To address these goals we used a next-generation sequencing specifically targeting the 3′-ends of mRNAs: PolyAdenylation Site Sequencing (PAS-Seq). PAS-Seq allows for quantitative gene expression measurements as well as transcriptome-wide assessment of APA 2,3. Using total RNA extracted from the villous tissue of 6 normal (N) and 11 PE patient placentae, we generated and sequenced 17 PAS-Seq libraries. Among the PE patients, 8 were early onset (eoPE = delivery <34 weeks).
By combining all counts from individual mRNA isoforms into a single measure (reads per million, RPM) for each gene and performing differential expression analysis (via DESeq2), we identified 288 up-regulated genes and 170 down-regulated genes in PE compared to N (padj 302161&_mathId=si1.gif&_user=111111111&_pii=S2210778916302161&_rdoc=1&_issn=22107789&md5=513301b6592b0327b95436ad1303e432" title="Click to view the MathML source">⩽0.05). Of these 458 differentially expressed genes, 200 (44%) overlap with the 1295 differentially expressed genes previously identified for severe PE upon meta-analysis of microarray data 4. However, only 15 (3%) of our set overlap with the 297 differentially expressed genes recently determined by RNA-Seq 5.Given the extreme heterogeneity of this disease [i.e., eoPE vs loPE; intrauterine growth restriction (IUGR) PE versus non-IUGR-PE] such a high degree of variability is perhaps unsurprising. Nonetheless, FLT1 was among the small set of differentially expressed genes common to all three datasets.
In our PAS-Seq data, FLT1 was the most up-regulated gene (2.8-fold in all PE samples vs. N; 4.2-fold in eoPE vs N). Among all detectable APA isoforms, the four most abundant were: sFlt1-e15a 302161&_mathId=si2.gif&_user=111111111&_pii=S2210778916302161&_rdoc=1&_issn=22107789&md5=b5ba80f33a101e4896490d72284932ee" title="Click to view the MathML source">> sFlt1-i13 long 302161&_mathId=si2.gif&_user=111111111&_pii=S2210778916302161&_rdoc=1&_issn=22107789&md5=b5ba80f33a101e4896490d72284932ee" title="Click to view the MathML source">> sFlt1-i13 short 302161&_mathId=si2.gif&_user=111111111&_pii=S2210778916302161&_rdoc=1&_issn=22107789&md5=b5ba80f33a101e4896490d72284932ee" title="Click to view the MathML source">> m-Flt1. All four isoforms were significantly higher (p 302161&_mathId=si1.gif&_user=111111111&_pii=S2210778916302161&_rdoc=1&_issn=22107789&md5=513301b6592b0327b95436ad1303e432" title="Click to view the MathML source">⩽0.05; t-test) in PE versus N; this suggests an overall increase in Flt1 gene transcription in PE. Along with this increased transcription, there also appears to be a switch toward more promoter-proximal sites. We are currently working to determine whether this switch to more promoter-proximal APA sites is a general phenomenon in PE placentae, or is solely a feature of the FLT1 gene.
Our PAS-Seq data indicate that the FLT1 gene is subject to both transcriptional upregulation and a shift toward promoter-proximal polyadenylation in PE. Further data analysis regarding global APA switches and pathway analysis will be presented at the conference.
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R.J. Levine, et al., N. Engl. J. Med. 350, (2004) 672–683.
P.J. Shepard, et al., RNA 17 (2011), 761–772.
A. Derti et al., Gen. Res. 22 (2012), 1173–1183.
K. Leavey, et al., PLoS One 10 (2015), e0116508.
S. Sõber, et al., Sci. Rep. (2015), 1–17.