ABSTRACT
ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.
Subject(s)
Adenosine Deaminase/metabolism , Peptidylprolyl Isomerase/metabolism , RNA Editing , Receptors, AMPA/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line , Fibroblasts/metabolism , Mice , NIMA-Interacting Peptidylprolyl Isomerase , RNA Polymerase II/metabolism , RNA-Binding Proteins , UbiquitinationABSTRACT
In Drosophila melanogaster, the expression of adenosine deaminase acting on RNA is regulated by transcription and alternative splicing so that at least four different isoforms are generated that have a tissue-specific splicing pattern. Even though dAdar has been extensively studied, the complete adult expression pattern has yet to be elucidated. In the present study, we investigate mature transcripts of dAdar arising from different promoters. Two predominant isoforms of dAdar are expressed in gonads and dAdar is transcribed from both the embryonic and the adult promoters. Furthermore, full-length transcripts containing the alternatively spliced exon-1 are expressed in a tissue-specific manner. The splicing factor B52/SRp55 binds within the alternative spliced exon 3a and plays a role in this alternative splicing event.
Subject(s)
Adenosine Deaminase/genetics , Alternative Splicing , Drosophila Proteins/genetics , Drosophila melanogaster/enzymology , Adenosine Deaminase/metabolism , Animals , Binding Sites , Cell Line , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Exons , Female , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Nuclear Proteins/metabolism , Oocytes/metabolism , Ovary/embryology , Ovary/metabolism , Phosphoproteins/metabolism , RNA Editing , RNA Splicing Factors , RNA, Messenger/metabolism , RNA-Binding Proteins , Testis/metabolismABSTRACT
Since its cloning in 1994, several studies have reported that thrombopoietin (THPO) presents several alternative splicing products that differ from the full-length protein in its 5' UTR, N- or C-terminal regions. Most of these splice variants are evolutionarily conserved and have been detected in different tissues as well as in cell lines. Although the possible functions of the THPO isoforms are still elusive, different clues link them to the peculiar mechanism that regulates THPO production. Moreover, novel fields to explore possible roles of the THPO variants are opened by observations that this hormone can influence the formation of hematopoietic progenitors and its expression occurs in some tumors as well as in tissues not directly related to the thrombopoiesis. In this review, we summarize the structure and functions of THPO through the published evidence on its splicing isoforms and discuss about their involvement with physiopathologic phenomena.
Subject(s)
Alternative Splicing , Thrombopoiesis/genetics , Thrombopoiesis/physiology , Thrombopoietin/genetics , Thrombopoietin/physiology , 5' Untranslated Regions , Amino Acid Sequence , Base Sequence , Exons , Humans , Molecular Sequence Data , Protein Biosynthesis , RNA, Messenger/genetics , Sequence Homology, Nucleic Acid , Thrombopoietin/chemistryABSTRACT
The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1-4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3' splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3-5 guanosines (namely, G1-G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7-G10 work in a combinatorial way to control the selection of the proper 3' splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3' splice site so providing functional evidence that this factor is involved in the selection of the authentic 3' splice site of THPO IVS2.
Subject(s)
Alternative Splicing , Guanosine/analysis , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/physiology , RNA Splice Sites , Thrombopoietin/genetics , Base Sequence , Binding Sites , Cell Line , Exons , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism , Humans , Introns , Molecular Sequence Data , RNA Interference , RNA, Messenger/chemistry , Thrombopoietin/metabolismABSTRACT
844ins68 is a frequent polymorphism of the cystathionine beta-synthase gene (CBS) that consists of a 68-bp insertion duplicating the 3' splice site of intron 7 and the 5'-end of exon 8. The presence of two identical 3' splice sites spaced by 68 bp should lead to either a selection of the proximal site or to at least two alternatively spliced CBS mRNA variants. Instead, an accurate selection of the distal 3' splice site is observed in the 844ins68 carriers. The duplication has generated a gene re-arrangement at the 3' splice site where two GGGG runs have been brought close to each other. Using a minigene system, we have investigated the effect this peculiar configuration might have on the selection of the 3' splice site of intron 7 in the CBS gene. Minimal disruption of the G runs resulted in a dramatic shift toward the proximal 3' splice site selection with inclusion of the 68-bp insertion and a consequent change of the reading frame. The insertional event created this peculiar configuration of two G repeats close to each other that subsequently acquired the ability to strongly bind heterogeneous nuclear ribonucleoprotein (hnRNP) H1, a specific trans-acting factor. The interaction of hnRNP H1 with G runs within the 844ins68 context might interfere with the recruitment of splicing factors to the proximal 3' splice site thus favoring the selection of the distal 3' splice site. Our results therefore suggest the possibility that the insertion was an evolutionary event that allowed the rescue of the wild-type sequence, so preserving protein function.
Subject(s)
Cystathionine beta-Synthase/biosynthesis , Cystathionine beta-Synthase/genetics , Polymorphism, Genetic , Alternative Splicing , Amino Acid Sequence , Base Sequence , Exons , Gene Deletion , Heterozygote , Humans , Introns , Models, Genetic , Molecular Sequence Data , RNA/metabolism , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Transfection , Ultraviolet RaysABSTRACT
En este trabajo se trata de establecer la eficacia de dos métodos para evitar la nefropatía asociada a contraste readiológico (N.A.C.). Se estudió en forma prospectiva y randomizada una muestra de 75 pacientes de los cuales 25 se asignaron al grupo Control: sin intervenciones, 25 al grupo Salina: solución salina 0.45 por ciento E.V., 1,5 cc/kg/hr, 6 horas antes y después del estudio angiográfico y 25 al grupo Dopa: igual al anterior más dopamina 2 mug/kg/min, 30 minutos antes del estudio y hasta su finalización. Se consideró como TO a la valoración realizada al momento del ingreso del paciente, T1 a las 24 y T2 a las 48 horas. En TO se registraron: edad; sexo; antecendetes patológicos; drogas y creatininemia. En T1: diuresis y creatininemia y en T2 creatininemia. Se consideró como N.A.C al aumento del 25 por ciento de la cratininemia em T2. Estadística: La edad fue analizada con Kruskal-Wallis H, las variables continuas con ANOVA y las nominales con el cálculo de X2 con corrección de Yates o Ficher según el tamaño e la muestra. La frecuencia de N.A.C en cada uno de los grupos se estudió mediante el análisis de tendencia lineal de proporciones. La N.A.C se presentó en 13/25 (OR:1) pacientes del grupo Control, 7/25 (OR 0,36) del grupo Salina y 5/25 (OR 0.23) del grupo Dopa (p=0.01). No se hallaron diferencias significativas en la diuresis ni en las cifras de creatininemia. Se concluye que la hidratación durante seis horas previas y posteriores al estudio con solución salina al 0.45 por ciento y el mismo plan con el agregado de dopamina son eficaces para prevenir la N.A.C.
Subject(s)
Female , Humans , Adult , Middle Aged , Acute Kidney Injury/chemically induced , Acute Kidney Injury/prevention & control , Contrast Media/adverse effects , Dopamine/therapeutic use , Saline Solution, Hypertonic/therapeutic use , Analysis of Variance , Creatinine/analysis , Evaluation Study , Prospective Studies , Random Allocation , Statistics, NonparametricABSTRACT
En este trabajo se trata de establecer la eficacia de dos métodos para evitar la nefropatía asociada a contraste readiológico (N.A.C.). Se estudió en forma prospectiva y randomizada una muestra de 75 pacientes de los cuales 25 se asignaron al grupo Control: sin intervenciones, 25 al grupo Salina: solución salina 0.45 por ciento E.V., 1,5 cc/kg/hr, 6 horas antes y después del estudio angiográfico y 25 al grupo Dopa: igual al anterior más dopamina 2 mug/kg/min, 30 minutos antes del estudio y hasta su finalización. Se consideró como TO a la valoración realizada al momento del ingreso del paciente, T1 a las 24 y T2 a las 48 horas. En TO se registraron: edad; sexo; antecendetes patológicos; drogas y creatininemia. En T1: diuresis y creatininemia y en T2 creatininemia. Se consideró como N.A.C al aumento del 25 por ciento de la cratininemia em T2. Estadística: La edad fue analizada con Kruskal-Wallis H, las variables continuas con ANOVA y las nominales con el cálculo de X2 con corrección de Yates o Ficher según el tamaño e la muestra. La frecuencia de N.A.C en cada uno de los grupos se estudió mediante el análisis de tendencia lineal de proporciones. La N.A.C se presentó en 13/25 (OR:1) pacientes del grupo Control, 7/25 (OR 0,36) del grupo Salina y 5/25 (OR 0.23) del grupo Dopa (p=0.01). No se hallaron diferencias significativas en la diuresis ni en las cifras de creatininemia. Se concluye que la hidratación durante seis horas previas y posteriores al estudio con solución salina al 0.45 por ciento y el mismo plan con el agregado de dopamina son eficaces para prevenir la N.A.C. (AU)
