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1.
Nat Commun ; 11(1): 2619, 2020 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-32457326

RESUMEN

DIS3L2-mediated decay (DMD) is a surveillance pathway for certain non-coding RNAs (ncRNAs) including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and RMRP. While mutations in DIS3L2 are associated with Perlman syndrome, the biological significance of impaired DMD is obscure and pathological RNAs have not been identified. Here, by ribosome profiling (Ribo-seq) we find specific dysregulation of endoplasmic reticulum (ER)-targeted mRNA translation in DIS3L2-deficient cells. Mechanistically, DMD functions in the quality control of the 7SL ncRNA component of the signal recognition particle (SRP) required for ER-targeted translation. Upon DIS3L2 loss, sustained 3'-end uridylation of aberrant 7SL RNA impacts ER-targeted translation and causes ER calcium leakage. Consequently, elevated intracellular calcium in DIS3L2-deficient cells activates calcium signaling response genes and perturbs ESC differentiation. Thus, DMD is required to safeguard ER-targeted mRNA translation, intracellular calcium homeostasis, and stem cell differentiation.


Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Exorribonucleasas/metabolismo , Macrosomía Fetal/microbiología , ARN Mensajero/metabolismo , Tumor de Wilms/microbiología , Animales , Señalización del Calcio/genética , Diferenciación Celular , Células Madre Embrionarias , Exorribonucleasas/deficiencia , Exorribonucleasas/genética , Macrosomía Fetal/enzimología , Macrosomía Fetal/genética , Regulación de la Expresión Génica , Humanos , Insulina/metabolismo , Ratones , Biosíntesis de Proteínas , ARN Citoplasmático Pequeño/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Uridina Monofosfato/metabolismo , Tumor de Wilms/enzimología , Tumor de Wilms/genética
2.
Mol Metab ; 35: 100959, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32244186

RESUMEN

OBJECTIVE: As diabetes develops, marked reductions of insulin secretion are associated with very modest elevations of glucose. We wondered if these glucose changes disrupt beta cell differentiation enough to account for the altered function. METHODS: Rats were subjected to 90% partial pancreatectomies and those with only mild glucose elevations 4 weeks or 10 weeks after surgery had major alterations of gene expression in their islets as determined by RNAseq. RESULTS: Changes associated with glucose toxicity demonstrated that many of the critical genes responsible for insulin secretion were downregulated while the expression of normally suppressed genes increased. Also, there were marked changes in genes associated with replication, aging, senescence, stress, inflammation, and increased expression of genes controlling both class I and II MHC antigens. CONCLUSIONS: These findings suggest that mild glucose elevations in the early stages of diabetes lead to phenotypic changes that adversely affect beta cell function, growth, and vulnerability.


Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglucemia/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Diferenciación Celular , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Tipo 2/etiología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Expresión Génica , Hiperglucemia/etiología , Insulina/metabolismo , Secreción de Insulina/genética , Trasplante de Islotes Pancreáticos/efectos adversos , Trasplante de Islotes Pancreáticos/métodos , Masculino , Pancreatectomía/efectos adversos , Pancreatectomía/métodos , ARN Mensajero/genética , Ratas , Ratas Endogámicas Lew
3.
Nat Struct Mol Biol ; 26(6): 490-500, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31160785

RESUMEN

Ribosomal RNA (rRNA) biogenesis is a multistep process requiring several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remain obscure. Here, using loss-of-function approaches in mouse embryonic stem cells (mESCs) and deep sequencing of rRNA intermediates, we investigate the requirements of exonucleases known to be involved in 5.8S maturation at nucleotide resolution and explore the role of the Perlman syndrome-associated 3'-5' exonuclease Dis3l2 in rRNA processing. We uncover a novel cytoplasmic intermediate that we name '7SB' rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligoadenylated by an unknown enzyme and/or oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide-level insight into the exonuclease requirements for mammalian rRNA processing.


Asunto(s)
Exorribonucleasas/metabolismo , ARN Ribosómico 5.8S/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Macrosomía Fetal/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Transporte de ARN , Ribosomas/metabolismo , Uridina/metabolismo , Tumor de Wilms/metabolismo
4.
Methods ; 155: 10-19, 2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30395968

RESUMEN

Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.


Asunto(s)
Biología Computacional/métodos , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Nuclear Pequeño/genética , Uridina/metabolismo , Región de Flanqueo 3' , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Exorribonucleasas/deficiencia , Exorribonucleasas/genética , Edición Génica/métodos , Ratones , Células Madre Embrionarias de Ratones , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo
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