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The GAR domain integrates functions that are necessary for the proper localization of fibrillarin (FBL) inside eukaryotic cells.

ABSTRACT: Fibrillarin (FBL) is an essential nucleolar protein that participates in pre-rRNA methylation and processing. The methyltransferase domain of FBL is an example of an extremely well-conserved protein domain in which the amino acid sequence was not substantially modified during the evolution from Archaea to Eukaryota. An additional N-terminal glycine-arginine-rich (GAR) domain is present in the FBL of eukaryotes. Here, we demonstrate that the GAR domain is involved in FBL functioning and integrates the functions of the nuclear localization signal and the nucleolar localization signal (NoLS). The methylation of the arginine residues in the GAR domain is necessary for nuclear import but decreases the efficiency of nucleolar retention via the NoLS. The presented data indicate that the GAR domain can be considered an evolutionary innovation that integrates several functional activities and thereby adapts FBL to the highly compartmentalized content of the eukaryotic cell.

SUBMITTER: Shubina MY

PROVIDER: S-EPMC7194090 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Fibrillarin Ribonuclease Activity is Dependent on the GAR Domain and Modulated by Phospholipids.

Project description:Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear shape, and cell cycle progression. We show that fibrillarin has an additional activity as a ribonuclease. The activity is affected by phosphoinositides and phosphatidic acid and insensitive to ribonuclease inhibitors. Furthermore, the presence of phosphatidic acid releases the fibrillarin-U3 snoRNA complex. We show that the ribonuclease activity localizes to the GAR (glycine/arginine-rich) domain conserved in a small group of RNA interacting proteins. The introduction of the GAR domain occurred in evolution in the transition from archaea to eukaryotic cells. The interaction of this domain with phospholipids may allow a phase separation of this protein in nucleoli.

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Project description:During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.

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The GAR Domain Determines Subcellular Localization of the Growth Controlling RNA Binding Protein Nucleolin

Project description:In this project we aimed to identify the spectrum of protein binders of nucleolin’s glycine arginine reach (GAR) domain. We did so by designing a 15 amino acid peptide derived from the GAR domain sequence, which includes 3 arginine-glycine-glycine (RGG) repeats and we call "R3". We conducted affinity purification with biotinylated R3 peptide, from mouse sciatic nerve axoplasm preparations, followed by mass spectrometry, in order to identify potential GAR interacting proteins.

2021-07-23 | PXD020804 | Pride

son is necessary for proper vertebrate blood development.

Project description:The gene SON is on human chromosome 21 (21q22.11) and is thought to be associated with hematopoietic disorders that accompany Down syndrome. Additionally, SON is an RNA splicing factor that plays a role in the transcription of leukemia-associated genes. Previously, we showed that mutations in SON cause malformations in human and zebrafish spines and brains during early embryonic development. To examine the role of SON in normal hematopoiesis, we reduced expression of the zebrafish homolog of SON in zebrafish at the single-cell developmental stage with specific morpholinos. In addition to the brain and spinal malformations we also observed abnormal blood cell levels upon son knockdown. We then investigated how blood production was altered when levels of son were reduced. Decreased levels of son resulted in lower amounts of red blood cells when visualized with lcr:GFP transgenic fish. There were also reduced thrombocytes seen with cd41:GFP fish, and myeloid cells when mpx:GFP fish were examined. We also observed a significant decrease in the quantity of T cells, visualized with lck:GFP fish. However, when we examined their hematopoietic stem and progenitor cells (HSPCs), we saw no difference in colony-forming capability. These studies indicate that son is essential for the proper differentiation of the innate and adaptive immune system, and further investigation determining the molecular pathways involved during blood development should elucidate important information about vertebrate HSPC generation, proliferation, and differentiation.

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Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse.

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GAR-Driven Subcellular Localization of the Growth Controlling RNA Binding Protein Nucleolin

Project description:This SuperSeries is composed of the SubSeries listed below.

2021-07-23 | GSE142576 | GEO

Fibrillarin-like proteins occur in the domain Archaea.

Project description:Fibrillarin is found in the nucleolus of Eucarya and associated with small nucleolar RNAs. It is involved in the processing of precursor rRNA. Two genes, encoding fibrillarin-like proteins from Methanococcus voltae and Methanococcus vannielii, have been isolated. The genes were named flpA (fibrillarin-like protein).

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The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

Project description:Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.

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A small conserved domain in the yeast Spa2p is necessary and sufficient for its polarized localization.

Project description:SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother-daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (a2p omolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150-amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.

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