Ceramide regulates SR protein phosphorylation during adenoviral infection

Kanj, Souha S.; Dandashi, Nadine; El-Hed, Aimee; Harik, Hisham; Maalouf, Maria; Kozhaya, Lina; Mousallem, Talal; Tollefson, Ann E.; Wold, William S.M.; Chalfant, Charles E.; Dbaibo, Ghassan

doi:10.1016/j.virol.2005.09.060

Cited by 24 publications

(27 citation statements)

References 58 publications

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“…The three RNA oligomers (r2, r3 and r6) derived from other regions in exon i9 did not or only minimally bind to SRSF3 protein (Supplementary Figure S3b). In agreement with the previous reports showing that the phosphorylation of SR splicing factors is required for their interaction with RNA, [63][64][65][66][67][68][69][70] dephosphorylation by treatment with calf intestine phosphatase abrogated the ability of SRSF3 to bind to RNA oligomers r1, r4 and r5 (data not shown). Introduction of mutations into the SRSF3 BS in RNA oligomers r1, r4 and r5 (r1*, r4* and r5*, Figure 4a) partially or fully abolished the binding of SRSF3 to these RNAs (Figure 4b).…”

Section: Srsf3 Regulatessupporting

confidence: 93%

Downregulation of splicing factor SRSF3 induces p53β, an alternatively spliced isoform of p53 that promotes cellular senescence

et al. 2012

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Most human pre-mRNA transcripts are alternatively spliced, but the significance and fine-tuning of alternative splicing in different biological processes is only starting to be understood. SRSF3 (SRp20) is a member of a highly conserved family of splicing factors that have critical roles in key biological processes, including tumor progression. Here, we show that SRSF3 regulates cellular senescence, a p53-mediated process to suppress tumorigenesis, through TP53 alternative splicing. Downregulation of SRSF3 was observed in normal human fibroblasts undergoing replicative senescence, and was associated with the upregulation of p53b, an alternatively spliced isoform of p53 that promotes p53-mediated senescence. Knockdown of SRSF3 by short interfering RNA (siRNA) in early-passage fibroblasts induced senescence, which was associated with elevated expression of p53b at mRNA and protein levels. Knockdown of p53 partially rescued SRSF3-knockdown-induced senescence, suggesting that SRSF3 acts on p53-mediated cellular senescence. RNA pulldown assays demonstrated that SRSF3 binds to an alternatively spliced exon uniquely included in p53b mRNA through the consensus SRSF3-binding sequences. RNA crosslinking and immunoprecipitation assays (CLIP) also showed that SRSF3 in vivo binds to endogenous p53 pre-mRNA at the region containing the p53b-unique exon. Splicing assays using a transfected TP53 minigene in combination with siRNA knockdown of SRSF3 showed that SRSF3 functions to inhibit the inclusion of the p53b-unique exon in splicing of p53 pre-mRNA. These data suggest that downregulation of SRSF3 represents an endogenous mechanism for cellular senescence that directly regulates the TP53 alternative splicing to generate p53b. This study uncovers the role for general splicing machinery in tumorigenesis, and suggests that SRSF3 is a direct regulator of p53.

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Section: Srsf3 Regulatessupporting

confidence: 93%

Downregulation of splicing factor SRSF3 induces p53β, an alternatively spliced isoform of p53 that promotes cellular senescence

et al. 2012

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“…As a possible mechanism, Kanj et al (25) reported recently that adenoviral infection causes an accumulation of ceramide, which results in the dephosphorylation of SR proteins. Because de novo ceramide synthesis by Fas activation or heat shock also causes dephosphorylation of SR proteins via the activation of PP1 (26), the viral infection may mimic the ceramide-medicated host pathway.…”

Section: Discussionmentioning

confidence: 99%

“…Flp-In293 cells contained a single, stably integrated Flp recognition target site at a transcriptionally active locus. Briefly, mouse SRPK2 (mSRPK2) cDNA (25) or Venus was inserted into pcDNA5͞Flp recognition target͞V5-His-TOPO (Invitrogen) and transfected into Flp-In293 cells with pOG44 (Flp recombinase expression vector), resulting in the targeting integration of the expression vector. Then Flp-In293 cells stably expressing mock (Venus) or SRPK2 (SRPK2-2 or SRPK2-3) were selected and established as instructed.…”

Section: Methodsmentioning

confidence: 99%

Utilization of host SR protein kinases and RNA-splicing machinery during viral replication

Fukuhara

Hosoya

Saki

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

204

192

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Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNAprocessing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serinearginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein͞activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNAprocessing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.HIV ͉ kinase inhibitor ͉ SR protein phosphorylation inhibitor 340 H IV-1 precursor RNA transcribed from proviral DNA integrated in the host cell genome contains all of the transcribed viral reading frames (1). Alternative splicing is essential for producing mRNAs encoding various viral proteins from the limited size of a single precursor mRNA (2). In the early phase of HIV expression, eight splice acceptor sites compete for the splicing machinery to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs (3). In the late phase of the virus life cycle, singly spliced longer RNA is translated to a polyprotein and then cleaved by HIV protease to generate gag and pol proteins. Several reports show that regulation of the complex splicing pattern can dramatically affect HIV-1 infectivity and pathogenesis (4-6). However, little is known about the molecular mechanism that links this alternative splicing regulation and the dynamics of virus propagation.Alternative splicing depends on the alternative utilization of four 5Ј splice sites and eight 3Ј splice sites (3). The combination of these splice sites are regulated by cis-regulatory elements, which bind cellular heterogeneous nucleoproteins (hnRNPs) of the A, B, and H groups and serine-arginine-rich (SR) proteins (7). SR proteins are highly conserved in eukaryotes and are characterized by having one or two RNA-recognition motifs at the amino termi...

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“…It has been reported that virus infection induces dephosphorylation and functional inactivation of SR proteins. As a possible mechanism, Kanj et al (13) have reported that adenoviral infection caused cellular accumulation of ceramide, which induces dephosphorylation of SR proteins by activation of the host pro- (32). Forty-eight hours after transfection, Western blotting was performed using anti-HA and anti-beta-actin antibodies.…”

Section: Vol 54 2010 Sr Protein Kinase Inhibitor Suppresses Hcv Repmentioning

confidence: 99%

Inhibition of Hepatitis C Virus Replication by a Specific Inhibitor of Serine-Arginine-Rich Protein Kinase

Karakama¹,

Sakamoto

Itsui

et al. 2010

Antimicrob Agents Chemother

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Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.Hepatitis C virus (HCV) infects up to 170 million people worldwide, and these infections frequently are characterized by chronic liver inflammation, leading to decompensated liver cirrhosis and hepatocellular cancers (1). Alpha and beta interferons are the mainstay of HCV therapeutics. However, the most effective pegylated interferon plus ribavirin combination therapies can eliminate HCV from around half of the patients only (6). These difficulties in eradicating HCV are compounded by the limited treatment options. For this reason, the development of safe and effective therapeutic agents against HCV has been a strong motivation in academia and industry (23).Serine-arginine-rich (SR) proteins are a family of non-small nuclear ribonucleoprotein particle (non-snRNP) splicing factors that are highly conserved throughout the eukaryotes. They harbor one or two RNA recognition motifs and an RS domain at the amino and carboxyl termini, respectively (29). RS domains consist of multiple consecutive Arg-Ser/Ser-Arg dipeptide repeats, in which the Ser residues are extensively phosphorylated by several kinases, including SR protein kinases (SRPKs). SRPK1 was the first SR protein kinase to be cloned, on the basis of its ability to phosphorylate SR proteins in vitro (8, 9), and two other structurally related kinases, SRPK2 and SRPK3, also have been shown to phosphorylate SR proteins (16,31). Although the precise physiological role of this phosphorylation remains unknown, it is expected that phosphorylation of SR proteins affects their protein-protein and protein-RNA interactions, intracellular localization and trafficking, and alternative splicing of pre-mRNA (21).As SRPK-dependent herpes simplex virus (HSV) splicing and SRPK-mediated phosphorylation of hepatitis B virus (HBV) core protein have been reported (4,25,33), it is reasonable to expect that SR...

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Ceramide regulates SR protein phosphorylation during adenoviral infection

Cited by 24 publications

References 58 publications

Downregulation of splicing factor SRSF3 induces p53β, an alternatively spliced isoform of p53 that promotes cellular senescence

Downregulation of splicing factor SRSF3 induces p53β, an alternatively spliced isoform of p53 that promotes cellular senescence

Utilization of host SR protein kinases and RNA-splicing machinery during viral replication

Inhibition of Hepatitis C Virus Replication by a Specific Inhibitor of Serine-Arginine-Rich Protein Kinase

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