scholarly journals ATPase/Helicase Activities of p68 RNA Helicase Are Required for Pre-mRNA Splicing but Not for Assembly of the Spliceosome

2005 ◽  
Vol 25 (17) ◽  
pp. 7484-7493 ◽  
Author(s):  
Chunru Lin ◽  
Liuqing Yang ◽  
Jenny J. Yang ◽  
Youliang Huang ◽  
Zhi-Ren Liu
Keyword(s):  
Functional Role ◽  
Rna Helicase ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Present Evidence ◽  
U1 Snrna ◽  
P68 Rna Helicase ◽  
Rna Unwinding

ABSTRACT We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5′ splice site (5′ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5′ss, while the mutants still interacted with the U1-5′ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5′ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.

scholarly journals p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

2002 ◽  
Vol 22 (15) ◽  
pp. 5443-5450 ◽  
Author(s):  
Zhi-Ren Liu
Keyword(s):  
Splice Site ◽  
Early Stage ◽  
Rna Helicase ◽  
Mrna Splicing ◽  
Cross Linking ◽  
Spliceosome Assembly ◽  
U1 Snrna ◽  
U1 Snrnp ◽  
P68 Rna Helicase

ABSTRACT Modulation of the interaction between U1 snRNP and the 5′ splice site (5′ss) is a key event that governs 5′ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5′ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5′ss during early stage of splicing. However, dissociation of the U1 from the 5′ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5′ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

Abstract 18382: Rna Splicing Regulated by A2bp1 is Essential for Cardiac Function in Zebrafish

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Karen S Frese ◽  
Benjamin Meder ◽  
Andreas Keller ◽  
Jan Haas ◽  
Steffen Just ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Rna Sequencing ◽  
Functional Role ◽  
Heart Function ◽  
Mrna Splicing ◽  
Complex Nature ◽  
Zebrafish Embryos ◽  
Splicing Regulator

Objective: Alternative splicing (AS) is one of the key mechanisms for the proteomic and functional diversity of eukaryotes. However, the complex nature of AS, its associated regulators and their targets are only partially understood. In the present study we investigated the transcriptomic diversity in the zebrafish heart using RNA-Sequencing and elucidated the functional role of the splicing regulator A2BP1 in vivo. Results: Using RNA-Sequencing we characterized the cardiac transcriptome of 48 hours post fertilization (hpf) old zebrafish embryos and compared the expression of genes and their isoforms to whole fish tissue. Besides the known cardiac genes, we found several previously described genes, highly expressed in cardiac tissue. The analysis of RNA-Seq data indicates that 14% of all genes expressed in the heart undergo AS by single exon-skipping/inclusion. To determine the effect of splicing factors on mRNA splicing we investigated the functional role of splicing regulator a2bp1 in vivo by using the zebrafish as a model organism. Morpholino-mediated a2bp1 knockdown in zebrafish embryos led to progressive cardiac contractile dysfunction, suggesting an important role of a2bp1 in maintenance of cardiac function. Splicing analysis revealed that loss of a2bp1 does not result in a completely splicing failure, but rather alters the splicing pattern of specific target genes. Here we identified novel spliceforms and potentialy novel targets of splicing factor a2bp1. Splice-junction blockage experiments showed that a balanced isoform expression of the targets actn3a, hug, ktn1, ptpla and camk2g is necessary for maintaining cardiac function in zebrafish. We assume, that the a2bp1-knockdown phenotype is not caused by missplicing of specific targets rather by the cumulative effect of many splicing abnormalities. Conclusion: Our study reveal a novel splicing regulator that is necessary for normal heart function. We showed that dysfunction of a2bp1 not only leads to heart failure, but show that a2bp1 mediates the splicing of different transcripts which might mediate the observed phenotype. Our results highlight the importance of balanced mRNA splicing in the heart and represents intriguing opportunities for novel therapeutic approaches.

scholarly journals Specific Role of the SR Protein Splicing Factor B52 in Cell Cycle Control in Drosophila

2006 ◽  
Vol 26 (9) ◽  
pp. 3468-3477 ◽  
Author(s):  
Vanya I. Rasheva ◽  
David Knight ◽  
Przemyslaw Bozko ◽  
Katherine Marsh ◽  
Maxim V. Frolov
Keyword(s):  
Cell Cycle Control ◽  
Mrna Splicing ◽  
Specific Role ◽  
Splicing Factor ◽  
Sr Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
Specific Regulation ◽  
Retinoblastoma Tumor

ABSTRACT E2F and retinoblastoma tumor suppressor protein pRB are important regulators of cell proliferation; however, the regulation of these proteins in vivo is not well understood. In Drosophila there are two E2F genes, an activator, de2f1, and a repressor, de2f2. The loss of de2f1 gives rise to the G1/S block accompanied by the repression of E2F-dependent transcription. These defects can be suppressed by mutation of de2f2. In this work, we show that the de2f1 mutant phenotype is rescued by the loss of the pre-mRNA splicing factor SR protein B52. Mutations in B52 restore S phase in clones of de2f1 mutant cells and phenocopy the loss of the de2f2 function. B52 acts upstream of de2f2 and plays a specific role in regulation of de2f2 pre-mRNA splicing. In B52-deficient cells, the level of dE2F2 protein is severely reduced and the expression of dE2F2-dependent genes is deregulated. Reexpression of the intronless copy of dE2F2 in B52-deficient cells restores the dE2F2-mediated repression. These results uncover a previously unrecognized role of the splicing factor in maintaining the G1/S block in vivo by specific regulation of the dE2F2 repressor function.

scholarly journals Loss of splicing factor IK impairs normal skeletal muscle development

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Hye In Ka ◽  
Hyemin Seo ◽  
Youngsook Choi ◽  
Joohee Kim ◽  
Mina Cho ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Development ◽  
Myogenic Differentiation ◽  
Mrna Splicing ◽  
C2c12 Cells ◽  
Splicing Factor ◽  
Skeletal Muscle Development

Abstract Background IK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo. Results The ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells. Conclusion This study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

scholarly journals WNT-5A regulates TGF-β-related activities in liver fibrosis

2017 ◽  
Vol 312 (3) ◽  
pp. G219-G227 ◽  
Author(s):  
Leonie Beljaars ◽  
Sara Daliri ◽  
Christa Dijkhuizen ◽  
Klaas Poelstra ◽  
Reinoud Gosens
Keyword(s):  
Liver Fibrosis ◽  
Functional Role ◽  
Collagen Type ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

scholarly journals Cathepsin G-dependent platelet stimulation by activated polymorphonuclear leukocytes and its inhibition by antiproteinases: role of P-selectin-mediated cell-cell adhesion

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2947-2957 ◽  
Author(s):  
V Evangelista ◽  
P Piccardoni ◽  
JG White ◽  
G de Gaetano ◽  
C Cerletti
Keyword(s):  
Functional Role ◽  
Polymorphonuclear Leukocytes ◽  
Platelet Adhesion ◽  
Inhibitory Effect ◽  
Serotonin Release ◽  
Cathepsin G ◽  
Inhibitory Antibody ◽  
Neuraminidase Treatment

Human PMN stimulated by fMLP are able to activate coincubated, autologous platelets. Cathepsin G, a neutral serine protease stored in the azurophilic granules of PMN, is the major platelet activator in this system. We previously proposed that shear-induced close PMN- platelet contact creates the conditions for which cathepsin G activity on platelets is protected against antiproteinases. The aim of this study was to investigate the adhesive mechanisms, possibly creating between PMN and platelet membranes the microenvironment in which cathepsin G, discharged from stimulated PMN onto adherent platelets, is protected against antiproteinases. Microscopic examination showed that under conditions of high shear, 71.3% +/- 6.1% of PMN were associated to platelets forming small clumps. This percentage decreased to 10% +/- 2% and 13% +/- 4%, respectively, in the presence of an inhibitory antibody to P-selectin or 20 mmol/L mannose-1-phosphate and to 10.8% +/- 3.7% when cells were not stirred. Similarly, PMN pretreatment with neuraminidase abolished PMN binding to platelets. These results indicate that P-selectin mediates PMN-platelet adhesion occurring before PMN stimulation. Prevention of PMN-platelet contact significantly potentiated the inhibitory effect of alpha 1-protease inhibitor on subsequent cathepsin G-induced platelet serotonin release. Because anti-P-selectin antibody, mannose-1-phosphate, and neuraminidase treatment of PMN did not modify PMN-induced platelet activation in the absence of antiproteinases, it is suggested that P- selectin-mediated PMN-platelet adhesion results in the formation of a sequestered microenvironment between cell membranes, in which higher amounts of antiproteinases are required to prevent the activity of released cathepsin G. These data add a new functional role to P- selectin-mediated PMN-platelet adhesion that could be important in vivo because of the presence of antiproteinases in plasma.

Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

2000 ◽  
Vol 279 (5) ◽  
pp. L835-L841 ◽  
Author(s):  
Olafur Baldursson ◽  
Herbert A. Berger ◽  
Michael J. Welsh
Keyword(s):  
Functional Role ◽  
Transmembrane Conductance Regulator ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Dependent Protein ◽  
Rat Thyroid ◽  
Cystic Fibrosis Transmembrane

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl− current. Mutation of four in vivo phosphorylation sites, Ser660, Ser737, Ser795, and Ser813 (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser660 or Ser813 alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser660 nor Ser813 alone increased current to wild-type levels; both residues were required. Changing Ser737 to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser737 may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

scholarly journals Integrin α10, a Novel Therapeutic Target in Glioblastoma, Regulates Cell Migration, Proliferation, and Survival

Cancers ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 587 ◽  
Author(s):  
Matilda Munksgaard Thorén ◽  
Katarzyna Chmielarska Masoumi ◽  
Cecilia Krona ◽  
Xiaoli Huang ◽  
Soumi Kundu ◽  
...  
Keyword(s):  
Cell Death ◽  
Therapeutic Target ◽  
Functional Role ◽  
Treatment Strategies ◽  
Antibody Drug Conjugate ◽  
Potential Therapeutic Target ◽  
Sphere Formation

New, effective treatment strategies for glioblastomas (GBMs), the most malignant and invasive brain tumors in adults, are highly needed. In this study, we investigated the potential of integrin α10β1 as a therapeutic target in GBMs. Expression levels and the role of integrin α10β1 were studied in patient-derived GBM tissues and cell lines. The effect of an antibody–drug conjugate (ADC), an integrin α10 antibody conjugated to saporin, on GBM cells and in a xenograft mouse model was studied. We found that integrin α10β1 was strongly expressed in both GBM tissues and cells, whereas morphologically unaffected brain tissues showed only minor expression. Partial or no overlap was seen with integrins α3, α6, and α7, known to be expressed in GBM. Further analysis of a subpopulation of GBM cells selected for high integrin α10 expression demonstrated increased proliferation and sphere formation. Additionally, siRNA-mediated knockdown of integrin α10 in GBM cells led to decreased migration and increased cell death. Furthermore, the ADC reduced viability and sphere formation of GBM cells and induced cell death both in vitro and in vivo. Our results demonstrate that integrin α10β1 has a functional role in GBM cells and is a novel, potential therapeutic target for the treatment of GBM.

Identifying the Functional Role of Martinotti Cells in Cortical Sensory Processing

2009 ◽  
Vol 102 (1) ◽  
pp. 9-11 ◽  
Author(s):  
James C. H. Cottam
Keyword(s):  
Sensory Processing ◽  
Pyramidal Cell ◽  
Functional Significance ◽  
Functional Role ◽  
Neural Computation ◽  
Inhibitory Interneurons ◽  
Local Circuit ◽  
Processing Function

Inhibitory interneurons are highly diverse, although the functional significance of their diversity is not yet well understood. This presents a barrier to understanding neural computation at the local circuit level. This review focuses on a recent study by Murayama et al. who used a novel in vivo technique in neocortex to demonstrate a specific sensory processing function of dendritic-targeting Martinotti interneurons. The function of Martinotti cells arises from their interaction with layer 5 pyramidal cell dendrites.

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