HIRIP3 is a nuclear phosphoprotein interacting with and phosphorylated by the serine-threonine kinase CK2

The HIRIP3 protein had been identified from its interaction with the HIRA histone chaperone. Experiments using anti-peptide antisera indicated that this 556-aa protein is nuclear throughout the cell cycle and excluded from condensed chromatin during mitosis. Based on its electrophoretic migration and sensitivity to phosphatase treatment, endogenous HIRIP3 was found to be heavily phosphorylated. HIRIP3 can be phosphorylated in vitro by a recombinant form of the serine-threonine kinase CK2. Moreover, HIRIP3 protein was found to co-purify with a CK2 activity. Together, these data prompt us to propose HIRIP3 as a new member of the growing list of CK2 substrates with a possible role in chromatin metabolism.     

Cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine UBP43 (Usp18)

The ubiquitin-specific proteases (UBP) are a family of enzymes that cleave ubiquitin from ubiquitinated protein substrates. We have recently cloned UBP43, a novel member of this family from AML1-ETO knock-in mice. To analyze the role of UBP43 in hematopoiesis and leukemogenesis, we have cloned a full-length human UBP43 cDNA by screening a human monocytic cDNA library as well as by 5'- and 3'-rapid amplification of cDNA ends analyses. This cDNA encodes a polypeptide of 372 amino acids with all of the structural motifs of a deubiquitinating enzyme. The human UBP43 mRNA is strongly expressed in human liver and thymus. Transfection analysis has demonstrated that UBP43 is a nuclear protein. Interestingly, the gene encoding human UBP43 maps to chromosome 22q11.2. This region, known as DiGeorge syndrome critical region, contains a minimal area of 2 Mb and is consistently deleted in DiGeorge syndrome and related disorders. The syndrome is marked by thymic aplasia or hypoplasia, parathyroid hypoplasia, or congenital cardiac abnormalities. Taken together, our results broaden the understanding of a new human ubiquitin-specific protease, UBP43, and suggest that this gene may also be related to DiGeorge syndrome.     

A human gene similar to Drosophila melanogaster peanut maps to the DiGeorge syndrome region of 22q11

A Drosophila-related expressed sequence tag (DRES) with sequence similarity to the peanut gene has previously been localized to human chromosome 22q11. We have isolated the cDNA corresponding to this DRES and show that it is a novel member of the family of septin genes, which encode proteins with GTPase activity thought to interact during cytokinesis. The predicted protein has P-loop nucleotide binding and GTPase motifs. The gene, which we call PNUTL1, maps to the region of 22q11.2 frequently deleted in DiGeorge and velo-cardio-facial syndromes and is particularly highly expressed in the brain. The mouse homologue, Pnutl1, maps to MMU16 adding to the growing number of genes from the DiGeorge syndrome region that map to this chromosome.     

Human CABIN1 is a functional member of the human HIRA/UBN1/ASF1a histone H3.3 chaperone complex

The mammalian HIRA/UBN1/ASF1a complex is a histone chaperone complex that is conserved from yeast (Saccharomyces cerevisiae) to humans. This complex preferentially deposits the histone variant H3.3 into chromatin in a DNA replication-independent manner and is implicated in diverse chromatin regulatory events from gene activation to heterochromatinization. In yeast, the orthologous complex consists of three Hir proteins (Hir1p, Hir2p, and Hir3p), Hpc2p, and Asf1p. Yeast Hir3p has weak homology to CABIN1, a fourth member of the human complex, suggesting that Hir3p and CABIN1 may be orthologs. Here we show that HIRA and CABIN1 interact at ectopic and endogenous levels of expression in cells, and we isolate the quaternary HIRA/UBN1/CABIN1/ASF1a (HUCA) complex, assembled from recombinant proteins. Mutational analyses support the view that HIRA acts as a scaffold to bring together UBN1, ASF1a, and CABIN1 into a quaternary complex. We show that, like HIRA, UBN1, and ASF1a, CABIN1 is involved in heterochromatinization of the genome of senescent human cells. Moreover, in proliferating cells, HIRA and CABIN1 regulate overlapping sets of genes, and these genes are enriched in the histone variant H3.3. In sum, these data demonstrate that CABIN1 is a functional member of the human HUCA complex and so is the likely ortholog of yeast Hir3p.     

Identification of an ubinuclein 1 region required for stability and function of the human HIRA/UBN1/CABIN1/ASF1a histone H3.3 chaperone complex

The mammalian HIRA/UBN1/CABIN1/ASF1a (HUCA) histone chaperone complex deposits the histone H3 variant H3.3 into chromatin and is linked to gene activation, repression, and chromatin assembly in diverse cell contexts. We recently reported that a short N-terminal fragment of UBN1 containing amino acids 1-175 is necessary and sufficient for interaction with the WD repeats of HIRA and attributed this interaction to a region from residues 120-175 that is highly conserved with the yeast ortholog Hpc2 and so termed the HRD for Hpc2-related domain. In this report, through a more comprehensive and refined biochemical and mutational analysis, we identify a smaller and more moderately conserved region within residues 41-77 of UBN1, which we term the NHRD, that is essential for interaction with the HIRA WD repeats; we further demonstrate that the HRD is dispensable for this interaction. We employ analytical ultracentrifugation studies to demonstrate that the NHRD of UBN1 and the WD repeats of HIRA form a tight 1:1 complex with a dissociation constant in the nanomolar range. Mutagenesis experiments identify several key residues in the NHRD that are required for interaction with the HIRA WD repeat domain, stability of the HUCA complex in vitro and in vivo, and changes in chromatin organization in primary human cells. Together, these studies implicate the NHRD domain of UBN1 as being an essential region for HIRA interaction and chromatin organization by the HUCA complex.     

GNB1L, a gene deleted in the critical region for DiGeorge syndrome on 22q11, encodes a G-protein beta-subunit-like polypeptide

CATCH 22 syndromes, which include DiGeorge syndrome and Velocardiofacial syndrome, are the most common cause of congenital heart disease which involve microdeletion of 22q11. Using a strategy including EST searching, PCR amplification and 5'-RACE, we have cloned a 1487 bp cDNA fragment from human heart cDNA library. The cloned GNB1L cDNA encodes a G-protein beta-subunit-like polypeptide, and the GNB1L gene is located in the critical region for DiGeorge syndrome. A comparison of GNB1L cDNA sequence with corresponding genomic DNA sequence revealed that this gene consists of seven exons and spans an approximately 60 kb genomic region. Northern blot analysis revealed GNB1L is highly expressed in the heart.     

Amino- and carboxy-terminal domains of the yeast Rab escort protein are both required for binding of Ypt small G proteins.

The Rab escort protein (REP) is an essential component of the heterotrimeric enzyme Rab geranylgeranyl transferase that modifies the carboxy-terminal cysteines of the Ras-like small G proteins belonging to the Rab/Ypt family. Deletions in the human CHM locus, encoding one of the two REPs known in humans, result in a retinal degenerative syndrome called choroideremia. The only known yeast homologue of the choroideremia gene product is encoded by an essential gene called MRS6. Besides three structurally conserved regions (SCRs) previously detected in the amino-terminal half of REPs and RabGDIs, three other regions in the carboxy-terminal domain (RCR 1-3) are here identified as being characteristic of REPs alone. We have performed the first mutational analysis of a REP protein to experimentally define the regions functionally important for Rab/Ypt protein binding, making use of the genetic system of the yeast Saccharomyces cerevisiae. This analysis has shown that the SCRs are necessary but not sufficient for Ypt1p binding by the yeast REP, the carboxy-terminal region also being required.     

Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein.

The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence of a ribosomal protein gene that affects translational ambiguity in a eucaryote. We show that the yeast omnipotent suppressor SUP44 encodes the yeast ribosomal protein S4. The gene exists as a single copy without an intron. The SUP44 protein is 26% identical (54% similar) to the well-characterized E. coli S5 ram protein. SUP44 is also 59% identical (78% similar) to mouse protein LLrep3, whose function was previously unknown (D.L. Heller, K.M. Gianda, and L. Leinwand, Mol. Cell. Biol. 8:2797-2803, 1988). The SUP44 suppressor mutation occurs near a region of the protein that corresponds to the known positions of alterations in E. coli S5 ram mutations. This is the first ribosomal protein whose function and sequence have been shown to be conserved between procaryotes and eucaryotes.     

HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis

The mammalian HIRA gene encodes a histone-interacting protein whose homolog in Xenopus laevis is characterized here. In vitro, recombinant Xenopus HIRA bound purified core histones and promoted their deposition onto plasmid DNA. The Xenopus HIRA protein, tightly associated with nuclear structures in somatic cells, was found in a soluble maternal pool in early embryos. Xenopus egg extracts, known for their chromatin assembly efficiency, were specifically immunodepleted for HIRA. These depleted extracts were severely impaired in their ability to assemble nucleosomes on nonreplicated DNA, although nucleosome formation associated with DNA synthesis remained efficient. Furthermore, this defect was largely corrected by reintroduction of HIRA along with (H3-H4)(2) tetramers. We thus delineate a nucleosome assembly pathway that depends on HIRA.     

PHF5A represents a bridge protein between splicing proteins and ATP-dependent helicases and is differentially expressed during mouse spermatogenesis

PHF5A is a highly conserved protein from yeast to man, and based on studies in yeast, it was suggested that the homologous protein RDS3P in S. cerevisiae takes part in the organization of U2 snRNP particles. By using the yeast two-hybrid assay we could demonstrate that PHF5A interacted both with ATP-dependent helicases EP400 and DDX1 and with arginine-serine (RS)-rich domains of splicing factors U2AF1 and SFRS5 in mouse. Furthermore, domain interaction studies revealed that PHF5A interaction with EP400 and DDX1 is restricted to the N-terminal part of PHF5A, whereas the C-terminal region of PHF5A was found to be responsible for the association with U2AF1 and SFRS5. By using the yeast three-hybrid assay, we could further show that both EP400 and DDX1 interacted only indirectly with U2AF1 and SFRS5 proteins via the bridge protein PHF5A. The subcellular localization of a PHF5A-GFP fusion protein was predominantly observed in the nucleus and, in addition, PHF5A co-localized with both U2AF1 and SFRS5 proteins in nuclear speckles of NIH3T3 cells. Moreover, expression analyses demonstrated that PHF5A and U2AF1 gene expression coincided in spermatocytes during murine spermatogenesis and interaction between these proteins was also detectable in the spermatocyte-specific cell line GC-4spc by using in vivo co-immunoprecipitation studies. Taken together, our results indicate that PHF5A resembles a protein which interacts with splicing factors U2AF1 and SFRS5 and helicases EP400 and DDX1 and functions as a bridge protein between these proteins.