FLT1 and transcriptome-wide polyadenylation site (PAS) analysis in preeclampsia

Maternal symptoms of preeclampsia (PE) are primarily driven by excess anti-angiogenic factors originating from the placenta. Chief among these are soluble Flt1 proteins (sFlt1s) produced from alternatively polyadenylated mRNA isoforms. Here we used polyadenylation site sequencing (PAS-Seq) of RNA from normal and PE human placentae to interrogate transcriptome-wide gene expression and alternative polyadenylation signatures associated with early-onset PE (EO-PE; symptom onset 34 weeks) cohorts. While we observed no general shift in alternative polyadenylation associated with PE, the EO-PE and LO-PE cohorts do exhibit gene expression profiles distinct from both each other and from normal placentae. The only two genes upregulated across all transcriptome-wide PE analyses to date (microarray, RNA-Seq and PAS-Seq) are NRIP1 (RIP140), a transcriptional co-regulator linked to metabolic syndromes associated with obesity, and Flt1. Consistent with sFlt1 overproduction being a significant driver of clinical symptoms, placental Flt1 mRNA levels strongly correlate with maternal blood pressure. For Flt1, just three mRNA isoforms account for > 94% of all transcripts, with increased transcription of the entire locus driving Flt1 upregulation in both EO-PE and LO-PE. These three isoforms thus represent potential targets for therapeutic RNA interference (RNAi) in both early and late presentations

Endogenous U2.U5.U6 snRNA complexes in S. pombe are intron lariat spliceosomes

Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2.U5.U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing

A Two-Pronged Approach to Preeclampsia: Understanding Gene Expression and Targeting sFlt1 using RNAi

Preeclampsia (PE) is a disorder affecting 2-10% of pregnancies worldwide. Clinical signs include high blood pressure (HBP) and proteinuria in the mother after the 20th week of pregnancy. Currently, the only cure for PE is delivery of the fetus, which is often necessary preterm and thus dangerous for both mother and fetus. Maternal symptoms of PE are caused by excess anti-angiogenic proteins of placental origin called soluble Flt1s (sFlt1s). sFlt1 mRNA isoforms are produced by alternative polyadenylation (APA) of full-length Flt1 (fl-Flt1) pre- mRNA. While fl-Flt1 encodes a transmembrane protein, sFlt1s encode truncated proteins that are soluble. Multiple sFlt1 isoforms exist, and their respective contribution to the pathophysiology of PE is unclear. Furthermore, it is unknown whether there is a genome-wide role for APA in PE. In my thesis research, I developed a polyadenylation site sequencing method, and used this method to simultaneously quantify transcriptome-wide polyadenylation site usage and gene expression levels in normal, early-onset PE, and late-onset PE human placentae. I observed distinct expression profiles amongst the three groups, with differential expression of genes in several functional categories, including angiogenesis. I found that three sFlt1 isoforms account for \u3e94% of all placental FLT1 transcripts, and that increased transcription of the entire FLT1 locus drives upregulation of both fl-Flt1 and sFlt1 in PE. I found that APA does not contribute substantially to PE pathophysiology. I also identified siRNAs that knock down sFlt1 mRNA efficiently in cell lines that pave the way for further development of novel RNAi based therapeutics to alleviate PE

Alterations in mRNA 3′ UTR Isoform Abundance Accompany Gene Expression Changes in Human Huntington’s Disease Brains

The huntingtin gene has two mRNA isoforms that differ in their 3′ UTR length. The relationship of these isoforms with Huntington’s disease is not established. We provide evidence that the abundance of huntingtin 3′ UTR isoforms differs between patient and control neural stem cells, fibroblasts, motor cortex, and cerebellum. Huntingtin 3′ UTR isoforms, including a mid-3′ UTR isoform, have different localizations, half-lives, polyA tail lengths, microRNA sites, and RNA-binding protein sites. Isoform shifts in Huntington’s disease motor cortex are not limited to huntingtin; 11% of alternatively polyadenylated genes change the abundance of their 3′ UTR isoforms. Altered expression of RNA-binding proteins may be associated with aberrant isoform abundance; knockdown of the RNA-binding protein CNOT6 in control fibroblasts leads to huntingtin isoform differences similar to those in disease fibroblasts. These findings demonstrate that mRNA 3′ UTR isoform changes are a feature of molecular pathology in the Huntington’s disease brain

RNAi modulation of placental sFLT1 for the treatment of preeclampsia

Preeclampsia is a placentally induced hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm preeclampsia result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1 without reducing levels of full-length FLT1 mRNA. Full chemical stabilization in the context of hydrophobic modifications enabled productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduced circulating sFLT1 in pregnant mice (up to 50%). In a baboon preeclampsia model, a single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of preeclampsia. Our results demonstrate RNAi-based extrahepatic modulation of gene expression with nonformulated siRNAs in nonhuman primates and establish a path toward a new treatment paradigm for patients with preterm preeclampsia

RNAi modulation of placental sFLT1 for the treatment of preeclampsia

Preeclampsia is a placentally induced hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm preeclampsia result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1 without reducing levels of full-length FLT1mRNA. Full chemical stabilization in the context of hydrophobic modifications enabled productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduced circulating sFLT1 in pregnant mice (up to 50%). In a baboon preeclampsia model, a single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of preeclampsia. Our results demonstrate RNAi-based extrahepatic modulation of gene expression with nonformulated siRNAs in nonhuman primates and establish a path toward a new treatment paradigm for patients with preterm preeclampsia

Staufen1 senses overall transcript secondary structure to regulate translation

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3\u27 untranslated regions (UTRs) or in \u27strongly distal\u27 3\u27 UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3\u27 UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins