The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males

Development ◽  
1999 ◽  
Vol 126 (13) ◽  
pp. 2841-2853 ◽  
Author(s):  
G. Deshpande ◽  
G. Calhoun ◽  
P.D. Schedl
Keyword(s):  
Fusion Protein ◽  
Rna Binding ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Terminal Domain ◽  
Binding Domains ◽  
Transcriptional Regulatory ◽  
Dominant Negative Activity ◽  
Female Specific

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.

In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1617-1630
Author(s):  
Suk-Won Jin ◽  
Nancy Arno ◽  
Adam Cohen ◽  
Amy Shah ◽  
Qijin Xu ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Germ Cell ◽  
Rna Binding ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Biochemical Tests ◽  
Cell Fates ◽  
Cytoplasmic Polyadenylation ◽  
Binding Domains ◽  
Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

Identification of the functional domain of p21WAF1/CIP1 that protects cells from cisplatin cytotoxicity

2005 ◽  
Vol 289 (3) ◽  
pp. F514-F520 ◽  
Author(s):  
Fang Yu ◽  
Judit Megyesi ◽  
Robert L. Safirstein ◽  
Peter M. Price
Keyword(s):  
Dominant Negative ◽  
Nuclear Antigen ◽  
Functional Domain ◽  
Binding Domains ◽  
Cdk2 Activity ◽  
Localization Signal ◽  
Induced Apoptosis ◽  
Proliferating Cell

The p21 cyclin-dependent kinase (cdk) inhibitor protects cells from cisplatin cytotoxicity in vivo and in vitro. However, the mechanism of protection is not known. Separate p21 domains are known to interact with several different proteins having proapoptotic functions. To investigate the mechanism of protection by p21, we have constructed adenoviruses encoding the different domains of p21. We were able to localize the protective activity to a region of 54 amino acids containing the cyclin-cdk interacting moiety. Other protein binding domains of p21, including the NH2-terminal procaspase-3 interactive region and the COOH-terminal region containing the proliferating cell nuclear antigen binding domain and the nuclear localization signal, had little protective effect on cisplatin cytotoxicity. The dependence of cisplatin cytotoxicity on cdk2 activity was also demonstrated because 1) cisplatin caused a marked increase in cdk2 activity, which was prevented by the p21 expression adenovirus, and 2) a cdk2 dominant-negative adenovirus also protected cells from cisplatin-induced apoptosis. Thus the data suggest that the mechanism of p21 protection is by direct inhibition of cdk2 activity and that cisplatin-induced apoptosis is caused by a cdk2-dependent pathway.

scholarly journals A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

scholarly journals Separable roles in vivo for the two RNA binding domains of a Drosophila A1-hnRNP homolog

RNA ◽  
1998 ◽  
Vol 4 (12) ◽  
pp. 1585-1598 ◽  
Author(s):  
KAI ZU ◽  
MARTHA L. SIKES ◽  
ANN L. BEYER
Keyword(s):  
Rna Binding ◽  
Binding Domains ◽  
Rna Binding Domains

scholarly journals Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000 ◽  
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

scholarly journals In vivo analysis of the role of aberrant histone deacetylase recruitment and RARα blockade in the pathogenesis of acute promyelocytic leukemia

2006 ◽  
Vol 203 (4) ◽  
pp. 821-828 ◽  
Author(s):  
Hiromichi Matsushita ◽  
Pier Paolo Scaglioni ◽  
Mantu Bhaumik ◽  
Eduardo M. Rego ◽  
Lu Fan Cai ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Histone Deacetylases ◽  
Retinoic Acid Receptor ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

The promyelocytic leukemia–retinoic acid receptor α (PML-RARα) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARα to inhibit RARα function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1–RARα fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARα mutants, as well as that of PML-RARα mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARα did act as a bona fide DN RARα mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARα mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML−/− and p53−/− compound mutants lends support to a model by which the RARα and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

scholarly journals Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

1999 ◽  
Vol 19 (8) ◽  
pp. 5441-5452 ◽  
Author(s):  
Sarah J. Lee ◽  
Susan J. Baserga
Keyword(s):  
Ribosomal Proteins ◽  
18S Rrna ◽  
Rna Binding ◽  
Specific Protein ◽  
Rrna Processing ◽  
Binding Domains ◽  
U3 Snorna ◽  
Genes Encoding ◽  
Protein Components

ABSTRACT The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.

scholarly journals Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas.

1994 ◽  
Vol 180 (6) ◽  
pp. 2413-2418 ◽  
Author(s):  
R P Bissonnette ◽  
A McGahon ◽  
A Mahboubi ◽  
D R Green
Keyword(s):  
T Cell ◽  
Apoptotic Cell ◽  
Tolerance Induction ◽  
Dominant Negative ◽  
Binding Partner ◽  
Dominant Negative Form ◽  
Dominant Negative Activity ◽  
Induced Apoptosis ◽  
Negative Form

T cell hybridomas respond to activation signals by undergoing apoptotic cell death, and this is likely to represent comparable events related to tolerance induction in immature and mature T cells in vivo. Previous studies using antisense oligonucleotides implicated the c-Myc protein in the phenomenon of activation-induced apoptosis. This role for c-Myc in apoptosis is now confirmed in studies using a dominant negative form of its heterodimeric binding partner, Max, which we show here inhibits activation-induced apoptosis. Further, coexpression of a reciprocally mutant Myc protein capable of forming functional heterodimers with the mutant Max can compensate for the dominant negative activity and restore activation-induced apoptosis. These results imply that Myc promotes activation-induced apoptosis by obligatory heterodimerization with Max, and therefore, by regulating gene transcription.

Signaling by the Granulocyte-Colony Stimulating Factor Receptor Involves Jak2-Dependent Phosphorylation of Gab2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2173-2173
Author(s):  
Lin Wang ◽  
Jia Xue ◽  
Seth J. Corey ◽  
Lisa J. Robinson
Keyword(s):  
Fusion Protein ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Erk Phosphorylation ◽  
Stimulating Factor

Abstract Granulocyte colony stimulating factor (G-CSF) is the major cytokine involved in neutrophil production. G-CSF has pleiotropic effects on myeloid cells, initially stimulating proliferation but later promoting differentiation. The specific signaling pathways that mediate the diverse effects of G-CSF remain incompletely understood. Recently, the scaffolding molecule Grb2-associated binder protein 2 (Gab2) was shown to play an important role in G-CSF induced myeloid differentiation (Zhu et al. Blood 2004). Ligand stimulation of the G-CSF receptor results in the rapid phosphorylation of Gab2, but the identity of the responsible kinases and the molecular events dependent on Gab2 phosphorylation remain unclear. Because Janus kinases (Jaks) play a central role in G-CSF signaling, we investigated the involvement of Jaks in G-CSF-stimulated Gab2 phosphorylation using the hematologic DT40 cell line stably transduced with the human G-CSF receptor (DT40GR). Antisense Jak1 and Jak2 constructs expressed in DT40GR cells each produced a marked reduction in their target Jak protein, but only antisense Jak2 reduced G-CSF-stimulated Gab2 phosphorylation. To determine whether Gab2 phosphorylation required Jak2 kinase activity, dominant negative Jak2 mutants lacking catalytic activity were expressed in the DT40GR cells. Expression of dominant negative Jak2 inhibited Gab2 phosphorylation in response to G-CSF. Similarly, treatment with the Jak2-selective kinase inhibitor AG490 markedly reduced G-CSF-dependent Gab2 phosphorylation. Co-immunoprecipitation studies further demonstrated a G-CSF- and Gab2 phosphorylation-dependent association of Jak2 with Gab2 in vivo, which was detectable by 30 seconds after G-CSF stimulation. To determine whether Gab2 was a direct substrate of Jak2, we performed in vitro phosphorylation studies using Gab2-GST fusion protein substrates. Jak2 immunoprecipitated from G-CSF-stimulated cells, but not from control cells, phosphorylated the Gab2 fusion protein. To identify potential Jak2 tyrosine phosphorylation sites in Gab2, we used site-directed mutagenesis to produce three Gab2 tyrosine mutants. Tyrosines 409, 452, and 476 were each replaced by phenylalanine (Y409F, Y452F, and Y476F). The Y452F and Y476F mutations of Gab2 each inhibited G-CSF-stimulated Jak2-dependent phosphorylation of Gab2, both in stably-transfected DT40GR cells and in transiently-transfected 293 cells also transduced with the G-CSF receptor. In contrast, G-CSF-stimulated Gab2 phosphorylation appeared unaffected by the Y409F mutation. We also evaluated downstream events in G-CSF signaling in cells expressing these Gab2 tyrosine- mutants. Akt and Erk phosphorylation following G-CSF stimulation was inhibited by both the Y452F and Y476F Gab2 mutations, but was unaffected by the Y409F mutation. These results suggest that Jak2 may mediate G-CSF differentiation signals through Stat-independent mechanisms.

In Vivo Analysis of the Role of C/EBPα in Acute Promyelocytic Leukemia Genesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1937-1937
Author(s):  
Barbara A.A. Santana-Lemos ◽  
Florence Guibal ◽  
Maria-Carolina Pintao ◽  
Priscila S. Scheucher ◽  
Rodrigo S. Abreu-Lima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Leucine Zipper ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Spleen Cells ◽  
Specific Subset

Abstract Acute promyelocytic leukemia (APL) is characterized by the infiltration of bone marrow (BM) and peripheral blood (PB) by leukemic cells presenting a block of differentiation at the stage of promyelocytes. At the cytogenetic level, APL is associated with the t(15;17) which causes the fusion of two genes: Retinoic Receptor α (RARα) and Promyelocytic Leukemia (PML), on chromosomes 17 and 15, respectively. C/EBPα is a leucine zipper transcription factor essential to normal granulopoiesis. Mutations in C/EBPα gene are detected in 6–10% of Acute Myeloid Leukemia (AML) cases and C/EBPα activity is down-regulated by AML1-ETO fusion protein associated with FAB M2 subtype. We decided to test whether PML-RARα interferes with C/EBPα function, thus contributing to the blockage of differentiation characteristic of APL. We generated mutant mice expressing PML-RARα and haploinsufficient for C/EBPα (PR C/EBPα+/−) by crossing hCG PML-RARα transgenic mice (PR TM) and C/EBPα+/−. Leukemia was not detected in WT (n=415) and C/EBPα+/− (n=47) mice after 800 days of follow up. In contrast, 8.2% PR TM (19/233) and 14.9% (13/87) PR C/EBPα+/− mice developed a form of leukemia that closely resembled human APL and identical to that developed by PR TM. The leukemia-free survival was significantly shorter in PR C/EBPα+/− compared to PR TM (644.3 days; 95% Confidence Interval, 95%C.I.: 586.4 – 702.2 vs 718.4 days; 95%C.I.: 689.3 – 747.5, P=0.02). Both groups presented a long latency for the development of the disease with a mean age (95%C.I.) at diagnosis of 399.9 days (184–673) and 495.8 days (215–757) in PR and PR C/EBPα+/−, respectively. PR and PR C/EBPα+/− leukemic mice presented similar WBC counts (107.1 ± 82.65x103vs 63.55 ± 57.82x103cells/ml, P=0.26), hemoglobin concentrations (10.87 ± 3.69 vs 9.92 ± 2.39 g/dl, P =0.57) and platelet counts (283.8 ± 188.7x103vs 177.8 ± 149.5x103platelets/ml, P =0.24). In both groups, the leukemic cells resembled promyelocytes, and expressed the phenotype CD11b+ Gr1+ CD34± c-Kit+, which represented 46.65 ± 26.89%; 32.72 ± 15.16% and 1.91 ± 1.42% of the spleen cells from PR; PR C/EBPα+/− and WT, respectively. In order to isolate PML-RARα leukemic cells and their normal counterparts to gene expression assessment, BM samples from WT mice (pooled from 2 mice), non leukemic PR C/EBPα+/− mice (pooled from 2 mice), and spleen cells from WT and leukemic PR C/EBPα+/− mice were first submitted to red cells lyses, stained with previously conjugated antibodies. Cells expressing CD16/32, CD11b, c-kit and CD34, but neither CD3 nor CD45/B220 were isolated. These cells presented medium to large size with a granular cytoplasm. C/EBPα and PML-RARα expression was analyzed by qRT-PCR in the sorted cells. Compared to WT cells, promyelocytes from leukemic and non leukemic PR C/EBPα+/− mice expressed significantly less C/EBPα, with the lowest levels detected in leukemic samples. As expected, PML-RARα was not detected in WT samples. Comparison between cells suspensions containing similar numbers of promyelocytes revealed that PML-RARα expression was higher in leukemic compared to non leukemic PR C/EBPα+/− mice. In conclusion, our data strongly suggest that PML-RARα fusion protein acts as a dominant negative product on C/EBPα gene expression in a specific subset of early myeloid cells and contribute to the pathogenesis of APL.

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