RESUMEN
Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is a prevalent and conserved RNA modification. While A-to-I RNA editing is essential in mammals, in Caenorhabditis elegans, it is not, making them invaluable for RNA editing research. In C. elegans, ADR-2 is the sole catalytic A-to-I editing enzyme, and ADR-1 is an RNA editing regulator. ADAR localization is well-studied in humans but not well-established in C. elegans. In this study, we examine the cellular and tissue-specific localization of ADR-2. We show that while ADR-2 is present in most cells in the embryo, at later developmental stages, its expression is both tissue- and cell-type-specific. Additionally, both ADARs are mainly in the nucleus. ADR-2 is adjacent to the chromosomes during the cell cycle. We show that the nuclear localization of endogenous ADR-2 depends on ADBP-1, not ADR-1. In adbp-1 mutant worms, ADR-2 is mislocalized, while ADR-1 is not, leading to decreased editing levels and de-novo editing, mostly in exons, suggesting that ADR-2 is also functional in the cytoplasm. Besides, mutated ADBP-1 affects gene expression. Furthermore, we show that ADR-2 targets adenosines with different surrounding nucleotides in exons and introns. Our findings indicate that ADR-2 cellular localization is highly regulated and affects its function.
RESUMEN
Adenosine deaminases that act on RNA (ADARs) are present in all animals and function to both bind double-stranded RNA (dsRNA) and catalyze the deamination of adenosine (A) to inosine (I). As inosine is a biological mimic of guanosine, deamination by ADARs changes the genetic information in the RNA sequence and is commonly referred to as RNA editing. Millions of A-to-I editing events have been reported for metazoan transcriptomes, indicating that RNA editing is a widespread mechanism used to generate molecular and phenotypic diversity. Loss of ADARs results in lethality in mice and behavioral phenotypes in worm and fly model systems. Furthermore, alterations in RNA editing occur in over 35 human pathologies, including several neurological disorders, metabolic diseases, and cancers. In this review, a basic introduction to ADAR structure and target recognition will be provided before summarizing how ADARs affect the fate of cellular RNAs and how researchers are using this knowledge to engineer ADARs for personalized medicine. In addition, we will highlight the important roles of ADARs and RNA editing in innate immunity and cancer biology.
Asunto(s)
Adenosina Desaminasa/metabolismo , Carcinogénesis/metabolismo , Inmunidad Innata , Neoplasias/metabolismo , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Animales , Desaminación , Humanos , Inosina/metabolismo , Edición de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis.
RESUMEN
Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is a prevalent and conserved RNA modification. While A-to-I RNA editing is essential in mammals, in Caenorhabditis elegans , it is not, making them invaluable for RNA editing research. In C. elegans , ADR-2 is the sole catalytic A-to-I editing enzyme, and ADR-1 is an RNA editing regulator. ADAR localization is well-studied in humans but not well-established in C. elegans . In this study, we examine the cellular and tissue-specific localization of ADR-2. We show that while ADR-2 is present in most cells in the embryo, at later developmental stages, its expression is both tissue- and cell-type-specific. Additionally, both ADARs are mainly in the nucleus. ADR-2 is adjacent to the chromosomes during the cell cycle. We show that the nuclear localization of endogenous ADR-2 depends on ADBP-1, not ADR-1. In adbp-1 mutant worms, ADR-2 is mislocalized, while ADR-1 is not, leading to decreased editing levels and de-novo editing, mostly in exons, suggesting that ADR-2 is also functional in the cytoplasm. Besides, mutated ADBP-1 affects gene expression. Furthermore, we show that ADR-2 targets adenosines with different surrounding nucleotides in exons and introns. Our findings indicate that ADR-2 cellular localization is highly regulated and affects its function.
RESUMEN
RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Characterization of gross fertility defects, such as sterility, has identified RNA binding proteins that are critical regulators of germline gene expression; however, broader screens for RNA binding proteins involved in specific reproductive processes are lacking. Here, a reverse genetic screen was performed to identify RNA binding proteins that impact yolk and embryo production in Caenorhabditis elegans hermaphrodites. This screen makes use of animals expressing a vitellogenin (yolk protein) fusion with green fluorescent protein, in a genetic background that corrects for a previously identified fertility defect in this strain. From this screen, we identified 23 RNA binding proteins that regulate embryo production in Caenorhabditis elegans. This screen lays groundwork for future interrogations into the molecular roles of these proteins in yolk production and embryogenesis. Additionally, the screen uncovered a genetic interaction between ADR-2, a member of the Adenosine DeAminase Acting on RNA (ADAR) family, and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family. Transcriptome-wide assessment in animals depleted of sqd-1 revealed over 8000 misregulated transcripts, suggesting SQD-1 is a major regulator of gene expression. Consistent with this, microscopy and reproductive assays reveal that sqd-1 is essential for oogenesis. In the absence of adr-2, the effects of loss of sqd-1 on gene expression are attenuated, as well as the defects in oogenesis. Together, these data indicate that both ADR-2 and SQD-1 are important regulators of germline gene expression and oogenesis.
RESUMEN
Vitellogenin::GFP fusion proteins have been used in several studies of the synthesis, endocytosis, and function of yolk in Caenorhabditis elegans. Here we report that one commonly used transgenic strain harboring a vit-2::gfp fusion displays defects in reproduction that lead to a significantly decreased embryo content and brood size in adult worms.