RNAi-Mediated PTB Depletion Leads to Enhanced Exon Definition

Wagner, Eric J.; García-Blanco, Mariano A.

doi:10.1016/s1097-2765(02)00645-7

Cited by 139 publications

(159 citation statements)

References 31 publications

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“…Interestingly, both PTBP1 and CSTF2 had some significant enrichment at distinct distances 5Ј of the exon, consistent with recruitment of the RNA binding protein to the transcription machinery before the exon is synthesized. Consistent with this interpretation are past findings that PTBP1 binds to intronic sequences both upstream of and downstream from exons (Wagner and Garcia-Blanco 2002).…”

Section: Genome Research 917supporting

confidence: 74%

“…ALY is an RNA binding protein that links splicing to mRNA export as a component of the exon junction complex (EJC) (Zhou et al 2000). PTBP1 represses alternative exons like those of FGFR1 and FGFR2, where PTBP1's influence yields receptors with higher affinities for fibroblast growth factor (Carstens et al 2000;Wagner and Garcia-Blanco 2002;Jin et al 2003). CSTF2 is the mammalian homolog of the yeast protein, Rna15p, and is a 3Ј end cleavage factor that can modulate alternative 3Ј end cleavage of transcripts (Takagaki and Manley 1998).…”

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confidence: 99%

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Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Swinburne

Meyer²,

Liu³

et al. 2006

Genome Res.

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Pre-mRNA processing often occurs in coordination with transcription thereby coupling these two key regulatory events. As such, many proteins involved in mRNA processing associate with the transcriptional machinery and are in proximity to DNA. This proximity allows for the mapping of the genomic associations of RNA binding proteins by chromatin immunoprecipitation (ChIP) as a way of determining their sites of action on the encoded mRNA. Here, we used ChIP combined with high-density microarrays to localize on the human genome three functionally distinct RNA binding proteins: the splicing factor polypyrimidine tract binding protein (PTBP1/hnRNP I), the mRNA export factor THO complex subunit 4 (ALY/THOC4), and the 3Ј end cleavage stimulation factor 64 kDa (CSTF2). We observed interactions at promoters, internal exons, and 3Ј ends of active genes. PTBP1 had biases toward promoters and often coincided with RNA polymerase II (RNA Pol II). The 3Ј processing factor, CSTF2, had biases toward 3Ј ends but was also observed at promoters. The mRNA processing and export factor, ALY, mapped to some exons but predominantly localized to introns and did not coincide with RNA Pol II. Because the RNA binding proteins did not consistently coincide with RNA Pol II, the data support a processing mechanism driven by reorganization of transcription complexes as opposed to a scanning mechanism. In sum, we present the mapping in mammalian cells of RNA binding proteins across a portion of the genome that provides insight into the transcriptional assembly of RNA-protein complexes.[Supplemental material is available online at www.genome.org and http://silver.med.harvard.edu/brodsky/.] RNA binding proteins regulate many stages of RNA metabolism to help determine gene expression programs (for reviews, see Keene and Tenenbaum 2002;Hieronymus and Silver 2004). To prepare mRNA for export to and translation in the cytoplasm, many events including splicing, cleavage of the 3Ј end, and editing occur cotranscriptionally (Kornblihtt et al. 2004;Aguilera 2005a). After transcription, the mRNA-protein complex may reorganize as it is exported to the cytoplasm and prepared for translation (Fairman et al. 2004). Thus, significant efforts have been made to determine how RNA binding proteins interact at genes and/or mRNAs at various stages of mRNA metabolism (Tenenbaum et al. 2000;Brown et al. 2001;Hieronymus and Silver 2003;Ule et al. 2003;Yu et al. 2004). These early genomic efforts suggested that, similar to DNA binding proteins, functional groupings of genes are being bound by RNA binding proteins perhaps as post-transcriptional operons (Keene and Tenenbaum 2002). However, it is not known at which stage of RNA processing these operons are established.Much of pre-mRNA processing is coupled to transcription both physically and functionally, potentially through interactions with the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II) (Mortillaro et al. 1996;Yuryev et al. 1996;Kim et al. 1997). The CTD is phosphorylated as transcription begins. Following...

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Section: Genome Research 917supporting

confidence: 74%

mentioning

confidence: 99%

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Swinburne

Meyer²,

Liu³

et al. 2006

Genome Res.

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“…PTBP1 serves as a repressor of alternative splicing in mammalian cells (24)(25)(26)(27)(28)(29) and contains RNA-binding domains, each of which binds to CU-rich elements (30). It is involved in polyadenylation of the pre-mRNA 3 0 end (31-33) and also plays an important role in translational regulation of a subset of RNA transcription through internal ribosome entry sites (21,(34)(35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer

Takahashi

Nishimura

Kagawa

et al. 2015

Molecular Cancer Therapeutics

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Polypyrimidine tract-binding protein (PTBP1) is an RNAbinding protein with various molecular functions related to RNA metabolism and a major repressive regulator of alternative splicing, causing exon skipping in numerous alternatively spliced pre-mRNAs. Here, we have investigated the role of PTBP1 in colorectal cancer. PTBP1 expression levels were significantly overexpressed in cancerous tissues compared with corresponding normal mucosal tissues. We also observed that PTBP1 expression levels, c-MYC expression levels, and PKM2: PKM1 ratio were positively correlated in colorectal cancer specimens. Moreover, PTBP1 expression levels were positively correlated to poor prognosis and lymph node metastasis. In analyses of colorectal cancer cells using siRNA for PTBP1, we observed that PTBP1 affects cell invasion, which was partially correlated to CD44 splicing, and this correlation was also confirmed in clinical samples. PTBP1 expression also affected anchorage-independent growth in colorectal cancer cell lines. PTBP1 expression also affected cell proliferation. Using timelapse imaging analysis, PTBP1 was implicated in prolonged G 2 -M phase in HCT116 cells. As for the mechanism of prolonged G 2 -M phase in HCT116 siPTBP1 cells, Western blotting revealed that PTBP1 expression level was correlated to CDK11 p58 expression level, which was reported to play an important role on progression to complete mitosis. These findings indicated that PTBP1 is a potential therapeutic target for colorectal cancer.

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“…The small interfering RNA (siRNA) of PTB targeting a 19-nt sequence corresponding to nt 1135-1153 of PTB open reading frame (ORF) [32] was chemically synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa). Nonspecific siRNA was purchased from Ambion (Austin, Tex).…”

Section: Rna Interference Analysismentioning

confidence: 99%

“…To further examine the role of PTB in HCV replication in vivo, we attempted to knock down the endogenous PTB expression using the RNA interference method [32]. Replicon cells were transfected with either PTB-specific or nonspecific siRNA.…”

Section: In Vivo Knockdown Of Ptb Inhibited Hcv Replicationmentioning

confidence: 99%

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

et al. 2006

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The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane proteinassociated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.

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RNAi-Mediated PTB Depletion Leads to Enhanced Exon Definition

Cited by 139 publications

References 31 publications

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

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