Changes in nuclear localization of An3, a RNA helicase, during oogenesis and embryogenesis in Xenopus laevis

Abstract

The immunolocalization of An3 protein, an ATP‐dependent RNA helicase and a member of the DEAD box family, was compared with the localization of fibrillarin, a protein essential for rRNA processing, and snRNPs, which are involved in mRNA splicing reactions, during oogenesis and embryogenesis in Xenopus laevis. Although An3 protein was detected in the cytoplasm of all stages of oocytes, in most stages An3 protein was also present in the nucleus. Prior to stage I An3 protein was uniformly dispersed throughout the entire germinal vesicle; from stages I to V it was in nucleoli. By stage VI nucleolar labeling with anti An3 disappeared and the protein was no longer present within nuclei. An3 reactivity was also present throughout the nuclei of follicle cells surrounding prestage I to stage VI oocytes. Both cytoplasmic and nuclear An3 staining were present in cells of stages 8 to 35 embryos; however, nuclear staining was punctate and uniformly distributed throughout the nucleoplasm. Fibrillarin was diffusely distributed throughout the entire germinal vesicle prior to stage I, localized exclusively to nucleoli of oocytes between stages I and VI and in nucleoli of stages 12 and 35 embryonic cells. Reactivity for snRNPs (anti‐Sm) in germinal vesicles of prestage I oocytes was diffuse, and similar to the distribution of An3 and fibrillarin; in later stage oocytes anti‐Sm staining was restricted to a population of granules, much fewer in number and more heterogeneous in size than nucleoli. Anti‐Sm activity was apparent in nuclei of embryonic cells of stages 8 to 35 embryos. Although colocalization of the Sm epitope and An3 was not observed in developing oocytes and in embryonic cells, Sm reactive material was frequently found in close association with An3‐positive nucleoli (oocytes) and nuclear deposits (embryonic cells). In stage IV and V oocytes treated with actinomycin D (4 μg/ml) to inhibit rRNA synthesis, nucleoli, which continued to possess fibrillarin, lacked An3; staining of follicle cell nuclei for An3 was unchanged. Treatment with 200 μg/ml actinomycin D to block mRNA synthesis, inhibited An3 but not fibrillarin staining in nuclei of prestage I oocytes and follicle cells. The changing patterns of An3 reactivity and the differential effects of actinomycin D on such localizations observed here are consistent with a role for An3 in the processing/production of RNA. © 1996 Wiley‐Liss, Inc.

Nuclear Export of the DEAD Box An3 Protein by CRM1 Is Coupled to An3 Helicase Activity

“…However, due to the shuttling behavior of An3, we anticipate that PL10 family members may have a nuclear function as well. Based on localization studies, Weeks and colleagues have suggested that An3 is involved in rRNA processing (29,31), but there is no direct evidence available that might provide clues as to which function An3 fulfils in the nucleus. This led us to look for a correlation between nuclear export and the enzymatic activities of An3.…”

“…However, other aspects of gene expression might be regulated by these proteins. In agreement with the immunolocalization experiments showing that An3 is a partly nuclear protein (29,31), we have recently shown that the An3 protein shuttles between the nucleus and the cytoplasm (40). Nuclear export is mediated by direct binding to the transport receptor CRM1 (41) via an N-terminal leucine-rich nuclear export signal (NES) and, interestingly, an alignment of the PL10 family reveals that the NES sequence is conserved in all members.…”

“…Moreover, probing of early-stage oocytes as well as embryonic cells with anti-An3 antibodies gives rise to both nuclear and cytoplasmic staining (29,31). Export from the nucleus is mediated by the export receptor CRM1, requiring an N-terminal NES (40) (Fig.…”

Molecular Method Based on Hydrolysis Probe Assays to Identify the Sex Chromosomes of Iberian Desman (Galemys pyrenaicus) Using Non‐Invasive Sampling

“…The DBX genes belong to a large family of DEAD-box genes that are expressed in oocytes of both mammalian and nonmammalian species. Such genes showed differential expression throughout oogenesis and in mature oocytes, suggesting a role in RNA production, processing and transport during oogenesis (Longo et al 1996). Expression studies of the homologous DBX gene in the female germline of drosophila, frogs, zebrafish, mice and humans strongly suggest that this function is conserved and most likely plays a functional role in folliculogenesis.…”

The DDX3 Subfamily of the DEAD Box Helicases: Divergent Roles as Unveiled by Studying Different Organisms and In Vitro Assays

“…Then, reminiscent of the appearance of organ rudiments in blastogenesis, some of the newly formed organs in the developed embryo, including the endostyle, digestive system, tail muscles, and adhesive organ, express elevated quantities of BS-PL10 protein [69]. Similarly, the An3 [123] protein in Xenopus laevis embryos is transcribed only after gastrulation (young embryos utilize An3 protein of maternal origin [124]) and remains active throughout embryogenesis. This result coincides with the observation that maternal inherited CRM1 protein (DDX3's associate in nucleo-cytoplasmic transport) is inactive until gastrula-neurula transition [125].…”