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Identification of the RNA-protein interactions of PAIP1, which plays a novel role in alternative mRNA splicing (RNA-Seq)

ABSTRACT: Poly (A) binding protein interacting protein 1 (PAIP1) has been shown to causally contribute to the development and progression of cancer. However, the biological function of PAIP1 in tumorigenesis remains largely unexplored. Here, we used RNA immunoprecipitation to map the interactions between PAIP1 and RNA on a genome wide level. A genome wide analysis showed that PAIP1 binding is specifically enriched in exon, intron and 3’ untranslated regions. The genes bound by PAIP1 were strongly associated with cancer-related pathways and we found that PAIP1 knockdown mediates the statistically significant regulation of alternative splice events. These data suggest that PAIP1 may influence alternative splicing and, using a VEGF-A minigene, we showed that overexpression of PAIP1 inhibits the expression of exon 6. Further analysis of PAIP1 showed that interacting proteins participate in splice factors, spliceosomes and exon junction complex assembly. Our work has demonstrated that PAIP1 has specific RNA binding properties that help to coordinate the isoform-specific expression of coding genes. These genes are associated with cancer, which suggests that PAIP1 is an important regulator of key cancer processes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE150779 | GEO | 2022/05/05

REPOSITORIES: GEO

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Identification of the RNA-protein interactions of PAIP1, which plays a novel role in alternative mRNA splicing (RIP-seq)

Project description:Poly (A) binding protein interacting protein 1 (PAIP1) has been shown to causally contribute to the development and progression of cancer. However, the biological function of PAIP1 in tumorigenesis remains largely unexplored. Here, we used RNA immunoprecipitation to map the interactions between PAIP1 and RNA on a genome wide level. A genome wide analysis showed that PAIP1 binding is specifically enriched in exon, intron and 3’ untranslated regions. The genes bound by PAIP1 were strongly associated with cancer-related pathways and we found that PAIP1 knockdown mediates the statistically significant regulation of alternative splice events. These data suggest that PAIP1 may influence alternative splicing and, using a VEGF-A minigene, we showed that overexpression of PAIP1 inhibits the expression of exon 6. Further analysis of PAIP1 showed that interacting proteins participate in splice factors, spliceosomes and exon junction complex assembly. Our work has demonstrated that PAIP1 has specific RNA binding properties that help to coordinate the isoform-specific expression of coding genes. These genes are associated with cancer, which suggests that PAIP1 is an important regulator of key cancer processes.

2022-05-05 | GSE150780 | GEO

PAIP1 promote the progress of liver cancer via regulating expression of immune and inflammatory response associated genes

Project description:Poly(A) binding protein interacting protein 1 (PAIP1) is a translation regulator and also regulate the decay of mRNA. PAIP1 has also been reported to be a marker of increased invasive potential of liver cancer. However, the underlying molecular mechanism of PAIP1 in liver cancer is still unclear. Here, the gene expression profile of HepG2 cells transfected with PAIP1 shRNA (shPAIP1) was compared with cells transfected with empty vector (shCtrl). The results showed that shPAIP1 extensively affects the transcriptional level of 893 genes in HepG2 cells. In particular, GO analysis showed that a large number of up-regulated genes were annotated with terms including cell cycle and WNT receptor signaling pathway. The down-regulated genes were enriched in some pathways including immune response and inflammatory response. qPCR confirmed that PAIP1 knockdown significantly negatively or positively affected the expression of selected DEGs including interleukin-10 signaling pathway genes in HepG2 cells. Expression analysis of TCGA revealed that PAIP1 had positive correlations with IL1R2 and PTAFR in liver tumor tissue, respectively. Our results demonstrated that PAIP1 was not only a translation regulator, but also a transcription regulator in liver cancer. Thus, our study provides important cues for further study on the regulatory mechanism of PAIP1 in liver cancer

2023-03-12 | GSE185866 | GEO

PTB CLIP-seq

2009-12-24 | GSE19323 | GEO

PTB CLIP-seq

Project description:Recent transcriptome analysis indicates that >90% of human genes undergoes alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now revealed that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells. Examination of PTB-RNA binding in Hela cells using CLIP-seq (Cross-Linking ImmunoPrecipitation coupled with high-throughput sequencing) method. Peaks: The four alignment files (linked as supplementary files on Sample records) were combined together for peak finding, as we found that most of the monomeric and dimeric tags are similarly distributed in the genome with high pearson correlation coefficient. The method to detect the peaks above gene-specific randomized background was similar to (Yeo et al., 2009) and described in the paper (Xue et al., 2009).

2009-12-24 | E-GEOD-19323 | biostudies-arrayexpress

Transcriptome analysis of control (y w) and Paip1 mutant third instar larval wing imaginal discs

Project description:We report the genome-wide RNA profiling of third instar larval wing imaginal discs upon Paip1 mutation

2023-07-31 | GSE146531 | GEO

Mapping hnRNP H binding sites on the RON wildtype minigene via iCLIP.

Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. We identified the RNA binding protein hnRNP H as an important regulator of RON exon 11 splicing. To map hnRNP H binding sites on the RON minigene, we performed hnRNP H iCLIP with RON wildtype minigene transfected HEK293T cells. iCLIP was performed according to a previously published protocol (PMID: 26463384). The iCLIP libraries were made from two replicates of HEK293T cells at 24 h after RON minigene transfection. The cells were irradiated with 150 mJ/cm2 UV light at 254 nm. For the immunoprecipitation step, we used 7.5 µg of a polyclonal rabbit anti-HNRNPH antibody from Abcam (AB10374). RNase digestion was performed by adding 10 µl of 1/100 diluted RNase I (Ambion) to each sample. We performed the sequencing on an Illumina HiSeq2000 (75-nt single-end reads).

2018-06-27 | E-MTAB-6220 | biostudies-arrayexpress

Mapping hnRNP H binding sites on RON wildtype minigene and RON mutant minigenes via iCLIP.

Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. We identified the RNA binding protein hnRNP H as an important regulator of RON exon 11 splicing. To map hnRNP H binding sites on the RON minigene with either wildtype or hnRNP H binding site mutant background, we performed hnRNP H iCLIP with RON wildtype or mutant minigene transfected HEK293T cells. iCLIP was performed according to a previously published protocol (PMID: 26463384). The iCLIP libraries were made from two (G331C and G348C) or three replicates (wildtype and G305A) of HEK293T cells at 24 h after RON minigene transfection. The cells were irradiated with 150 mJ/cm2 UV light at 254 nm. For the immunoprecipitation step, we used 7.5 µg of a polyclonal rabbit anti-HNRNPH antibody from Abcam (AB10374). RNase digestion was performed by adding 10 µl of 1/100 diluted RNase I (Ambion) to each sample. We performed the sequencing on an Illumina MiSeq or NextSeq500 with 75-nt single-end reads.

2018-06-27 | E-MTAB-6221 | biostudies-arrayexpress

Saturation mutagenesis reveals manifold determinants of exon definition

Project description:To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts we conducted saturation mutagenesis of a 51 nt internal exon in a 3-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high throughput genetics, 5560 minigene molecules were assayed for splicing in HEK293 cells. Over 70% of mutations produced substantial (>2X) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements only a single 15 nt stem-loop, showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1Aa, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51 nt test exon simultaneously. Surprisingly, such correlations extended to weak relative protein-sequence affinities. These myriad protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.

2017-11-27 | GSE105785 | GEO

Exon arrays from HeLa cells transfected with GFP-ZRANB2 vector

Project description:Human ZRANB2 mRNA is alternatively spliced to give two variants with different 3M-bM-^@M-^Y ends. Both isoforms are present in the nucleus of human cells. ZRANB2 binds to mRNA, as well as the essential splicing factors U170K and U2AF35, and the novel splicing component SFRS17A (formerly known as XE7). It has been shown to alter splicing patterns of Tra2M-NM-21 minigene primary transcripts in a dose-dependent manner. It is still unclear what role ZRANB2 plays in the regulation of alternative splicing. To determine the endogenous transcripts that are targeted by ZRANB2 at the genome wide level, we decided to perform exon microarray studies and analyzed the suitability of this method to examine splicing differences in human cells overexpressing a splicing factor. GFP-ZRANB2 construct containing isoform 2 (short form, SF) of ZRANB2 was used. This construct includes exon 1 through to alternative exon 10. Isoform 2 was chosen since it has been shown previously to affect alternative splicing of a Tra2M-NM-21 minigene. HeLa cells were transfected with GFP-ZRANB2, control vector GFP or were left untransfected. RNA was extracted using a Qiagen RNA extraction kit. Experiments were run in 5 replicates.

2012-12-15 | E-GEOD-32932 | biostudies-arrayexpress

Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome

Project description:The U2AF heterodimer has been well studied for its role in defining functional 3’ splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3’ splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3’ splice site associated with either the alternative exon to cause exon skipping or with the competing constitutive exon to induce exon inclusion. We further demonstrate partial functional impairment with mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states.

2014-10-08 | GSE61603 | GEO

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