- Analysis of splice variants of the human protein disulfide isomerase ( P4HB ) gene. - PDIA1 is coded by the P4HB gene. - Structural features suggest that except for P4HB-021 , other splice variants are unlikely to exert thiol isomerase activity at the endoplasmic reticulum. - Extensive analyses using FANTOM5, ENCODE Consortium and GTEx project databases as RNA-seq data sources were performed. - P4HB- 02, P4HB- 027 and P4HB- 021 were relatively more expressed across each database, the latter particularly in vascular smooth muscle. - The most consistently expressed splice variant was P4HB- 021 in human mammary artery vascular smooth muscle which, together with canonical P4HB gene, had its expression enhanced by serum starvation.. - P4HB -021 splice variant deserves further investigation in vascular smooth muscle cells.. - Some PDIs exhibit an un- folded protein response-sensitive element in their pro- moter region, but this is not the case of P4HB [13]. - 1 P4HB gene and protein organization. - The coding sequence of P4HB -027 includes the 3 ′ UTR, which does not contain a stop codon. - However, there is essentially no information with respect to alternative splicing variants of P4HB gene.. - Alternative splice variants of P4HB gene. - P4HB - 02 and P4HB -021 are the only to display the intact C- terminus with the KDEL motif, indicating that eventual protein products generated from other isoforms may not be retrievable to the endoplasmic reticulum.. - Taking advantage of CAGE tags to determine expression levels, we analyzed an upstream region of P4HB -02 and P4HB genes. - We also checked for the presence of CAGE tags upstream of P4HB gene using the same data and the result was similar. - Expression profiling of P4HB splice variants in FANTOM5 database. - We next addressed an overview of P4HB gene and spliced variant expression profiling in different cell lines and tissues, using a number of distinct databases:. - We used information of all FANTOM5 RNA-seq libraries (70 samples) [30], in order to prospectively analyze P4HB splice junctions. - Figure 2b represents the percentage of splice variant abun- dance in this set of samples from FANTOM5, show- ing that almost 30% of total isoform fraction is represented by P4HB-021 . - The variant P4HB -021 was significantly repre- sented, particularly in aortic smooth muscle cell, followed in this cell type by P4HB -019, P4HB- 023, P4HB -027 and P4HB -02. - The most fre- quently expressed splice variant across all the 70 samples was P4HB -02, present in 28 samples, while P4HB -021 and P4HB -027 depicted the highest splice junction TPMs. - The isoform P4HB -021 had its highest level of expression in aortic smooth muscle cells (Fig. - S2 illustrates the splicing event in the middle of P4HB -027 exon 3.. - 2e there is a plot for P4HB -021 displaying the splicing event. - Expression profiling of P4HB splice variants in RNA-seq ENCODE database. - has a set of different types of experiments such as Exon Arrays, Chip-Seq and RNA-seq analysis, avail- able at http://www.encodeproject.org. - In this graph, the most representative (i.e., expressed in most samples) was P4HB -029, but the isoforms most expressed (in SJ TPM) were P4HB -02 and P4HB -021. - Isoform P4HB -02 is well expressed in aortic adventi- tial fibroblasts, P4HB -021 in fibroblasts and 2 types of endothelial cells and P4HB -024 in two other endothe- lial cell types.. - We next applied the same pipeline above to identify and count the splice junction TPM (tag per million) to investi- gate P4HB gene expression in polyA RNA-seq ENCODE human datasets (https://www.encodeproject.org/) from donors (primary cell). - Expression profiling of P4HB splice variants in GTEx database. - number of transcriptomic data, including RNA-seq from various tissues. - Here, we used 11,690 RNA-seq data from different tissues and conditions listed in Table S3.. - 2 Distributions of P4HB splice variant expression in RNA-seq from FANTOM5. - Splice Junction Tag per Million (SJ TPM) is a unit to count the number of a specific isoform junction normalized by the total number of reads for each RNA-seq dataset. - b Fraction of expression of P4HB splice variants in FANTOM5. - c The expression of P4HB -02 and P4HB -021 in three types of cell: primary cell, tissue and cell line.. - d Representative diagram of P4HB -021 expression in all analyzed samples. - e Visualization of splicing event of P4HB -021. - The blue diagram at the bottom indicates a part of P4HB gene, in reverse direction from exon 1 to exon 4. - The total number of RNA-seq data was 70 samples. - P4HB -02 and P4HB -027 displayed slightly higher expres- sion when compared to P4HB -021. - The fractional ex- pression of variant P4HB -021 in heart was higher compared to other tissues. - 3 Distribution of splice variant expression in RNA-seq from ENCODE. - Splice Junction Tag per Million (SJ TPM) is a unit to count the number of specific isoform junctions normalized by the total number of reads for each RNA-seq dataset. - b Distribution of P4HB splice variant expression, showing 7 detected splice variants. - c Fractional distribution of P4HB splice variants expressed in distinct cell types (HUVEC, HAoEC, HaoAF). - d SJ TPM of P4HB gene and splice variants in a set of 12 different cell types. - e SJ TPM of P4HB gene and splice variants of SMC from pulmonary artery from 2 different donors. - In this specific subset, P4HB -027, P4HB -021 and P4HB -02 were represented in arterial cells (aorta, coronary and tibial), with slightly higher prevalence of P4HB -027.. - P4HB splice variants are highly expressed in smooth muscle cell. - 4 Quantification of P4HB splice variants ( P4HB -02, P4HB -021 and P4HB -027) as splice junction tags per million (SJ TPM) relative to P4HB expression.. - Similarly, in VSMC from proximal aorta isoforms P4HB -02 and P4HB - 021 were more representative in physiological conditions.. - We next performed the validation of P4HB splice variant expression in distinct cell types using PCR. - expression levels and tissue specificity (above data), namely P4HB -02, P4HB -021 and P4HB -027.. - 5 Analysis of RNA-seq data from a study [31] using VSMC (vascular smooth muscle cells) mimicking pathologic (stiff) and physiologic conditions (soft). - a P4HB ( P4HB -001) gene expression. - Also shown is the expression of P4HB and its splice variants in (b) VSMC from coronary artery and (c) VSMC of proximal aorta. - Im- portant, in accordance with previous results from data- banks, P4HB -021 was detected in VSMC at baseline and upregulated, together with the expression of P4HB , after 24 h serum starvation compared with 16 h (Fig. - Ex- posure to CoCl 2 , however, did not significantly affect the expression of P4HB and its variants in the conditions of our experiments (Fig. - We also assessed the effects of tunicamycin, a potent inhibitor of N -linked glycosylation, in HEK-293 cell line to investigate the influence of ensuing endoplas- mic reticulum stress in the expression of P4HB gene and its splice variants. - Cells were incubated for 16 h and 40 h with 3 tunicamycin concentrations and the expression of P4HB and its splicing variants analyzed (Fig. - S4), expressions of P4HB -02 and P4HB-027 (but not of P4HB or P4HB-021 ) decreased vs. - 6 PCR amplification of the splice junction of splice variants P4HB -02, P4HB -021 and P4HB -027. - The specific splice junction of P4HB -02 (89 bp) and 027 (211 bp) were amplified in HCT-116 cells and SK-N-SH cells. - For the amplification of P4HB -021, the expected fragment was 148 bp. - The amplification of this fragment was performed using a vector pUC57 with the P4HB -021 cloned as template to PCR reaction. - 2) loss of the isomerase function of P4HB , which re- quires all 4 domains, so most PDIA1 alternative splicing isoforms are unlikely to display thiol isomerase activity, with possible exception of P4HB -021. - a Expression of P4HB gene and P4HB -021 in primary VSMC (from human mammary artery) submitted to serum starvation during 16 h and 24 h. - b Expression of P4HB gene and three P4HB isoforms in VSMC treated with CoCl 2 . - When all such aspects are considered together, most PDIA1 isoforms are unlikely to exert thiol isomerase ac- tivity at the ER, with the possible exception of P4HB - 021. - Likely, they may exert other types of activity at dis- tinct subcellular locations, greatly expanding the func- tional reach of P4HB gene products. - 8 Predicted protein modeling of P4HB -021. - (C-D) P4HB -021 splice variant shows a different conformation, particularly in the b domain, as compared to PDIA1. - Our results further corroborate that the expression pattern of P4HB isoforms is consistent with multiple specific functions, since the expression is distinct among the different cell types and tissues. - P4HB -021 depicts truncation of a 44-amino acid stretch at the transition between a and b domains. - The absence of exon 3 at the a and b domain transition of P4HB -021 promotes the absence of one α-helix in a domain, two β-sheets and one α-helix in b domain and one α-helix in b’. - For this ana- lysis, we used a subset of 70 human samples from FAN- TOM5 for which RNA-seq data were available [31].. - The RNA-seq libraries from ENCODE repository (https://www.encodeproject.org/) are publicly available.. - The accession number for VSMC RNA-seq data [31] is GSE100081.. - To determine the landscape of P4HB splice variant expression, we investigated RNA-seq data across FANTOM5, ENCODE Project and GTEX database.. - RNA-seq pipeline analyses to detect splice-junction of P4HB gene in FANTOM5, ENCODE and GTEx data- base were developed. - The only difference was that for GTEx database we used the raw data obtained, meaning the splice junction value for each splice variant and nor- malized by the number of reads of P4HB gene. - RNA-seq libraries of human samples. - RNA-seq data from FANTOM5, corresponding to 70 samples, includes a diverse set of human biological samples. - The RNA-seq from [31], was sequenced in Illumina Hi-Seq 2500, 100 bp paired-end.. - For GTEx, RNA-seq data was obtained from https://gtex- portal.org/home/datasets. - The RNA-seq was performed using the Illumina TruSeq library construction preparation and the sequencing produced 76 bp paired ended reads . - Visualization of alternative splicing in P4HB isoforms To visualize the RNA-seq data we use the Integrative Genomics Viewer (IGV) browser [47], using as input spliced alignments (in BAM files format) and gene model annotation in GFF format [48]. - The BAM files are from FANTOM5 RNA-seq data.. - P4HB -021 (exon 2 fwd 5′ TATCCCACCATCAAGT TCTTCAG 3′. - Cloning of novel human P4HB splice variants. - The sequence of P4HB -021 as used as template for isoform validation.. - P4HB gene and splice variant information.. - Table with information about GTEx RNA- seq data is presented as dataset.. - Quantification of P4HB splice variants to detect the fraction of isoform abundance normalized by P4HB gene. - (A) Fraction of P4HB -02, P4HB -021 and P4HB -027 in blood vessels of three subtypes: aorta ( n = 299), coronary artery ( n = 172) and tibial artery ( n = 400) (B) Fraction of P4HB splice variants in heart with two sub-regions:. - Expression of P4HB gene and variants P4HB- 02, P4HB- 021 and P4HB- 027. - RNA-seq: RNA sequencing. - DB, JFP, KH, DK contributed to RNA-seq analysis. - Quantitative visualization of alternative exon expression from RNA-seq data.
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