Functional and Clinical Relevance of Splicing Factor SRSF1 in Multiple Myeloma (MM)

Abstract The mechanisms modifying expression and function of genes, such as alternative pre-mRNA splicing or microRNA (miRNA) activity, need to be considered in order to provide a more accurate genomic framework for clinical correlation, as well as for high value therapeutic target discovery. Aberrant splicing of numerous genes has been reported in other malignancies, including a small number of genes reported in MM. The emerging focus therefore, has been to understand the molecular mechanisms driving alternate splicing. Several studies provide evidence that an abnormally expressed splicing factor can have oncogenic properties by impacting alternative splicing of cancer-associated genes. Based on our previously described significant role of E2F1 and its heterodimerization partner Dp1 in myeloma, we evaluated their chromatin binding targets related with splicing factors. Using genome wide chromatin and transcription landscape mapping techniques, we observed both transcription factors bound to the promoter of several splicing factors including Serine Arginine-Rich Splicing Factor 1 (SRSF1). Further evaluation in our gene expression profile from 170 newly-diagnosed myeloma patients, identified high expression of SRSF1 in MM compared to normal plasma cells, with a significant difference in the non-hyperdiploid subtype. Importantly, we have observed significant coexpression of Dp1 and SRSF1 in this dataset suggesting a mechanism for SRSF1 upregulation in tumors with elevated Dp1. SRSF1 has been shown to be overexpressed in human tumors with elevated Myc. No correlation between Myc with SRSF1 was observed in myeloma suggesting that SRSF1 expression is mediated by Myc-independent mechanism. We next explored the functional role of SRSF1 in MM. In gain-of-function experiments, enforced expression of SRSF1-eGFP in MM1S significantly increased proliferation. Conversely, downregulation of SRSF1 with specific shRNAs in MM cell lines significantly inhibited MM cell proliferation and cell survival. These data underscore the oncogenic potential of SRSF1 in MM. A significant reduction in SRSF1 at mRNA and protein levels was observed after E2F1 and/or Dp1 gene silencing. Moreover, peptide-based strategy to abrogate interaction between Dp1-E2F1 led to decreased SRSF1 expression levels. Importantly, the increased expression of SRSF1 was linked with overall survival in 2 independent MM datasets, highlighting for the first time the clinical relevance of splicing related factors in myeloma. In conclusion our results indicate a functional role and clinical significance of a gene involved in regulation of gene splicing. This study highlights the need to further understand the splicing pattern in myeloma and also supports the emerging concept that splicing programs, together with transcriptional programs participate in the altered cellular function during tumor initiation and progression. Disclosures Anderson: Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.

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BAG3 contributes to HGF-mediated cell proliferation, migration, and invasion via the Egr1 pathway in gastric cancer

Tumori Journal ◽

10.1177/0300891618811274 ◽

2018 ◽

Vol 105 (1) ◽

pp. 63-75

Author(s):

Jae Chang Lee ◽

Sung Ae Koh ◽

Kyung Hee Lee ◽

Jae-Ryong Kim

Keyword(s):

Gastric Cancer ◽

Cell Proliferation ◽

Cell Invasion ◽

Molecular Mechanisms ◽

Migration And Invasion ◽

Human Gastric Cancer ◽

Protein Levels ◽

Dependent Pathway

Introduction: Bcl2-associated athanogene 3 (BAG3) is elevated in several types of cancers. However, the role of BAG3 in progression of gastric cancer is unknown. Therefore, the present study aims to find out the role of BAG3 in hepatocyte growth factor (HGF)–mediated tumor progression and the molecular mechanisms by which HGF regulates BAG3 expression. Methods: BAG3 mRNA and protein were measured using reverse transcription polymerase chain reaction and Western blot in the 2 human gastric cancer cell lines, NUGC3 and MKN28, treated with or without HGF. The effects of BAG3 knockdown on cell proliferation, cell invasion, and apoptosis were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the in vitro 2-chamber invasion assay, and flow cytometry in BAG3 short hairpin RNA (shRNA)–transfected cells and control cells. The signaling pathways involved in BAG3 that are regulated by HGF were analyzed. The chromatin immunoprecipitation assay was used to determine binding of Egr1 to the BAG3 promoter. Results: BAG3 mRNA and protein levels were increased following treatment with HGF. HGF-mediated BAG3 upregulation increased cell proliferation and cell invasion; however, it decreased apoptosis. HGF-mediated BAG3 upregulation is regulated by an ERK and Egr1-dependent pathway. BAG3 may have an important role in HGF-mediated cell proliferation and metastasis in gastric cancer through an ERK and Egr1-dependent pathway. Conclusion: This pathway may provide novel therapeutic targets and provide information for further identification of other targets of therapeutic significance in gastric cancer.

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Nox4 Mediates RANKL-induced ER-Phagy and Osteoclastogenesis via Activating ROS/PERK/eIF-2α/ATF4 Pathway

10.21203/rs.3.rs-34288/v1 ◽

2020 ◽

Author(s):

Jing Sun ◽

wugui chen ◽

Songtao Li ◽

Sizhen Yang ◽

Ying Zhang ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Bone Resorption ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Nuclear Factor Κb ◽

Protein Levels ◽

Unfolded Protein ◽

Regulatory Effects ◽

Protein Response

Abstract Background: Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Methods: Osteoclastogenesis was evaluated by number of TRAP-positive multinuclear (≥3) osteoclasts, bone resorption pits and expression levels of related genes. Autophagy activity were evaluated by LC3-II/LC3-I ratio, number of autophagic vacuoles and adenovirus-mRFP-GFP-tagged LC3 reporting system; Inhibitor chloroquine (CQ) was used to verified the role of autophagy in RANKL-induced osteoclastogenesis; Via downregulating Nox4 with inhibitor (DPI) and retrovirus-conveyed shRNA, we further explored the importance of Nox4 in RANKL-induced autophagy and osteoclastogenesis, as well as the regulatory effects of Nox4 on nonmitochondrial reactive oxygen species (ROS) and PERK/eIF-2α/ATF4 pathway. Intracellular ROS scavenger (NAC), mitochondrial-targeted antioxidant (MitoTEMPO) and inhibitor of PERK (GSK2606414) were also employed to investigate the role of ROS and PERK/eIF-2α/ATF4 pathway in RANKL-induced autophagy and osteoclastogenesis. Results: RANKL markedly increased autophagy, while CQ treatment caused reduction of RANKL-induced autophagy and osteoclastogenesis. Consistent with the increased autophagy, the protein levels of Nox4 were significantly increased, and Nox4 was selectively localized within the endoplasmic reticulum (ER) after RANKL stimulation. DPI and shRNA efficiently decreased the protein level and (or) activity of Nox4 in the ER and inhibited RANKL-induced autophagy and osteoclastogenesis. Mechanistically, we found that Nox4 regulates RANKL-induced autophagy activation and osteoclastogenesis by stimulating the production of nonmitochondrial ROS. Additionally, Nox4-derived nonmitochondrial ROS dramatically activate PERK/eIF-2α/ATF4, which is a critical unfolded protein response (UPR)-related signaling pathway during ER stress. Blocking the activation of the PERK/eIF-2α/ATF4 signaling pathway either by Nox4 shRNA, ROS antioxidant or PERK inhibitor (GSK2606414) treatment significantly inhibited endoplasmic reticulum autophagy (ER-phagy) during RANKL-induced osteoclastogenesis. Conclusions: Our findings provide new insights into the processes of RANKL-induced osteoclastogenesis and will help the development of new therapeutic strategies for osteoclastogenesis-related diseases.

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Splicing factor SRSF1 limits IFN-γ production via RhoH and ameliorates experimental nephritis

Rheumatology ◽

10.1093/rheumatology/keaa300 ◽

2020 ◽

Vol 60 (1) ◽

pp. 420-429

Author(s):

Takayuki Katsuyama ◽

Hao Li ◽

Suzanne M Krishfield ◽

Vasileios C Kyttaris ◽

Vaishali R Moulton

Keyword(s):

T Cells ◽

T Cell ◽

Molecular Mechanisms ◽

Elevated Serum ◽

Splicing Factor ◽

Type I ◽

Human T Cells ◽

Experimental Nephritis ◽

Ifn Γ

Abstract Objective CD4 T helper 1 (Th1) cells producing IFN-γ contribute to inflammatory responses in the pathogenesis of SLE and lupus nephritis. Moreover, elevated serum type II IFN levels precede the appearance of type I IFNs and autoantibodies in patient years before clinical diagnosis. However, the molecules and mechanisms that control this inflammatory response in SLE remain unclear. Serine/arginine-rich splicing factor 1 (SRSF1) is decreased in T cells from SLE patients, and restrains T cell hyperactivity and systemic autoimmunity. Our objective here was to evaluate the role of SRSF1 in IFN-γ production, Th1 differentiation and experimental nephritis. Methods T cell-conditional Srsf1-knockout mice were used to study nephrotoxic serum-induced nephritis and evaluate IFN-γ production and Th1 differentiation by flow cytometry. RNA sequencing was used to assess transcriptomics profiles. RhoH was silenced by siRNA transfections in human T cells by electroporation. RhoH and SRSF1 protein levels were assessed by immunoblots. Results Deletion of Srsf1 in T cells led to increased Th1 differentiation and exacerbated nephrotoxic serum nephritis. The expression levels of RhoH are decreased in Srsf1-deficient T cells, and silencing RhoH in human T cells leads to increased production of IFN-γ. Furthermore, RhoH expression was decreased and directly correlated with SRSF1 in T cells from SLE patients. Conclusion Our study uncovers a previously unrecognized role of SRSF1 in restraining IFN-γ production and Th1 differentiation through the control of RhoH. Reduced expression of SRSF1 may contribute to pathogenesis of autoimmune-related nephritis through these molecular mechanisms.

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Characterization of bacterial communities of rhizosphere and rhizoplane of Early Zhukovsky potato

E3S Web of Conferences ◽

10.1051/e3sconf/202022202050 ◽

2020 ◽

Vol 222 ◽

pp. 02050

Author(s):

Marat Lutfulin ◽

Darya Zaripova ◽

Oksana Moiseeva ◽

Semen Vologin ◽

Ayslu Mardanova

Keyword(s):

Bacterial Communities ◽

Functional Role ◽

Agricultural Crop ◽

Potato Root ◽

Bacterial Microbiota ◽

16S R Rna ◽

Significant Difference ◽

Dominant Bacteria

Identification of patterns of formation of bacterial communities of the rhizosphere and rhizoplane of potato (Solanum tuberosum L.), the most important agricultural crop, is necessary for the introduction and maintenance of sustainable organic farming. The purpose of this work was the study of the biodiversity of the bacterial microbiota of the rhizosphere and rhizoplane of Early Zhukovsky potato, cultivated on gray forest soils. Comparative analysis based on sequencing of the 16S R RNA gene showed a significant difference in the representation of different groups of bacteria in these potato root compartments. Thus, the proportions of the dominant bacteria in the rhizosphere and rhizoplane of the Proteobacteria phylum reach 47.66% ± 7.22 % and 86.35 % ± 0.53%, respectively (P < 0.05). In contrast, the representation of phylum Bacteroidetes and Firmicutes in the rhizosphere is significantly higher and reaches 41.45 % ± 10.42% and 6.49 % ± 3.23%, respectively, compared to the rhizoplane (7.84 % ± 1.24 % and 0.43 % ± 0.48 %, (P < 0.05). At the same time, Actinobacteria phylum bacteria are present in both compartments in approximately equal amounts (4.40 % ± 1.81% in the rhizosphere and 5.37 % ± 1.42% in the rhizoplane). Thus, it was found that potato forms different bacterial communities in the rhizosphere and rhizoplane in quantitative proportions, which is probably determined by the functional role of these microorganisms in the plant physiology.

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Urinary ACE2 in healthy adults and patients with uncomplicated type 1 diabetes

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2014-0065 ◽

2014 ◽

Vol 92 (8) ◽

pp. 703-706 ◽

Cited By ~ 13

Author(s):

David Z.I. Cherney ◽

Fengxia Xiao ◽

Joseph Zimpelmann ◽

Ronnie L.H. Har ◽

Vesta Lai ◽

...

Keyword(s):

Type 1 Diabetes ◽

Enzyme Activity ◽

Angiotensin Converting Enzyme ◽

Functional Role ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Protein Levels ◽

Clinical Complications

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be renoprotective. We determined whether urinary ACE2 enzyme activity and protein levels (ELISA), as well as angiotensinogen and ACE, are elevated during clamped euglycemia (4–6 mmol·L–1) in patients with uncomplicated type 1 diabetes (T1D, n = 58) compared with normoglycemic controls (n = 21). We also measured the effect of clamped hyperglycemia (9–11 mmol·L–1) on each urinary factor in T1D patients. Urinary ACE2 activity and protein levels were higher during clamped euglycemia in T1D compared with the controls (p < 0.0001). In contrast, urinary angiotensinogen levels (p = 0.27) and ACE excretion (p = 0.68) did not differ. In response to clamped hyperglycemia in T1D, urinary ACE2 protein decreased (p < 0.0001), whereas urinary ACE2 activity as well as angiotensinogen and ACE levels remained unchanged. Urinary ACE2 activity and protein expression are increased in T1D patients prior to the onset of clinical complications. Further work is required to determine the functional role of urinary ACE2 in early T1D.

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Role of Plasma Presepsin, Procalcitonin and C-reactive Protein Levels in Determining the Severity and Mortality of Community-Acquired Pneumonia in the Emergency Department

10.22514/sv.2020.16.0034 ◽

2020 ◽

Keyword(s):

Emergency Department ◽

Community Acquired Pneumonia ◽

C Reactive Protein ◽

Pneumonia Severity ◽

Protein Levels ◽

Reactive Protein ◽

Imaging Results ◽

Severity Scores ◽

Significant Difference

Objective: In this study, we aimed to explore the role of the plasma presepsin level in patients with community-acquired pneumonia during admission to the emergency department in assessing the diagnosis, severity, and prognosis of the disease. In addition, we wanted to investigate the relationship of presepsinin with procalcitonin, C-reactive protein and pneumonia severity scores. Methods: One hundred twenty-three patients over the age of 18 who presented with a diagnosis of pneumonia to the emergency department were included in the study. The vital signs, symptoms, examination findings, background information, laboratory results, and radiological imaging results of the patients were recorded. The 30-day mortality rates of the patients were determined. Results: A statistically significant difference was found between the presepsin levels of the patients diagnosed with pneumonia and those of healthy subjects (p < 0.05). The plasma presepsin levels of the patients who died (8.63 ± 6.46) were significantly higher than those of the patients who lived (5.82 ± 5.97) (p < 0.05). The plasma procalcitonin and C-reactive protein levels of the dead patients were significantly higher than those living (p < 0.05). A presepsin cut-off value of 3.3 ng/mL for 30-day mortality was established (AUROC, 0.65; specificity, 45%; sensitivity, 82%). Procalcitonin is the most successful biomarker in the determination of mortality (AUROC, 0.70). A significant correlation was available between presepsin and lactate, C-reactive protein and procalcitonin (p < 0.05). There was a significant correlation between the Pneumonia Severity Index values and presepsin levels (p < 0.001, r = 0.311). Conclusion: The plasma presepsin level can be utilized for diagnosing community-acquired pneumonia. Plasma presepsin, procalcitonin and C-reactive protein levels can be used to predict the severity and mortality of community-acquired pneumonia.

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Human Blood-Derived Lymphoblastoid Cybrids for Assessment of Mitochondrial tRNAs Deficiency by MTO1 Silence

10.21203/rs.3.rs-208835/v2 ◽

2021 ◽

Author(s):

Chao Chen ◽

Qi Wang ◽

Maerhaba Aishanjiang ◽

Yuping Wei ◽

Zewen Gao ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Functional Role ◽

Cardiac Tissue ◽

White Blood Cells ◽

High Energy ◽

Mitochondrial Translation ◽

Protein Levels ◽

Energy Demands ◽

Translation Optimization

Abstract Background: Mutations in the mitochondrial translation optimization 1 (MTO1) gene can cause hypertrophic cardiomyopathy. Although the functional role of MTO1 deficiency in certain cells is gradually confirmed, the phenotype of MTO1 deficiency in a lymphoblastoid cybrid line is not yet reported. In this study, we characterized changes of mitochondrial function in MTO1 silenced cybrid cells derived from human lymphoblastoids, immature white blood cells that give rise to lymphocytes. Results: We showed that MTO1 silence decreased the levels of 2-thiourylation of mitochondrial tRNALys, tRNAGlu, and tRNAGln, nevertheless, the aminoacylation efficiency of tRNALys and the steady state of mitochondrial tRNAs were elevated. These aberrant tRNA changes caused a significant decrease in protein levels of oxidative phosphorylation complex subunits including complex I, III, IV and V. Furthermore, dysfunctional mitochondria promoted apoptosis in stress, evidenced by elevated ratios of apoptotic cells and increased levels of apoptosis-activated proteins in the MTO1 knockdown cell lines, as compared to the controls. Conclusions: Our data provide new insights into the important functional role of MTO1 in lymphoblastoid mitochondria. We envision the cybrid cell line approach we have established provides an alternative model for the cardiac tissue of high-energy demands characteristics, and they hold promises for the diagnosis and drug screening for the therapeutic agents of hypertrophic cardiomyopathy caused by MTO1 dysfunction.

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A Role for Splicing Factors and R-Loop Formation in the Pathophysiology of Fanconi Anemia.

Blood ◽

10.1182/blood.v116.21.3371.3371 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3371-3371

Author(s):

Allison M Green ◽

Natalie Collins ◽

Simonne Longerich ◽

Patrick Sung ◽

Gary M Kupfer

Keyword(s):

Genomic Instability ◽

Splicing Factor ◽

Splicing Factors ◽

Loop Formation ◽

Knock Down ◽

Protein Levels ◽

In Vitro Binding ◽

Vitro Binding ◽

Cellular Sensitivity

Abstract Abstract 3371 Introduction: Fanconi anemia(FA) is an autosomal and X-linked recessive genetic disorder characterized by congenital defects, aplastic anemia, and a predisposition to cancer. At the cellular level, patients with FA display hypersensitivity to DNA crosslinking agents and increased levels of chromosomal instability. Because of these cellular phenotypes, the FA pathway has been thought to function in DNA maintenance and repair. Our data suggest that RNA and R-loop formation may account for at least part of the genomic instability seen in FA cells. Methods: Nuclear extracts were incubated with ribohomopolymers to determine whether FANCD2 is able to bind RNA. In vitro binding assays were performed to determine whether FANCD2 is able to bind R-loop nucleic acid structures. siRNA transfections were used to reduce protein levels of splicing factors. Survival assays were performed to determine cell sensitivity to the interstrand crosslinking drug mitomycin C (MMC) and the topoisomerase I inhibiting drug camptothecin (CPT). Result: In vitro binding studies with ribohomopolymers revealed that the non-ubiquitylated form of FANCD2 is able to bind polyG RNA preferentially. Because G-rich RNA is associated with R-loop formation at IgG switch regions (Yu et al, Nat. Immunology, 2003), we wanted to determine whether FANCD2 is also able to bind R-loops. A human recombinant protein complex containing FANCD2, FANCI, and FANCE was shown to bind R-loop structures in vitro. Since depletion of the splicing factor ASF/SF2 has been demonstrated to increase cellular genomic instability through the formation of increased levels of R-loop structures (Li et al, Cell, 2005), we next wanted to determine what effect depletion of ASF/SF2 would have on the FA pathway. siRNA mediated knock down of ASF/SF2 protein levels resulted in decreased levels of FANCD2 ubiquitylation following treatment with MMC. As FANCD2 ubiquitylation is often considered a marker of activation of the FA pathway, this result suggests that depletion of ASF/SF2 inhibits activation of the FA pathway. siRNA mediated knock down of ASF/SF2 also increased cellular sensitivity to MMC and CPT. siRNA mediated knock down of the splicing factor SC35 yielded a similar result, inducing an increase in sensitivity to MMC and CPT and reducing levels of FANCD2 ubiquitylation. However, siRNA mediated knock down of a third splicing factor, SRp55, had no effect on FANCD2 ubiquitylation or cellular sensitivity to MMC and CPT. Conclusion: These results suggest that R-loop formation may be a source of some of the genomic instability present in FA cells. We also demonstrate that splicing factors may play a role in activation of the FA pathway. These findings may potentially provide new targets for therapeutic treatments for FA. Disclosures: No relevant conflicts of interest to declare.

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Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00346.2014 ◽

2015 ◽

Vol 308 (4) ◽

pp. G298-G312 ◽

Cited By ~ 72

Author(s):

Diptadip Dattaroy ◽

Sahar Pourhoseini ◽

Suvarthi Das ◽

Firas Alhasson ◽

Ratanesh Kumar Seth ◽

...

Keyword(s):

Oxidative Stress ◽

Nadph Oxidase ◽

Nonalcoholic Steatohepatitis ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Micro Rna ◽

Protein Levels ◽

Significant Research ◽

Human Livers

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-β signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-β, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-β signaling and fibrogenesis in experimental and human NASH.

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

Molecular and Cellular Biology ◽

10.1128/mcb.25.17.7484-7493.2005 ◽

2005 ◽

Vol 25 (17) ◽

pp. 7484-7493 ◽

Cited By ~ 47

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.

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