Caroline Lemieux

Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor -1 and -2 heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A165 inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A165 promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI2). However, it is unknown whether VEGF-A165 is mediating PGI2 synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A165 activity. In this study, PGI2 synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F1α, 6-keto PGF1α) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A165 (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A121 (VEGFR-1 and VEGFR-2 agonist) (up to 10–9 m), increased PGI2 synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI2 synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI2 release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI2 release mediated by VEGF-A165 and VEGF-A121, and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI2 synthesis mediated by VEGF-A165 and VEGF-A121 up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI2 synthesis mediated by VEGF-A165 and VEGF-A121, which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A165 to NRP-1 potentiates its capacity to promote PGI2 synthesis.

The Nuclear Poly(A)-Binding Protein Interacts with the Exosome to Promote Synthesis of Noncoding Small Nucleolar RNAs

Poly(A)-binding proteins (PABPs) are important to eukaryotic gene expression. In the nucleus, the PABP PABPN1 is thought to function in polyadenylation of pre-mRNAs. Deletion of fission yeast pab2, the homolog of mammalian PABPN1, results in transcripts with markedly longer poly(A) tails, but the nature of the hyperadenylated transcripts and the mechanism that leads to RNA hyperadenylation remain unclear. Here we report that Pab2 functions in the synthesis of noncoding RNAs, contrary to the notion that PABPs function exclusively on protein-coding mRNAs. Accordingly, the absence of Pab2 leads to the accumulation of polyadenylated small nucleolar RNAs (snoRNAs). Our findings suggest that Pab2 promotes poly(A) tail trimming from pre-snoRNAs by recruiting the nuclear exosome. This work unveils a function for the nuclear PABP in snoRNA synthesis and provides insights into exosome recruitment to polyadenylated RNAs.

A Pre-mRNA Degradation Pathway that Selectively Targets Intron-Containing Genes Requires the Nuclear Poly(A)-Binding Protein

General discard pathways eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. In contrast to such general pre-mRNA decay, we describe here a nuclear pre-mRNA degradation pathway that controls the expression of select intron-containing genes. We show that the fission yeast nuclear poly(A)-binding protein, Pab2, and the nuclear exosome subunit, Rrp6, are the main factors involved in this polyadenylation-dependent pre-mRNA degradation pathway. Transcriptome analysis and intron swapping experiments revealed that inefficient splicing is important to dictate susceptibility to Pab2-dependent pre-mRNA decay. We also show that negative splicing regulation can promote the poor splicing efficiency required for this pre-mRNA decay pathway, and in doing so, we identified a mechanism of cross-regulation between paralogous ribosomal proteins through nuclear pre-mRNA decay. Our findings unveil a layer of regulation in the nucleus in which the turnover of specific pre-mRNAs, besides the turnover of mature mRNAs, is used to control gene expression.

Regulation of the Nuclear Poly(A)-binding Protein by Arginine Methylation in Fission Yeast

Two structurally different poly(A)-binding proteins (PABP) bind the poly(A) tract of mRNAs in most mammalian cells: PABPC in the cytoplasm and PABP2/PABPN1 in the nucleus. Whereas yeast orthologs of the cytoplasmic PABP are characterized, a gene product homologous to mammalian PABP2 has not been identified in yeast. We report here the identification of a homolog of PABP2 as an arginine methyltransferase 1 (RMT1)-associated protein in fission yeast. The product of the Schizosaccharomyces pombe pab2 gene encodes a nonessential nuclear protein and demonstrates specific poly(A) binding in vitro. Consistent with a functional role in poly(A) tail metabolism, mRNAs from pab2-null cells displayed hyperadenylated 3′-ends. We also show that arginine residues within the C-terminal arginine-rich domain of Pab2 are modified by RMT1-dependent methylation. Whereas the arginine methylated and unmethylated forms of Pab2 behaved similarly in terms of subcellular localization, poly(A) binding, and poly(A) tail length control; Pab2 oligomerization levels were markedly increased when Pab2 was not methylated. Significantly, Pab2 overexpression reduced growth rate, and this growth inhibitory effect was exacerbated in rmt1-null cells. Our results indicate that the main cellular function of Pab2 is in poly(A) tail length control and support a biological role for arginine methylation in the regulation of Pab2 oligomerization.

Negative Regulation of Meiotic Gene Expression by the Nuclear Poly(a)-binding Protein in Fission Yeast

Meiosis is a cellular differentiation process in which hundreds of genes are temporally induced. Because the expression of meiotic genes during mitosis is detrimental to proliferation, meiotic genes must be negatively regulated in the mitotic cell cycle. Yet, little is known about mechanisms used by mitotic cells to repress meiosis-specific genes. Here we show that the poly(A)-binding protein Pab2, the fission yeast homolog of mammalian PABPN1, controls the expression of several meiotic transcripts during mitotic division. Our results from chromatin immunoprecipitation and promoter-swapping experiments indicate that Pab2 controls meiotic genes post-transcriptionally. Consistently, we show that the nuclear exosome complex cooperates with Pab2 in the negative regulation of meiotic genes. We also found that Pab2 plays a role in the RNA decay pathway orchestrated by Mmi1, a previously described factor that functions in the post-transcriptional elimination of meiotic transcripts. Our results support a model in which Mmi1 selectively targets meiotic transcripts for degradation via Pab2 and the exosome. Our findings have therefore uncovered a mode of gene regulation whereby a poly(A)-binding protein promotes RNA degradation in the nucleus to prevent untimely expression.

Crossing the borders: poly(A)-binding proteins working on both sides of the fence.

The addition of a 3’ poly(A) tail is a pre-requisite for the maturation of the majority of eukaryotic transcripts. In most eukaryotic species, RNA poly(A) tails are bound by two important poly(A)-binding proteins (PABPs): PABPC1 and PABPN1 that localize to the cytoplasm and the nucleus, respectively. Such steady state localization for PABPN1 and PABPC1 led to a model whereby PABPN1-bound nuclear mRNAs are remodelled during or after nuclear export so that PABPN1 is replaced by PABPC1 to allow robust cap-dependent translation in the cytoplasm. Here we discuss evidence that challenge the view in which PABPN1 and PABPC1 function solely in the nucleus and cytoplasm, respectively. We discuss accumulating evidence that support nuclear roles for PABPC1 in mRNA biogenesis as well as cytoplasmic roles for PABPN1 in translational control. Because 3’ poly(A) tails can also act as a degradation mark via the exosome complex of 3’5’ exonucleases, we also discuss recent results that involve the nuclear PABP in posttranscriptional gene regulation.

Cotranscriptional recruitment of the nuclear poly(A)-binding protein Pab2 to nascent transcripts and association with translating mRNPs

Synthesis of the pre-mRNA poly(A) tail in the nucleus has important consequences on the translational activity of the mature mRNA in the cytoplasm. In most eukaryotes, nuclear polyadenylation of pre-mRNAs is thought to require the nuclear poly(A)-binding protein (PABP2/PABPN1) for poly(A) tail synthesis and ultimate length control. As yet, however, the extent of the association between PABP2 and the exported mRNA remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) assays to show that the fission yeast ortholog of mammalian PABP2 (Pab2) is cotranscriptionally recruited to active genes. Notably, the association of Pab2 to genes precedes that of a typical 3′-processing/polyadenylation factor, suggesting that Pab2 recruitment during the transcription cycle precedes polyadenylation. The inclusion of an RNase step in our ChIP and immunoprecipitation assays suggests that Pab2 is cotranscriptionally recruited via nascent mRNA ribonucleoprotein (mRNPs). Tandem affinity purification coupled with mass spectrometry also revealed that Pab2 associates with several ribosomal proteins as well as general translation factors. Importantly, whereas previous results suggest that the nuclear poly(A)-binding protein is not present on cytoplasmic mRNAs, we show that fission yeast Pab2 is associated with polysomes. Our findings suggest that Pab2 is recruited to nascent mRNPs during transcription and remains associated with translated mRNPs after nuclear export.