Glycine-arginine-rich Research Articles

Continuous assay measures methyltransferase activity: Defining the substrate specificity of rat Protein Arginine Methyltransferase 1 (PRMT1)

Since the discovery of protein arginine methyltransferases (PRMTs), the substrate specificity for all PRMT isoforms remains ill-defined due to the absence of a convenient activity assay. Research has shown that PRMT1 prefers to methylate arginine residues located in glycine/arginine rich (GAR) areas of a protein or RXR motifs. However, PRMT1 methylates Arg 3 of histone H4 in vivo, which does not contain a GAR or RXR motif. Thus far, PRMT1 activity has been assayed using labeled S-adenosyl methionine. We have developed a continuous spectrophotometric assay to measure methyltransferase activity that eliminates the cost and time of the radioactive method. The continuous assay utilizes two coupling enzymes to break down S-adenosylhomocysteine (SAH), a product and potent inhibitor of methyltransferases. SAH is converted into adenine and S-ribosyl homocysteine by MTA nucleosidase. Adenine is converted into hypoxanthine by adenine deaminase which is associated with a decrease in the absorbance at 265 nm. Kinetic parameters have been obtained for PRMT1 with peptide substrates derived from two in vivo substrates, fibrillarin and H4. The objective of this research is to characterize PRMT1 substrate specificity. A detailed knowledge of PRMT1 substrate specificity will help in the design of specific inhibitors, the creation of isoform specific antibodies, and the prediction of protein substrates. Funded by USU New Faculty Funds

53BP1 Oligomerization is Independent of its Methylation by PRMT1

p53 binding protein 1 (53BP1) participates in the repair of DNA double stranded breaks (DSBs)where it is recruited to or near sites of DNA damage. Although little is known about thebiochemical functions of 53BP1, the protein possesses several motifs that are likely important for itsrole as a DNA damage response element. This includes two BRCA1 C-terminal repeats, tandemTudor domains, and a variety of phosphorylation sites. Here we show that a glycine-arginine rich(GAR) stretch of 53BP1 lying upstream of the Tudor motifs is methylated. We demonstrate thatarginine residues within this region are important for asymmetric methylation by the PRMT1methyltransferase. We further show that sequences upstream of the Tudor domains that do notinclude the GAR stretch are sufficient for 53BP1 oligomerization in vivo. Thus, although Tudordomains bind methylated proteins, 53BP1 homo-oligomerization occurs independently of Tudorfunction. Lastly, we find that deficiencies in 53BP1 generate a “hyper-rec” phenotype. Collectively,these data provide new insight into 53BP1, an important component in maintaining genomicstability.

The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylarginine-specific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few ?-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and is the site of methylation by PRMT1.

PRMT8, a New Membrane-bound Tissue-specific Member of the Protein Arginine Methyltransferase Family

Protein arginine methylation is a common post-translational modification that has been implicated in signal transduction, RNA processing, transcriptional regulation, and DNA repair. A search of the human genome for additional members of the protein arginine N-methyltransferase (PRMT) family of enzymes has identified a gene on chromosome 12 that we have termed PRMT8. This novel enzyme is most closely related to PRMT1, although it has a distinctive N-terminal region. The unique N-terminal end harbors a myristoylation motif, and we have shown here that PRMT8 is indeed modified by the attachment of a myristate to the glycine residue after the initiator methionine. The myristoylation of PRMT8 results in its association with the plasma membrane. The second singular property of PRMT8 is its tissue-specific expression pattern; it is largely expressed in the brain. A glutathione S-transferase fusion protein of PRMT8 has type I PRMT activity, catalyzing the formation of omega-NG-monomethylated and asymmetrically omega-NG,NG-dimethylated arginine residues on a recombinant glycine- and arginine-rich substrate. PRMT8 is thus an active arginine methyltransferase that is membrane-associated and tissue-specific, two firsts for this family of enzymes.

Unusual features of fibrillarin cDNA and gene structure in Euglena gracilis: evolutionary conservation of core proteins and structural predictions for methylation-guide box C/D snoRNPs throughout the domain Eucarya

Box C/D ribonucleoprotein (RNP) particles mediate O2′-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution.

Methylation of MRE11 Regulates its Nuclear Compartmentalization

The cellular response to DNA damage includes the orderly recruitment of many proteincomplexes to DNA lesions. The MRE11-RAD50-NBS1 (MRN) complex is well knownto localize early to sites of DNA damage, but the post-translational modificationsrequired to mobilize it to DNA damage sites are poorly understood. Recently, we haveshown that MRE11 is arginine methylated in a C-terminal glycine-arginine rich (GAR)domain by protein arginine methyltransferase 1 (PRMT1). Arginine methylation isrequired for the exonuclease activity of MRE11 and the intra-S phase DNA damageresponse. Herein, we report that cells treated with methylase inhibitors failed to relocalizeMRE11 from PML nuclear bodies to sites of DNA damage and formed few ?-H2AX foci. We also demonstrate that PRMT1 is a component of PML nuclear bodieswhere it co-localizes with MRE11. Using cellular fractionation, we demonstrate thatmethylated MRE11 is predominantly associated with nuclear structures and that MRE11methylated arginines were required for this association. These results suggest thatMRE11 methylation regulates its association with nuclear structures such as PML nuclearbodies and sites of DNA damage.

Novel Checkpoint Response to Genotoxic Stress Mediated by Nucleolin-Replication Protein A Complex Formation

Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.

The Gly-Arg-rich C-terminal domain of pea nucleolin is a DNA helicase that catalytically translocates in the 5′- to 3′-direction

Nucleolin is a major nucleolar phosphoprotein of exponentially growing eukaryotic cells. Here we report the cloning, purification, and characterization of the C-terminal glycine/arginine-rich (GAR) domain of pea nucleolin. The purified recombinant protein (17 kDa) shows ATP-/Mg 2+-dependent DNA helicase and ssDNA-/Mg 2+-dependent ATPase activities. The enzyme unwinds DNA in the 5′- to 3′-direction, which is the first report in plant for this directional activity. It unwinds forked/non-forked DNA with equal efficiency. The anti-nucleolin antibodies immunodepleted the activities of the enzyme. The DNA interacting ligands nogalamycin, daunorubicin, actinomycin C1, and ethidium bromide were inhibitory to DNA unwinding (with K i values of 0.40, 2.21, 8.0, and 9.0 μM, respectively) and ATPase (with K i values of 0.43, 1.65, 4.6, and 7.0 μM, respectively) activities of the enzyme. This study confirms that the unwinding and ATPase activities of pea nucleolin resided in the GAR domain. This study should make important contribution to our better understanding of DNA transaction in plants, mechanism of DNA unwinding, and the mechanism by which these ligands can disturb genome integrity.

Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins

Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10. The assembly and molecular interactions of the components of H/ACA RNPs are unknown. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following. NOP10 associates with NAP57 as a prerequisite for NHP2 binding. Although NHP2 on its own binds RNA nonspecifically, this NAP57-NOP10-NHP2 core trimer specifically recognizes H/ACA RNAs. GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs. In contrast to other RNPs whose assembly is initiated by protein-RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs. Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis.

Deletion and site-specific mutagenesis of nucleolin's carboxy GAR domain.

Vertebrate nucleolin is an abundant RNA-binding protein in the dense fibrillar component of active nucleoli. Nucleolin is modular in composition. Its amino-terminal third contains alternating acidic and basic domains, its middle section contains four consensus RNA-binding domains (cRBDs), and its carboxy-terminus contains a distinctive glycine/arginine-rich (GAR) domain with several RGG motifs. The arginines within these motifs are asymmetrically dimethylated. Several laboratories have shown that the GAR domain is necessary but not sufficient for the efficient localization of nucleolin to nucleoli. We examined the distribution of endogenous fibrillarin, Nopp140, and B23 when full-length and DeltaGAR nucleolin were expressed exogenously as enhanced green fluorescent protein (EGFP)-tagged fusions. Only B23 redistributed when DeltaGAR-EGFP was expressed at moderate to high levels, suggesting an in vivo interaction between nucleolin and B23. Next we substituted all ten arginines within the GAR domain of Chinese hamster ovary (CHO) nucleolin with lysines to test the hypothesis that methylation of the carboxy GAR domain is necessary for the nucleolar association of nucleolin. The lysine-substituted mutant was not an in vitro substrate for the yeast protein methyltransferase, Hmt1p/Rmt1. It was, however, able to associate properly with interphase nucleoli and with interphase pre-nucleolar bodies upon recovery from hypotonic shock. We conclude, therefore, that although the GAR domain is necessary for the efficient localization of nucleolin to nucleoli, methylation of this domain is not required for proper nucleolar localization.

Role for Nucleolin/Nsr1 in the Cellular Localization of Topoisomerase I

Nucleolin functions in ribosome biogenesis and contains an acidic N terminus that binds nuclear localization sequences. In previous work we showed that human nucleolin associates with the N-terminal region of human topoisomerase I (Top1). We have now mapped the topoisomerase I interaction domain of nucleolin to the N-terminal 225 amino acids. We also show that the Saccharomyces cerevisiae nucleolin ortholog, Nsr1p, physically interacts with yeast topoisomerase I, yTop1p. Studies of isogenic NSR1(+) and Deltansr1 strains indicate that NSR1 is important in determining the cellular localization of yTop1p. Moreover, deletion of NSR1 reduces sensitivity to camptothecin, an antineoplastic topoisomerase I inhibitor. By contrast, Deltansr1 cells are hypersensitive to the topoisomerase II-targeting drug amsacrine. These findings indicate that nucleolin/Nsr1 is involved in the cellular localization of Top1 and that this localization may be important in determining sensitivity to drugs that target topoisomerases.

Identification of genes in the genome of the archaeon Methanosarcina mazeii that code for homologs of nuclear eukaryotic molecules involved in RNA processing

A 2.6 kb fragment of chromosomal DNA from the archaeon Methanosarcina mazeii was sequenced and analyzed, and it was found to contain coding regions for three proteins that were 321, 234, and 193 amino acids (aa) in length. Homologs of the 321-aa protein were found in all archaeal genomes examined, but not in eukaryotic or bacterial genomes, with one exception in the latter. The protein with 234 aa (named PrpM) was most similar to the putative protein Prp31p from Methanobacterium thermoautotrophicum, while the 193-aa protein (named FibM) was identified as an archaeal fibrillarin homolog. Prp and fibrillarin proteins are involved in RNA processing in eukaryotes, but their functions in archaea are not yet understood. The M. mazeii PrpM was also similar to three proteins from Saccharomyces cerevisiae: Prp31p, Nop56p, and Nop58p. Prp31p is a pre-mRNA processing protein, while Nop56p and Nop58p are involved in rRNA processing and interact with fibrillarin. No homologs of either protein were found in bacteria. The archaeal fibrillarin was shorter than its eukaryotic counterpart because it lacked the N-terminal glycine-arginine-rich (GAR) domain, present in most eukaryal homologs. The archaeal prp and fibrillarin gene homologs were found adjacent to each other, whereas in eukarya these genes are on separate chromosomes. Sequence signatures typical of the eukaryal molecules were identified in the M. mazeii and the other archaeal molecules studied. The close proximity of the prp and fib genes raises the possibility of a Prp–fibrillarin interaction in archaea.

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2.

Chemical modifications and processing of the 18S, 5.8S, and 25S ribosomal RNAs from the 35S pre-ribosomal RNA depend on an important set of small nucleolar ribonucleoprotein particles (snoRNPs). Genetic depletion of yeast Gar1p, an essential common component of H/ACA snoRNPs, leads to inhibition of uridine isomerizations to pseudo-uridines on the 35S pre-rRNA and of the early pre-rRNA cleavages at sites A1 and A2, resulting in a loss of mature 18S rRNA synthesis. To identify Gar1p functional partners, we screened for mutations that are synthetically lethal with a gar1 mutant allele encoding a Gar1p mutant protein lacking its two glycine/arginine-rich (GAR) domains. We identified a previously uncharacterized Saccharomyces cerevisiae open reading frame, YDR083W (now designated RRP8), that encodes a highly conserved protein containing motifs found in methyltransferases. Rrp8p localizes to the nucleolus. A yeast strain lacking this protein is viable at 30 degrees C but displays strong growth impairment at lower temperatures. In this strain, cleavage of the pre-rRNA at site A2 is strongly affected whereas cleavages at sites A0 and A1 are only slightly inhibited or delayed.

NopA64, a novel nucleolar phosphoprotein from proliferating onion cells, sharing immunological determinants with mammalian nucleolin.

Five major soluble nuclear proteins associated with cell proliferation were identified in Allium cepa L. root cells. One of them, of 64 kDa, was revealed by Western blotting with anti-mammalian nucleolin antibodies. A polyclonal antibody raised against this protein, which we have named NopA64, localised it in the nucleolus as well as in nuclear coiled bodies. Together with NopA64, the antibody also revealed a smaller form, called NopA61. Both proteins were present in the soluble ribonucleoprotein fraction and in the nuclear matrix of proliferating cells, but NopA61 was the only form revealed in differentiated cells. NopA64 contained epitopes also present in other plants, in mammalian nucleolin and in its yeast homologue, gar2. In mammals, the highest homology was with 50-kDa nucleolin fragments containing the RNA-binding motifs and the glycine-arginine-rich (GAR) domain. NopA64 was moderately phosphorylated in vitro by exogenous casein kinase II and cdc2 kinase, whereas NopA61 was highly phosphorylated by casein kinase II. Furthermore, NopA61 was the only band detected after dephosphorylation as well as after endoproteolysis of NopA64. This protein could be one of the various functional homologues of mammalian nucleolin in plant cells.