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1.
Proc Natl Acad Sci U S A ; 121(3): e2315259121, 2024 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-38194449

RESUMEN

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity toward canonical snRNAs through their Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.


Asunto(s)
Exonucleasas , ARN Nuclear Pequeño , ARN Nuclear Pequeño/genética , ARN , Mutación , Control de Calidad
2.
bioRxiv ; 2023 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-37645788

RESUMEN

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity towards canonical snRNAs by recognizing Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.

3.
Genes Dev ; 34(13-14): 989-1001, 2020 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-32499401

RESUMEN

Polymerases and exonucleases act on 3' ends of nascent RNAs to promote their maturation or degradation but how the balance between these activities is controlled to dictate the fates of cellular RNAs remains poorly understood. Here, we identify a central role for the human DEDD deadenylase TOE1 in distinguishing the fates of small nuclear (sn)RNAs of the spliceosome from unstable genome-encoded snRNA variants. We found that TOE1 promotes maturation of all regular RNA polymerase II transcribed snRNAs of the major and minor spliceosomes by removing posttranscriptional oligo(A) tails, trimming 3' ends, and preventing nuclear exosome targeting. In contrast, TOE1 promotes little to no maturation of tested U1 variant snRNAs, which are instead targeted by the nuclear exosome. These observations suggest that TOE1 is positioned at the center of a 3' end quality control pathway that selectively promotes maturation and stability of regular snRNAs while leaving snRNA variants unprocessed and exposed to degradation in what could be a widespread mechanism of RNA quality control given the large number of noncoding RNAs processed by DEDD deadenylases.


Asunto(s)
Proteínas Nucleares/metabolismo , Procesamiento de Término de ARN 3'/genética , Estabilidad del ARN/genética , ARN Nuclear Pequeño/genética , Línea Celular , Núcleo Celular/metabolismo , Eliminación de Gen , Células HeLa , Humanos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/metabolismo , ARN Nuclear Pequeño/biosíntesis
4.
J Biol Chem ; 294(40): 14674-14685, 2019 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-31395656

RESUMEN

Human Schlafen 11 (SLFN11) is an interferon-stimulated gene (ISG) that we previously have demonstrated to ablate translation of HIV proteins based on the virus's distinct codon preference. Additionally, lack of SLFN11 expression has been linked to the resistance of cancer cells to DNA-damaging agents (DDAs). We recently resolved the underlying mechanism, finding that it involves SLFN11-mediated cleavage of select tRNAs predominantly employed in the translation of the ATR and ATM Ser/Thr kinases, thereby establishing SLFN11 as a novel tRNA endonuclease. Even though SLFN11 is thus involved in two of the most prominent diseases of our time, cancer and HIV infection, its regulation remained thus far unresolved. Using MS and bioinformatics-based approaches combined with site-directed mutagenesis, we show here that SLFN11 is phosphorylated at three different sites, which requires dephosphorylation for SLFN11 to become fully functionally active. Furthermore, we identified protein phosphatase 1 catalytic subunit γ (PPP1CC) as the upstream enzyme whose activity is required for SLFN11 to cleave tRNAs and thereby act as a selective translational inhibitor. In summary, our work has identified both the mechanism of SLFN11 activation and PPP1CC as the enzyme responsible for its activation. Our findings open up future studies of the PPP1CC subunit(s) involved in SLFN11 activation and the putative kinase(s) that inactivates SLFN11.


Asunto(s)
Daño del ADN/genética , Proteínas Nucleares/genética , Biosíntesis de Proteínas , Proteína Fosfatasa 1/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Biología Computacional , Infecciones por VIH/genética , Infecciones por VIH/patología , Humanos , Interferones/genética , Mutagénesis Sitio-Dirigida , Mutación , Neoplasias/genética , Neoplasias/patología , Fosforilación/genética , ARN de Transferencia/genética
5.
Nat Genet ; 49(3): 457-464, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28092684

RESUMEN

Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg2+-dependent 3'-end RNases with substrate specificity that is mostly unknown. Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia. We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo. Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3' genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3'-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3'-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3' ends.


Asunto(s)
Enfermedades Cerebelosas/genética , Exonucleasas/genética , Mutación/genética , Proteínas Nucleares/genética , ARN Nuclear Pequeño/genética , Alelos , Animales , Femenino , Humanos , Masculino , Ratones , Enfermedades Neurodegenerativas/genética , ARN Mensajero/genética , Empalmosomas/genética , Pez Cebra
6.
Neuron ; 92(4): 780-795, 2016 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-27773581

RESUMEN

HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide crosslinking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within ∼2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1. VIDEO ABSTRACT.


Asunto(s)
Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/genética , Supervivencia Celular/genética , Fibroblastos/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Neuronas Motoras/metabolismo , Transporte de Proteínas/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Estudios de Casos y Controles , D-Aminoácido Oxidasa/genética , D-Aminoácido Oxidasa/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Perfilación de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Ratones , Mutación , Poliadenilación
7.
Proc Natl Acad Sci U S A ; 111(8): 3062-7, 2014 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-24516132

RESUMEN

Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Conformación Proteica , Empalme del ARN/fisiología , ARN Nuclear Pequeño/química , Proteínas de Unión al ARN/genética , Empalmosomas/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Intestinos/anomalías , Hígado/anomalías , Análisis por Micromatrices , Datos de Secuencia Molecular , Páncreas/anomalías , Mutación Puntual/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Empalmosomas/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/metabolismo
8.
RNA ; 16(3): 516-28, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20089683

RESUMEN

Eukaryotic pre-mRNA splicing is a complex process requiring the precise timing and action of >100 trans-acting factors. It has been known for some time that the two steps of splicing chemistry require three DEAH-box RNA helicase-like proteins; however, their mechanism of action at these steps has remained elusive. Spliceosomes arrested in vivo at the three helicase checkpoints were purified, and first step-arrested spliceosomes were functionally characterized. We show that the first step of splicing requires a novel ATP-independent conformational change. Prp2p then catalyzes an ATP-dependent rearrangement displacing the SF3a and SF3b complexes from the branchpoint within the spliceosome. We propose a model in which SF3 prevents premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine prior to the first transesterification. When the spliceosome attains the proper conformation and upon the function of Prp2p, SF3 is displaced from the branchpoint allowing first step chemistry to occur.


Asunto(s)
Precursores del ARN/metabolismo , Empalme del ARN , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Hidróxidos/metabolismo , Intrones , Modelos Químicos , Precursores del ARN/química , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/química
9.
Am J Physiol Regul Integr Comp Physiol ; 292(4): R1603-12, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17158267

RESUMEN

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30 degrees C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25 degrees C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30 degrees C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 +/- 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10 degrees C. The K(m) for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30 degrees C, is 20 +/- 4 microM for phosphorylated fibers and 192 +/- 40 microM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30 degrees C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF(3) and no inhibition in the presence of 0.25 mM AlF(4). Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue.


Asunto(s)
Contracción Muscular/efectos de los fármacos , Fibras Musculares de Contracción Rápida/fisiología , Músculo Esquelético/fisiología , Cadenas Ligeras de Miosina/metabolismo , Vanadatos/metabolismo , Compuestos de Aluminio/farmacología , Animales , Berilio/farmacología , Fluoruros/farmacología , Contracción Isométrica/efectos de los fármacos , Cinética , Modelos Biológicos , Fosforilación , Conejos , Temperatura , Vanadatos/farmacología
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