Vandivier, Lee E

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2012 - 20155
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  • Publication
    Detection of Pol IV/RDR2-Dependent Transcripts at the Genomic Scale in Arabidopsis Reveals Features and Regulation of siRNA Biogenesis
    (2015-02-01) Vandivier, Lee E; Li, Shaofang; Tu, Bin; Gao, Lei; Won, So Youn; Li, Shengben; Gregory, Brian D; Zheng, Binglian; Chen, Xuemei
    Twenty-four-nucleotide small interfering (si)RNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes and maintains DNA methylation at transposable elements to ensure genome stability in plants. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is believed to transcribe RdDM loci to produce primary transcripts that are converted to double-stranded RNAs (dsRNAs) by RDR2 to serve as siRNA precursors. Yet, no such siRNA precursor transcripts have ever been reported. Here, through genome-wide profiling of RNAs in genotypes that compromise the processing of siRNA precursors, we were able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci. We show that Pol IV/RDR2-dependent transcripts correspond to both DNA strands, whereas the RNA polymerase II (Pol II)-dependent transcripts produced upon derepression of the loci are derived primarily from one strand. We also show that Pol IV/RDR2-dependent transcripts have a 5′ monophosphate, lack a poly(A) tail at the 3′ end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Like Pol II-transcribed genic regions, Pol IV-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes, which highlights similarities in Pol II- and Pol IV-mediated transcription. Computational analysis of siRNA abundance from various mutants reveals differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation via two different DNA methyltransferases. These findings begin to reveal features of Pol IV/RDR2-mediated transcription at the heart of genome stability in plants.
  • Publication
    The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins
    (2015-12-01) Li, Ning; Yousefi, Maryam; Nakauka-Ddamba, Angela; Vandivier, Lee; Naqvi, Ammar S; Rao, Shilpa; Tobias, John; Cedeno, Ryan J; Valvezan, Alexander James; Shankar, Sheila; Li, Fan; Klein, Peter S; Parada, Kimberly; Jensen, Shane T; Woo, Dong-Hun; Wang, Shan; Gregory, Brian D; Lengner, Christopher J; Minuesa, Gerard; Y, Katz; Barlowe, Trevor S; Deering, Raquel R; Kharas, Michael G; Yu, Zhengquan
    Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.
  • Publication
    Essential Role for Endogenous siRNAs during Meiosis in Mouse Oocytes
    (2015-02-19) Cárdenas, Fabián L; Davydenko, Olga; Stein, Paula; Vandivier, Lee; Rozhkov, Nikolay V; Gregory, Brian D; Li, Fan; Schultz, Richard M; Hannon, Gregory J
    In animals, the three main classes of small RNAs are microRNAs, short interfering RNAs, and PIWI-interacting RNAs. All three RNA species silence gene expression post-transcriptionally through interaction with the ARGONAUTE family of proteins. In mammals in particular, microRNAs are ubiquitously expressed, are essential for development, and perform numerous functions in a variety of cells and tissues. piRNAs are expressed almost exclusively in the germline, and are essential for male fertility and defense against transposons. Endogenous siRNAs are only expressed in germ cells and embryonic stem cells and have not been ascribed a functional role. By engineering a mouse that expresses a modified ARGONAUTE protein, we disrupt the function of endo-siRNAs exclusively in oocytes and find that females are infertile. Oocytes with an impaired siRNA pathway fail to complete meiosis I, and display severe spindle formation and chromosome alignment defects. Their transcriptome is widely perturbed and expression of the most abundant transposon is increased. These findings indicate that endo-siRNAs are essential for female fertility in mouse, are required for spindle formation, chromosome congression, and defense against transposons. This study unequivocally demonstrates an essential function for siRNAs in mammals, mediated through endonucleolytic cleavage of targets, and provides an explanation for the selective pressure that one AGO protein retains catalytic activity.
  • Publication
    Chemical Modifications Mark Alternatively Spliced and Uncapped Messenger RNAs in Arabidposis
    (2015-11-01) Vandivier, Lee E; Kuksa, Pavel P; Wang, Li-San; Campos, Rafael; Gregory, Brian D; Silverman, Ian M
    Posttranscriptional chemical modification of RNA bases is a widespread and physiologically relevant regulator of RNA maturation, stability, and function. While modifications are best characterized in short, noncoding RNAs such as tRNAs, growing evidence indicates that mRNAs and long noncoding RNAs (lncRNAs) are likewise modified. Here, we apply our high-throughput annotation of modified ribonucleotides (HAMR) pipeline to identify and classify modifications that affect Watson-Crick base pairing at three different levels of the Arabidopsis thaliana transcriptome (polyadenylated, small, and degrading RNAs). We find this type of modifications primarily within uncapped, degrading mRNAs and lncRNAs, suggesting they are the cause or consequence of RNA turnover. Additionally, modifications within stable mRNAs tend to occur in alternatively spliced introns, suggesting they regulate splicing. Furthermore, these modifications target mRNAs with coherent functions, including stress responses. Thus, our comprehensive analysis across multiple RNA classes yields insights into the functions of covalent RNA modifications in plant transcriptomes.
  • Publication
    Regulatory Impact of RNA Secondary Structure across the Arabidopsis Transcriptome
    (2012-11-01) Li, Fan; Vandivier, Lee E; Gregory, Brian D; Willmann, Matthew R; Chen, Ying
    The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, and function. However, the global influence of this feature on plant gene expression is still largely unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of rRNA-depleted (RNA sequencing), small RNA, and ribosome-bound RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis thaliana. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing evidence that secondary structure is necessary for these RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We further reveal that RNA folding is significantly anticorrelated with overall transcript abundance, which is often due to the increased propensity of highly structured mRNAs to be degraded and/or processed into small RNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. These findings provide a global assessment of RNA folding and its significant regulatory effects in a plant transcriptome.