Critical Role of Pcid2 in B Cell Survival through the Regulation of MAD2 Expression

Abstract

MaterialDNA damage occurring during proliferation is detected by a sensor mechanism, and the subsequent cell cycling is regulated by checkpoint proteins. Mitotic checkpoint proteins, such as MAD2, MAD1, and BUBR1, strictly regulate the cell-cycle progression of DNA-damaged cells by arresting kinetochore tension during mitosis (10). Thus, the expression of mitotic checkpoint proteins might be altered according to the various B cell differentiation stages.The GC-associated nuclear protein GANP is homologous to Saccharomyces SAC3, which is a component of the ribonucleoprotein Thp1/SAC3 complex required for mRNA export in yeast cells (11). The lack of mammalian GANP does not affect the expression levels of cell-cycle-associated proteins, such as MAD2, BUBR1, and cohesin, but selectively impairs Shugoshin-1 expression, which is required to protect cohesion during mitosis (12), suggesting that the expression of mammalian mRNAs, particularly mRNAs essential for genomic stability, is selectively regulated during accelerated cell proliferation. Mammalian Pcid2 is a homolog of yeast Thp1 based on structural similarity with PAM (proteasome, COP9, initiation factor/PINT-associated module) and PINT (proteasome, Int-6, Nip-1, and TRIP-15)-like regions (13). Saccharomyces Thp1 is associated with RNAs as a ribonucleoprotein complex and is recruited to the nuclear pore docking proteins with SAC3 (14). We hypothesized that Pcid2 interacts with ribonucleoprotein complex in a manner similar to GANP and plays a role in B cell proliferation or survival.The findings of this study demonstrated that mammalian Pcid2 was selectively involved in the export of MAD2 mRNA and regulated the mitotic checkpoint function during cell proliferation. Pcid2 was expressed at high levels in pre-B, immature B, T1, and Materials and Methods Cell surface staining and sortingLymphoid cells from the bone marrow and spleen were stained by FITCconjugated mAb, PE-conjugated mAb, and biotin-labeled mAb with PerCPconjugated streptavidin (GE Healthcare, Buckinghamshire, U.K.), and analyzed by FACSCalibur (BD Biosciences, Mountain View, CA) using FlowJo software (Tree Star, Ashland, OR). mAbs were obtained from eBioscience (San Diego, CA) for B220, CD21, CD23, CD24, IgM, IgD, and CD93 (AA4.1). Anti-CD43 mAb was purchased from BD Biosciences. B220 + cells enriched by MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) were purified using a JSAN cell sorter (BayBioscience, Kobe, Japan) (15). For each fraction, 1.5 3 10 5 cells were collected with the purity of .95% by postsorting analysis. Real-time RT-PCRReal-time RT-PCR was performed using a LightCycler (Roche Molecular Biochemicals, Basel, Switzerland) with cDNAs after RT reaction using total RNA, oligo-dT primer (Invitrogen, Carlsbad, CA), and SuperScript III (Invitrogen). The data are presented as the relative ratio compared with control GAPDH. Specific oligonucleotide primers and probes of hPcid2, hMAD2, mPcid2, mMAD2, and GAPDH were purchased from Nihon Gene Research Laboratories (Sendai, Japan) (Supple...

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“…Knockdown of PolQ expression suppresses the DNA repair mechanism (46). Interestingly in our study, we witnessed a remarkable down-regulation of PolQ (47) which is involved in recruiting mitotic checkpoint proteins and regulating mitosis during DNA damage (48). In our study PCID2 was highly down-regulated which can be correlated to the inability of cells to overcome cell cycle arrest in the mitotic phase.…”

Pcid2 Inactivates Developmental Genes in Human and Mouse Embryonic Stem Cells to Sustain Their Pluripotency by Modulation of EID1 Stability

“…Pcid2 flox/flox mice were generated by homologous recombination as described . Pcid2 + /− mice were produced by crossing Pcid2 flox/flox mice with EIIa‐Cre transgenic mice.…”

“…Saccharomyces Thp1 is present in the ribonucleoprotein complex to participate in export of mRNAs from a nucleus to a cytoplasm . We recently reported that murine Pcid2 is selectively involved in the transport of MAD2 mRNA that regulates the mitotic checkpoint during B‐cell proliferation . A genome‐scale RNA interference (RNAi) screen identified Pcid2 as one of the genes that modulate self‐renewal of mouse ESCs (mESCs) .…”

Normal and malignant megakaryopoiesis

“…Genes found to be required for spindle checkpoint control include the kinases BUB1 and BUBR1, the WD40 repeat containing protein BUB3, which directs the localization of the complex to kinetochores, and the APC inhibitors MAD2/CDC20. For example, loss-of-function of BUB1 , BUB3 and MAD2 results in increased polyploidy (32, 33). Moreover, although homozygous loss of Bubr1 leads to embryonic lethality, Bubr1 heterozygous mutant mice displayed splenomegaly and extramedullary megakaryopoiesis with a marked increase in polyploidization (34).…”