Modulation of alternative pre-mRNA splicing in vivo by pinin

Pre-mRNA splicing occurs in a large macromolecular RNA-protein complex called the spliceosome. The major components of the spliceosome include snRNP and SR proteins. We have previously identified an SR-like protein, pinin (pnn), which is localized not only in nuclear speckles but also at desmosomes. The nuclear localization of pnn is a dynamic process because pnn can be found not only with SR proteins in nuclear speckles but also in enlarged speckles following treatment of cells with RNA polymerase II inhibitors, DRB, and alpha-amanitin. Using adenovirus E1A and chimeric calcitonin/dhfr construct as a splicing reporter minigene in combination with cellular cotransfection, we found that pnn regulates alternative 5(') and 3(') splicing by decreasing the use of distal splice sites. Regulation of 5(') splice site choice was also observed for RNPS1, a general splicing activator that interacts with pnn in nuclear speckles. The regulatory ability of pnn in alternative 5(') splicing, however, was not dependent on RNPS1 and a pnn mutant, lacking the N-terminal 167 amino acids, behaved like a dominant negative species, inhibiting E1A splicing when applied in splicing assays. These results provide direct evidence that pnn functions as a splicing regulator which participates itself directly in splicing reaction or indirectly via other components of splicing machinery.     

Molecular characterization of two novel isoforms and a soluble form of mouse CLEC-2

CLEC-2 was first identified by sequence similarity to C-type lectin-like molecules with immune functions. Recently, human CLEC-2 has been reported as a receptor for the platelet-aggregating snake venom toxin rhodocytin and the endogenous sialoglycoprotein podoplanin. It has also been reported to facilitate the capture of HIV-1. However, investigation of mouse CLEC-2 (mCLEC-2) has little progressed after its identification. In this study, we identified two novel splicing variants of mCLEC-2 derived from omission of exon 2 and 2/4, respectively. These two variants had different expression profiles and subcellular localization from full-length mCLEC-2. Moreover, we observed that full-length mCLEC-2 could be cleaved probably by proteases sensitive to aprotinin and PMSF into a soluble form that partially existed as a disulfide-linked homodimer. The results presented here represent a further advancement toward the understanding of mCLEC-2.     

SRrp37, a novel splicing regulator located in the nuclear speckles and nucleoli,interacts with SC35 and modulates alternative pre‐mRNA splicing in vivo

We report here the identification and characterization of a novel SR‐related protein, referred to as SRrp37, based on its apparent molecular weight and subcellular location. SRrp37 was identified through a yeast two‐hybrid screen during the course of searching for proteins interacting with pNO40, a ribosomal 60S core subunit. SRrp37 exhibited two alternative spliced isoforms generated by differential usage of the translation start site with the longer one, SRrp37, initiating at first exon and the shorter, SRrp37‐2, starting from exon 2. Three distinct motifs can be discerned in the SRrp37 protein: (1) a serine–arginine (SR) dipeptide enriched domain, (2) a polyserine stretch, and (3) a potential nucleolar localization signal comprising a long array of basic amino acids. SRrp37's message was translated in tissue‐specific patterns with both isoforms expressed at comparable levels in tissues showing expression. Indirect immunofluorescence analysis with an anti‐SRrp37 antibody, as well as an experiment using myc‐tagged proteins, demonstrated that SRrp37 was localized in nucleoli and nuclear speckles. GST pull‐down assay showed that SRrp37 interacted physically with SC35. Using adenovirus E1A and chimeric calcitonin/dhfr constructs as splicing reporter minigenes, we found that SRrp37 modulated alternative 5′ and 3′ splicing in vivo. Together, SRrp37 may participate directly in splicing regulation or indirectly through interaction with SC35. Studies on this novel splicing regulator may provide new information on the intricate splicing machinery as related to the RNA metabolism involving processing of mRNA and rRNA. J. Cell. Biochem. 108: 304–314, 2009. © 2009 Wiley‐Liss, Inc.     

Identification and characterization of a novel alternative splice variant of mouse GMx33alpha/GPP34

We have cloned and characterized a novel splice variant of mouse GMx33alpha/Golgi-associated protein of 34 kDa (GPP34), hereby designated GMx33alphaV/GPP34V. This splice variant skips the second and third exons, and the resulting frame shift generates a stop codon in the fourth exon. GMx33alphaV/GPP34V is comprised of 81 amino acid residues derived from the N-terminal end of the full length protein and corresponds to approximately one-third of the full length GMx33alpha/GPP34 sequence with a calculated molecular mass of 8900. In contrast to GMx33alpha/GPP34 mRNA which is expressed at similar levels in various tissues, GMx33alphaV/GPP34V mRNA was differentially expressed when examined by RT-PCR. Compared to other tissues, skeletal muscle showed relatively strong expression of GMx33alphaV/GPP34V mRNA. This splice variant cDNA was also detected in a human cell line.     

cDNA cloning and characterization of a novel nucleolar protein.

In an initial study of anti-nuclear antibodies in the chronic inflammatory bladder disease interstitial cystitis, we reported that 7% of interstitial cystitis patients studied had autoantibodies to the nucleolus. We now report that, using an autoimmune serum from a patient with interstitial cystitis, we have identified and partially characterized a novel protein with an M(r) of approximately 55 kDa (hereafter referred to as No55) localized to the granular component of the nucleolus. No55 was initially characterized by diffuse nucleolar immunofluorescence staining in interphase cells and by Western blotting as a 55-kDa doublet on whole-cell extracts. During mitosis, No55 was associated with chromosomes and appeared in prenucleolar bodies during telophase, but it did not colocalize with p80-coilin in coiled bodies. Immunoelectron microscopy revealed that No55 was localized uniformly throughout the granular component of the nucleolus compared with a more peripheral localization of nucleolar granular component protein B23. On segregation of the nucleolus with actinomycin D, No55 remained with the granular component of the segregated nucleolus, whereas protein B23 was found predominantly in the nucleoplasm. Finally, a cDNA expression library was screened with the human autoantibody against No55, and a 2.4-kb insert was isolated, subcloned to homogeneity, and then sequenced. Analysis of this sequence showed an open reading frame of approximately 1.3 kb coding for 437 amino acids with a predicted molecular weight of 50 kDa. A search of the gene sequence database indicated homology with SC65, a rat synaptonemal complex protein. Therefore, on the basis of molecular weight, nucleolar sublocalization, response to actinomycin D, and cDNA sequence determination, No55 is a novel protein of the interphase nucleolus.     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

Identification and characterization of novel NuMA isoforms

The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.     

人端粒保护蛋白hPot1的一种新选择性剪接体的克隆及鉴定

hPot1是端粒单链结合蛋白,在维持染色体末端的稳定性中发挥着重要作用。从前列腺癌细胞C4-2中提取总RNA,以反转录得到的cDNA为模板,扩增全长的hPot1 cDNA,发现hPot1基因的一种新的剪接形式。这种新的剪接形式缺失了野生型hPot1基因的第2个外显子,并且造成了读码框架的改变,使翻译提前终止,表达出一段有45个氨基酸残基的短肽。进一步检测表明,这一hPot1 mRNA新剪接体广泛存在于多种组织来源的细胞中,提示这一剪接形式可能是细胞调控hPot1功能的一种调节机制。     

NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated α-tubulin, suggesting that it plays a role in maintaining or enhancing stability of α-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated α-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.     

Rat homolog of PinX1 is a nucleolar protein involved in the regulation of telomere length

Human PinX1 involves in regulation of telomere length. Here, we describe the function of a rat homolog of PinX1. Rat PinX1 (rPinX1) was cloned from WB-F344, a rat hepatic stem-like epithelial cell. It encodes a protein of 331 amino acids with 70% homology to human PinX1 and 91% homology to mouse. Northern analysis revealed that rPinX1 is expressed in both somatic and germ tissues, most abundantly in heart, liver and testis. Co-localization with a nucleolar protein, fibrillarin, showed that rPinX1 resides in the nucleolus. Analysis of truncated mutants revealed that an internal K,E/D region seems to be important for nucleolar localization. A stable cell line expressing rPinX1 was established in NIH3T3, a mouse-transformed embryonic fibroblast cell line, and stable cells were subcultured for more than 150 population doublings. The growth of stable rPinX1 cells slowed down at late passages, and a fraction of these cells exhibited increased size and stained positively for senescence-associated β-galactosidase. Overexpression of rPinX1 in NIH3T3 cells resulted in gradual telomere shortening over successive passages. However, the telomeric 3′ overhang was not altered by PinX1 expression. This study demonstrates that a rat homolog of human PinX1 is a nucleolar protein, and that overexpression of rPinX1 induces cellular senescence and telomere shortening, but has no effect on 3′ overhang length. The function of PinX1 in regulating telomere length is conserved in rodents, and this study may provide insight into the mechanism by which a nucleolar protein can regulate telomere length.     

Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans

The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPases in C. elegans.