these genes that the isoform induced by CD3/CD28 costimulation promotes resistance to apoptosis, and that changes in all three genes together function combinatorially to further promote cell viability. Finally, we show that the JNK signaling pathway, induced downstream of CD3/CD28 costimulation, is required for each of these splicing events, further highlighting their co-regulation. Together, these findings demonstrate that alternative splicing is a key mechanism by which costimulation of CD28 promotes viability of activated T cells.


Asunto(s)
Antígenos CD28 , Linfocitos T , Humanos , Linfocitos T/metabolismo , Antígenos CD28/metabolismo , Empalme Alternativo , Supervivencia Celular , Receptores de Antígenos de Linfocitos T/metabolismo , Apoptosis
7.
Elife ; 92020 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-33269701

RESUMEN

Viral infection induces the expression of numerous host genes that impact the outcome of infection. Here, we show that infection of human lung epithelial cells with influenza A virus (IAV) also induces a broad program of alternative splicing of host genes. Although these splicing-regulated genes are not enriched for canonical regulators of viral infection, we find that many of these genes do impact replication of IAV. Moreover, in several cases, specific inhibition of the IAV-induced splicing pattern also attenuates viral infection. We further show that approximately a quarter of the IAV-induced splicing events are regulated by hnRNP K, a host protein required for efficient splicing of the IAV M transcript in nuclear speckles. Finally, we find an increase in hnRNP K in nuclear speckles upon IAV infection, which may alter accessibility of hnRNP K for host transcripts thereby leading to a program of host splicing changes that promote IAV replication.


Asunto(s)
Empalme Alternativo , Núcleo Celular/virología , Células Epiteliales/virología , Virus de la Influenza A/crecimiento & desarrollo , Pulmón/virología , Replicación Viral , Células A549 , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Epiteliales/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Interacciones Huésped-Patógeno , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/patogenicidad , Pulmón/metabolismo
8.
Nat Struct Mol Biol ; 24(9): 700-707, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28759050

RESUMEN

We describe a novel approach to separate two ribosome populations from the same cells and use this method in combination with RNA-seq to identify mRNAs bound to Saccharomyces cerevisiae ribosomes with and without Rps26, a protein linked to the pathogenesis of Diamond-Blackfan anemia (DBA). These analyses reveal that Rps26 contributes to mRNA-specific translation by recognition of the Kozak sequence in well-translated mRNAs and that Rps26-deficient ribosomes preferentially translate mRNA from select stress-response pathways. Surprisingly, exposure of yeast to these stresses leads to the formation of Rps26-deficient ribosomes and to the increased translation of their target mRNAs. These results describe a novel paradigm: the production of specialized ribosomes, which play physiological roles in augmenting the well-characterized transcriptional stress response with a heretofore unknown translational response, thereby creating a feed-forward loop in gene expression. Moreover, the simultaneous gain-of-function and loss-of-function phenotypes from Rps26-deficient ribosomes can explain the pathogenesis of DBA.


Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Unión Proteica
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