scholarly journals Testis-specific peroxiredoxin 4 variant is not absolutely required for spermatogenesis and fertility in mice

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takujiro Homma ◽  
Toshihiro Kurahashi ◽  
Naoki Ishii ◽  
Nobuyuki Shirasawa ◽  
Junichi Fujii
Keyword(s):  
Male Mice ◽  
Normal Numbers ◽  
Double Knockout ◽  
Exon 2 ◽  
Protein Levels ◽  
Oxidative Protein Folding ◽  
Upstream Promoter ◽  
Exon 1 ◽  
Normal Spermatogenesis

Abstract PRDX4, a member of peroxiredoxin family, is largely concentrated in the endoplasmic reticulum (ER) and plays a pivotal role in the redox relay during oxidative protein folding as well as in peroxidase reactions. A testis-specific PRDX4 variant transcript (PRDX4t) lacks the conventional exon 1, which encodes the signal peptide that is required for entry into the ER lumen, but instead carries alternative exon 1, which is transcribed from the upstream promoter in a testis-specific manner and results in the PRDX4t protein being localized in the cytosol. However, the potential roles of PRDX4t in male genital action remain unknown. Using a CRISPR/Cas9 system, we first disrupted the testis-specific promoter/exon 1 and generated mice that were specifically deficient in PRDX4t. The resulting PRDX4t knockout (KO) mice underwent normal spermatogenesis and showed no overt abnormalities in the testis. Mating PRDX4t KO male mice with wild-type (WT) female mice produced normal numbers of offspring, indicating that a PRDX4t deficiency alone had no effect on fertility in the male mice. We then generated mice lacking both PRDX4 and PRDX4t by disrupting exon 2, which is communal to these variants. The resulting double knockout (DKO) mice were again fertile, and mature sperm isolated from the epididymis of DKO mice exhibited a normal fertilizing ability in vitro. In the meantime, the protein levels of glutathione peroxidase 4 (GPX4), which plays an essential role in the disulfide bond formation during spermatogenesis, were significantly increased in the testis and caput epididymis of the DKO mice compared with the WT mice. Based on these results, we conclude that the disruption of the function of PRDX4t in the spermatogenic process appears to be compensated by other factors including GPX4.

Effect of 5' splice site mutations on splicing of the preceding intron

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

In-Vitro Cytotoxicity of Tipifarnib (Zarnestra TM) in Pediatric AML and ALL Samples Is Independent of RAS Mutations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1172-1172 ◽  
Author(s):  
Bianca F. Goemans ◽  
Christian M. Zwaan ◽  
Gertjan J.L. Kaspers ◽  
Karel Hählen ◽  
Dirk Reinhardt ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Direct Sequencing ◽  
Farnesyltransferase Inhibitors ◽  
Exon 2 ◽  
Ras Mutations ◽  
Lc50 Value ◽  
Significant Difference ◽  
Exon 1 ◽  
Pediatric Aml

Abstract The farnesyltransferase inhibitor tipifarnib (Zarnestra™) was originally developed to target malignancies harbouring RAS mutations. In the first clinical studies with tipifarnib, in adults with leukemia, it was found that patients who responded did not harbour any RAS mutations, suggesting a different mechanism of response. In a previous study we showed that 18% of 150 untreated pediatric AML patients harbour mutations in RAS, of which 30% were CBF-AML. We now studied 44 untreated and 13 relapsed pediatric AML, as well as 22 untreated ALL samples for mutations in RAS, using D-HPLC and direct sequencing. In vitro tipifarnib resistance was determined by a 4-day MTT assay (concentration 0.016-51μM, kindly provided by Janssen Research). The LC50 value, the concentration at which 50% of cells are killed by tipifarnib, was used as a measure of resistance. Patient characteristics were; for untreated AML: 64% boys; median age 9.3 years; median WBC 74.8x109/L; FAB 2xM0, 2xM1, 8xM2, 3xM3, 16xM4, 8xM5, 5x unclassified; for relapsed AML: 77% boys; median age 4.0 years; median WBC 41.6x109/L; FAB 2xM0, 2xM2, 3xM4, 2xM5, 2xM7, 2x unclassified; for untreated ALL: 73%boys; median age 6.0 years; median WBC 10.2x109/L; 15 B-cell precursor (BCP) ALL and 7 T-ALL. We found RAS mutations in 14 (32%) untreated AML samples (N-RAS : 8 samples exon 1, 1 sample exon 2; K-RAS: 5 samples exon 1 mutations). In relapsed AML 2 samples showed an N-RAS exon 1 mutation (15.4%). In ALL 18.2% had a RAS mutation: an N-RAS exon 1 mutation was found in 2 patients (9.1%) and a K-RAS exon 1 mutation in another 2 patients (9.1%). The distribution of tipifarnib sensitivity was similar in RAS mutated- and non-mutated untreated AML patients [median LC50 RAS mutated 7.1μM (P25-P75: 6.0-9.6μM) vs. non-mutated 4.9μM (P25-P75 2.3-8.2μM); p=0.199]. When we compared N-RAS mutated samples with K-RAS mutated samples there was no statistically significant difference in sensitivity to tipifarnib (median LC50 [p25-p75] 3.2μM [2.9-3.9μM] and 4.9μM [3.7-23.1μM], p=0.20), and comparing them separately with non-mutated AML did not show differences in sensitivity to tipifarnib (p=0.172 and p=0.463 respectively). One out of 9 (11%) N-RAS mutated and 3 out of 5 (60%) K-RAS mutated samples had an LC50 value above the 75th percentile for non-mutated AML and were considered resistant. Within relapsed AML the 2 RAS mutated samples had LC50 values of 0.83 and 6.3μM, versus a median value of 6.9μM for non-mutated relapsed AML. In ALL, we found similar results [median LC50 RAS mutated 7.8μM (P25-75: 4.1-12.8μM) vs. non-mutated 17.4μM (P25-75: 4.5-22.9μM), p=0.3], but the groups were very small. In conclusion, primary pediatric AML and ALL samples withRAS mutations show similar distributions of tipifarnib sensitivity as samples withoutRAS mutations. Hence, some RAS mutated samples may be relatively in-vitro resistant to tipifarnib, and some non-mutated samples may be relatively sensitive. Therefore, clinical studies with these compounds should not be restricted to RAS-mutated leukemia. Further studies are necessary to determine the molecular targets of farnesyltransferase inhibitors.

scholarly journals Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.

1988 ◽  
Vol 8 (2) ◽  
pp. 814-821 ◽  
Author(s):  
M Zillmann ◽  
M L Zapp ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Splice Junction ◽  
Rnase H ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
Complementary Oligonucleotide ◽  
Exon 1 ◽  
Precursor Rna ◽  
Splice Junctions

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.

Transcription initiation of the rat insulin-like growth factor-I gene in hepatocyte primary culture

1996 ◽  
Vol 151 (2) ◽  
pp. 215-223 ◽  
Author(s):  
A Y Krishna ◽  
C-I Pao ◽  
P M Thulé ◽  
B C Villafuerte ◽  
L S Phillips
Keyword(s):  
Transcription Initiation ◽  
Rat Hepatocytes ◽  
Regulatory Mechanisms ◽  
Exon 2 ◽  
Factor I ◽  
Igf I ◽  
Exon 1 ◽  
Normal Rat

Abstract Transcription initiation in the insulin-like growth factor-I (IGF-I) gene is complex, involving multiple sites in two exons. While most transcripts are initiated in exon 1 in vivo, critical regulatory mechanisms are difficult to assess in intact animals. To examine the impact of insulin and growth hormone (GH) under more controlled conditions, we have studied the utilization of different exon 1 and exon 2 transcription-initiation sites in normal rat hepatocytes in primary culture. Normal rat hepatocytes were cultured for 48 h in serum-free medium, with insulin at 10−6 or 10−11 m, and with or without human GH 200 ng/ml. Relative abundance of IGF-I transcripts was evaluated by the RNase-protection assay, using a probe which permitted identification of initiation in exon 1 (site 1 (−380 bp from the 3′ end of exon 1), site 2 (−343 bp), site 3 (−242 bp), sites 1 and 2 spliced, and site 4 (−32 bp)), as well as in exon 2. After normalization of signal intensity to adjust for differences in length of protected probe, the utilization of initiation sites in vitro was remarkably similar to that in vivo: 1, 14, 6, 23, 19 and 37% for sites 1, 2, 3, 1 and 2 spliced, 4 and exon 2 respectively in the cultured hepatocytes, compared with 1, 12, 8, 21, 18 and 40% for these sites in normal liver. Insulin alone increased transcripts initiated from exon 1, site 2 by over 3 times, and both sites 1 and 2 spliced and exon 2 transcripts by about 5 times. GH alone had similar effects, producing a 4–5 times increase in transcripts from these initiation sites. Addition of both insulin and GH had additive effects, increasing transcripts from exon 1, sites 2, 3 and 4 by 4–6 times, and from exon 1, sites 1 and 2 spliced, and exon 2 by over 8 times. Of the total IGF-I mRNA transcripts, 37% were initiated from sites 2 and/or sites 1 and 2 spliced, and 37% from exon 2. Analysis of the relative contribution of individual initiation sites revealed hormone-induced increases which were statistically significant only for exon 2, in the presence of insulin alone and in combination with GH. In conclusion, in cultured hepatocytes, insulin or GH alone produced a coordinated increase in all exon 1 transcripts, and the effect of the combination of insulin and GH was additive for these transcripts. Exon 2 appeared to be more sensitive to insulin alone, and to GH in the presence of insulin, than exon 1. Since utilization of initiation sites in hepatocytes mimics that found in liver, this in vitro system should be useful for examining underlying transcriptional regulatory mechanisms. Journal of Endocrinology (1996) 151, 215–223

scholarly journals Effect of 5' splice site mutations on splicing of the preceding intron.

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305 ◽  
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

Generation of CD4+ Regulatory T Cells by Retroviral Transduction of CD4+CD25− T Cells Either with the Full-Length or with a Common Exon-2 Negative Variant of Human Foxp3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3315-3315
Author(s):  
Tuna Mutis ◽  
Leo F. Verdonck ◽  
Tineke Aarts-Riemens ◽  
Maarten Emmelot
Keyword(s):  
T Cells ◽  
Marker Gene ◽  
Retroviral Vectors ◽  
Treg Cells ◽  
Full Length ◽  
Double Knockout ◽  
Exon 2 ◽  
Foxp3 Gene ◽  
Expression Levels

Abstract The forkhead/winged helix transcription factor, Foxp3 is a key element for the differentiation of CD4+CD25+ regulatory T (Treg) cells. While murine Foxp3 gene is expressed as a single full-length transcript, we observed that transcription of human Foxp3 gene usually reveals two different mRNA products with ~ 100 base pair difference. Sequencing of these products revealed that one transcript represents the full-length Foxp3, while the other transcript appears to be an alternative splicing product that lacks the exon-2. Using a specific primer set that amplifies the region between exon-1 and exon-3 we found that both exon-2pos and exon-2neg variants are preferentially expressed in CD4+CD25+ cells with high levels of expression in CD4+CD25hi cells. Analysis of individual Treg clones generated by limiting dilution of CD4+CD25hi cells revealed that both Foxp3 variants are simultaneously expressed in Treg clones. However, quantitative-real time PCR analyses, performed using a primer set that amplifies only the full-length Foxp3 and another set that amplifies both exon-2neg and exon-2pos variants, indicated that expression levels of the full-length Foxp3 gene do not correlate with expression levels of total Foxp3 in individual Treg clones, in PBMC of healthy donors and in leukemia patients after donor lymphocyte infusions. Remarkably, some donors expressed little or no full-length Foxp3 while expressing considerable amounts of total Foxp3, suggesting a predominant expression of the exon-2neg variant in CD4+CD25+ T cells of these donors. To determine the role of individual Foxp3 isoforms in Treg cell-differentiation, the full-length and the exon-2neg variants of the Foxp3 gene were cloned into retroviral vectors and transduced separately into highly purified CD4+CD25− cells. After a brief in vitro culture, T cells transduced with the Foxp3 isoforms were sorted to high purity by flow cytometry using the GFP marker gene and expanded to high quantities by nonspecific stimulation with allogeneic feeder cells, PHA and IL-2. Phenotypical and functional analyses of expanded cells revealed that T cells transduced with both Foxp3 variants expressed high levels of CD25, intacellular CTLA-4 and CD62L; were anergic to stimulation via CD3 and suppressed the CD3/CD28 triggered proliferation of autologous and allogeneic CD4+CD25− cells. These results reveal that both isoforms of human Foxp3 are functionally intact and can convert CD4+CD25− cells into Treg cells. Current efforts are focused on testing the in vivo capacity of in vitro expanded, Foxp3 transduced T cells to prevent Graft versus Host Disease (GvHD) in a xenogeneic model where GvHD is induced by administration of human T cells in Rag2, gamma chain double knockout mice.

scholarly journals Effect of training and growth hormone suppression on insulin-like growth factor I mRNA in young rats

1994 ◽  
Vol 76 (5) ◽  
pp. 2204-2209 ◽  
Author(s):  
S. Zanconato ◽  
D. Y. Moromisato ◽  
M. Y. Moromisato ◽  
J. Woods ◽  
J. A. Brasel ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Exercise Training ◽  
Insulin Like Growth Factor ◽  
Exon 2 ◽  
Protein Levels ◽  
Factor I ◽  
Igf I ◽  
Exon 1 ◽  
Growth Factor I

The growth hormone (GH)-insulin-like growth factor I (IGF-I) axis plays a role in the adaptation to exercise training, but IGF-I gene expression in response to exercise training and GH suppression has not been studied. Twenty female rates underwent a 4-wk treadmill training program begun in the prepubertal period (day 14 of life). In 10 of the training rats, GH production was suppressed by anti-GH-releasing hormone antibodies (GH suppressed). IGF-I mRNA and protein levels were measured in liver and hindlimb skeletal muscle. GH suppression reduced IGF-I mRNA expression in the liver to a much greater extent than in the muscle. In the GH control rats, training induced significant increases in hepatic exon 1-derived IGF-I mRNA (mean increase 30%; P < 0.05) and muscle exon 2-derived mRNA (mean increase 35%; P < 0.05). In the GH-suppressed rats, only muscle exon 1-derived transcripts were significantly increased by training (55%; P < 0.05) and this was associated with a significant increase in muscle IGF-I protein levels (P < 0.05). We speculate that the anabolic response to training may involve both GH-dependent increases in IGF-I mRNA in the liver and GH-independent increases in the muscle.

Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles

1988 ◽  
Vol 8 (2) ◽  
pp. 814-821
Author(s):  
M Zillmann ◽  
M L Zapp ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Splice Junction ◽  
Rnase H ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
Complementary Oligonucleotide ◽  
Exon 1 ◽  
Precursor Rna ◽  
Splice Junctions

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.

scholarly journals LIM domain proteins Pinch1/2 regulate chondrogenesis and bone mass in mice

Bone Research ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Yiming Lei ◽  
Xuekun Fu ◽  
Pengyu Li ◽  
Sixiong Lin ◽  
Qinnan Yan ◽  
...  
Keyword(s):  
Bone Mass ◽  
Adipogenic Differentiation ◽  
Mutant Mice ◽  
Lim Domain ◽  
Double Knockout ◽  
Single Mutant ◽  
Protein Levels ◽  
Proliferative Zone ◽  
Column Formation

Abstract The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell–extracellular matrix interaction and adhesion. Here, we report that deleting Pinch1 in limb mesenchymal stem cells (MSCs) and Pinch2 globally (double knockout; dKO) in mice causes severe chondrodysplasia, while single mutant mice do not display marked defects. Pinch deletion decreases chondrocyte proliferation, accelerates cell differentiation and disrupts column formation. Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone (PZ) chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone (HZ) chondrocytes. Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes, reduces bone formation, and increases bone resorption, leading to low bone mass. In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes. Through its C-terminal region, Pinch1 interacts with Smad2/3 proteins. Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells (BMSCs). Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs. Interestingly, compared to those from single mutant mice, BMSCs from dKO mice express dramatically lower protein levels of β-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity. Finally, ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb shortening. Collectively, our findings demonstrate critical roles for Pinch1/2 and a functional redundancy of both factors in the control of chondrogenesis and bone mass through distinct mechanisms.

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