Amy E Pasquinelli

University of California, San Diego, San Diego, California, United States

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Publications (49)691.4 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate gene expression in many multicellular organisms. They are encoded in the genome and transcribed into primary (pri-) miRNAs before two processing steps that ultimately produce the mature miRNA. In order to generate the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is essential. Here we identify the Period protein homolog LIN-42 as a new regulator of miRNA biogenesis in Caenorhabditis elegans. We mapped a spontaneous suppressor of the normally lethal let-7(n2853) allele to the lin-42 gene. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at most time points when mature let-7 miRNA is normally expressed. Levels of pri-let-7 and a let-7 transcriptional reporter were also increased in lin-42(n1089) worms. These results indicate that LIN-42 normally represses pri-let-7 transcription and thus the accumulation of let-7 miRNA. This inhibition is not specific to let-7, as pri- and mature levels of lin-4 and miR-35 were also increased in lin-42 mutants. Furthermore, small RNA-seq analysis showed widespread increases in the levels of mature miRNAs in lin-42 mutants. Thus, we propose that the period protein homolog LIN-42 is a global regulator of miRNA biogenesis.
    Developmental Biology 01/2014; · 3.87 Impact Factor
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    ABSTRACT: Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a complement and extension to the WormBook chapter Biochemistry and molecular biology, which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry.
    WormBook : the online review of C. elegans biology. 01/2014;
  • Amy E Pasquinelli
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    ABSTRACT: The let-7 miRNA (microRNA) is an essential regulator of development from nematode worms to humans. Altered expression of let-7 results in larval arrest or lethality in Caenorhabditis elegans. Likewise, under- or over-expression of let-7 in human cells can result in cellular overproliferation or halted cell division respectively. Thus the biogenesis of this critical miRNA is controlled at multiple levels. An unexpected mechanism for regulating the initial processing of let-7 was recently found to involve the let-7 miRNA itself. The mature let-7 miRNA along with its effector protein, Argonaute, were shown to bind to a site in the primary transcripts produced by the let-7 gene. This interaction enhances processing through a novel auto-regulatory feedback loop. This discovery highlights a new role for the miRNA complex in regulating miRNA biogenesis and enriches the classes of RNAs targeted by Argonaute.
    Biochemical Society Transactions 08/2013; 41(4):821-4. · 2.59 Impact Factor
  • James P Broughton, Amy E Pasquinelli
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    ABSTRACT: The identification of endogenous targets remains an important challenge in understanding microRNA (miRNA) function. Past approaches using in silico methods and reporter constructs lack biological context that may enhance or inhibit target recognition. To address these limitations, several labs have utilized crosslinking and immunoprecipitation (CLIP) of Argonaute (Ago) proteins to identify miRNA targets. Recently, the Ule Lab introduced individual-nucleotide resolution CLIP (iCLIP) to increase the sensitivity of identifying protein-RNA interaction sites. Here we adapt the iCLIP protocol for use in Caenorhabditis elegans to identify endogenous sites targeted by the worm Argonaute (ALG-1) primarily responsible for miRNA function.
    Methods 04/2013; · 3.64 Impact Factor
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    ABSTRACT: The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7-dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.
    PLoS Genetics 03/2013; 9(3):e1003353. · 8.52 Impact Factor
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    Katlin B Massirer, Amy E Pasquinelli
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    ABSTRACT: A recent study by Massirer et al. in the nematode C. elegans has shown that a family of microRNAs (miRNAs), miR-35-41, regulates the efficiency of RNA interference (RNAi), revealing a new connection between these small RNA pathways. In this commentary, we discuss the potential mechanisms for cross regulation in the miRNA and RNAi pathways and the implications for gene expression. While miRNAs are genetically encoded, the small interfering RNAs (siRNAs) that function in RNAi can originate from processing of exogenous dsRNA (exo-RNAi) or from the production of siRNAs from endogenous transcripts (endo-RNAi). These small RNA pathways involve Dicer and Argonaute proteins and typically use antisense base pairing to target mRNAs for downregulated expression. The discovery that loss of miR-35-41 results in enhanced exo-RNAi sensitivity and reduced endo-RNAi effectiveness suggests that these miRNAs normally help balance the RNAi pathways. The effect of mir-35-41 on RNAi is largely through lin-35, the C. elegans homolog of the tumor suppressor Retinoblastoma (Rb) gene. lin-35/Rb previously has been shown to regulate RNAi sensitivity through unclear mechanisms and the new finding that accumulation of LIN-35/Rb protein is dependent on miR-35-41 adds another layer of complexity to this process. The utilization of miRNAs to control the responsiveness of RNAi exemplifies the cross-regulation embedded in small RNA-directed pathways.
    Worm. 01/2013; 2(1):e21835.
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    ABSTRACT: The let-7 microRNA (miRNA) is highly conserved across animal phyla and generally regulates cellular differentiation and developmental timing pathways. In C. elegans, the mature let-7 miRNA starts to accumulate in the last stages of larval development where it directs cellular differentiation programs required for adult fates. Here we show that expression of the let-7 gene in C. elegans is under complex transcriptional control. The onset of let-7 transcription begins as early as the first larval stage in some tissues, and as late as the third larval stage in others, and is abrogated at the gravid adult stage. Transcription from two different start sites in the let-7 promoter oscillates during each larval stage. We show that transcription is regulated by two distinct cis-elements in the promoter of let-7, the previously described temporal regulatory element (TRE), and a novel element downstream of the TRE that we have named the let-7 transcription element (LTE). These elements play distinct and redundant roles in regulating let-7 expression in specific tissues. In the absence of the TRE and LTE, transcription of let-7 is undetectable and worms exhibit the lethal phenotype characteristic of let-7 null mutants. We also identify several genes that affect the transcription of let-7 generally and tissue-specifically. Overall, spatio-temporal regulation of let-7 transcription is orchestrated by multiple cis- and trans-acting factors to ensure appropriate expression of this essential miRNA during worm development.
    Developmental Biology 11/2012; · 3.87 Impact Factor
  • Emily F Finnegan, Amy E Pasquinelli
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    ABSTRACT: MicroRNAs (miRNAs) function as 21-24 nucleotide guide RNAs that use partial base-pairing to recognize target messenger RNAs and repress their expression. As a large fraction of protein-coding genes are under miRNA control, production of the appropriate level of specific miRNAs at the right time and in the right place is integral to most gene regulatory pathways. MiRNA biogenesis initiates with transcription, followed by multiple processing steps to produce the mature miRNA. Every step of miRNA production is subject to regulation and disruption of these control mechanisms has been linked to numerous human diseases, where the balance between the expression of miRNAs and their targets becomes distorted. Here we review the basic steps of miRNA biogenesis and describe the various factors that control miRNA transcription, processing, and stability in animal cells. The tremendous effort put into producing the appropriate type and level of specific miRNAs underscores the critical role of these small RNAs in gene regulation.
    Critical Reviews in Biochemistry and Molecular Biology 11/2012; · 5.58 Impact Factor
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    Amy E Pasquinelli
    The EMBO Journal 09/2012; 31(19):3790-1. · 9.82 Impact Factor
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    ABSTRACT: MicroRNAs (miRNAs) comprise a large family of small RNA molecules that post-transcriptionally regulate gene expression in many biological pathways. Most miRNAs are derived from long primary transcripts that undergo processing by Drosha to produce ~65-nucleotide precursors that are then cleaved by Dicer, resulting in the mature 22-nucleotide forms. Serving as guides in Argonaute protein complexes, mature miRNAs use imperfect base pairing to recognize sequences in messenger RNA transcripts, leading to translational repression and destabilization of the target messenger RNAs. Here we show that the miRNA complex also targets and regulates non-coding RNAs that serve as substrates for the miRNA-processing pathway. We found that the Argonaute protein in Caenorhabditis elegans, ALG-1, binds to a specific site at the 3′ end of let-7 miRNA primary transcripts and promotes downstream processing events. This interaction is mediated by mature let-7 miRNA through a conserved complementary site in its own primary transcript, thus creating a positive-feedback loop. We further show that ALG-1 associates with let-7 primary transcripts in nuclear fractions. Argonaute also binds let-7 primary transcripts in human cells, demonstrating that the miRNA pathway targets non-coding RNAs in addition to protein-coding messenger RNAs across species. Moreover, our studies in C. elegans reveal a novel role for Argonaute in promoting biogenesis of a targeted transcript, expanding the functions of the miRNA pathway in gene regulation. This discovery of autoregulation of let-7 biogenesis establishes a new mechanism for controlling miRNA expression.
    Nature 06/2012; 486(7404):541-4. · 38.60 Impact Factor
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    Antti P Aalto, Amy E Pasquinelli
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    ABSTRACT: During the past decade, it has become evident that small non-coding RNAs (ncRNAs) participate in widespread and essential regulatory mechanisms in most eukaryotic cells. Novel classes of small RNAs, their biogenesis pathways and cellular effects are continuously being described, and new properties of already established ncRNAs are still being discovered. As the list of small RNA molecules and their roles becomes more and more extensive, one can get lost in the midst of new information. In this review, we attempt to bring order to the small ncRNA transcriptome by covering some of the major milestones of recent years. We go through many of the new properties that have been attributed to already familiar RNA molecules, and introduce some of the more recent novel classes of tiny ncRNAs.
    Current opinion in cell biology 03/2012; 24(3):333-40. · 14.15 Impact Factor
  • Amy E Pasquinelli
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    ABSTRACT: MicroRNAs (miRNAs) have emerged as key gene regulators in diverse biological pathways. These small non-coding RNAs bind to target sequences in mRNAs, typically resulting in repressed gene expression. Several methods are now available for identifying miRNA target sites, but the mere presence of an miRNA-binding site is insufficient for predicting target regulation. Regulation of targets by miRNAs is subject to various levels of control, and recent developments have presented a new twist; targets can reciprocally control the level and function of miRNAs. This mutual regulation of miRNAs and target genes is challenging our understanding of the gene-regulatory role of miRNAs in vivo and has important implications for the use of these RNAs in therapeutic settings.
    Nature Reviews Genetics 03/2012; 13(4):271-82. · 41.06 Impact Factor
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    ABSTRACT: RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo-RNAi) or natural endogenous (endo-RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo-RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo-siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs.
    PLoS Genetics 03/2012; 8(3):e1002536. · 8.52 Impact Factor
  • Amy E Pasquinelli
    Nature Structural & Molecular Biology 02/2012; 19(2):133-4. · 11.90 Impact Factor
  • Vanessa Mondol, Amy E Pasquinelli
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    ABSTRACT: Noncoding RNAs have emerged as an integral part of posttranscriptional gene regulation. Among that class of RNAs are the microRNAs (miRNAs), which posttranscriptionally regulate target mRNAs containing complementary sequences. The broad presence of miRNAs in lower eukaryotes, plants, and mammals highlights their importance throughout evolution. MiRNAs have been shown to regulate many pathways, including development, and disruption of miRNA function can lead to disease (Ivey and Srivastava, 2010; Jiang et al., 2009). Although the first miRNA genes were discovered in the nematode, Caenorhabditis elegans, almost 20 years ago, the field of miRNA research began when they were found in multiple organisms a little over a decade ago (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001; Lee et al., 1993; Pasquinelli et al., 2000; Wightman et al., 1993). Here, we review one of the first characterized miRNAs, let-7, and describe its role in development and the intricacies of its biogenesis and function.
    Current Topics in Developmental Biology 01/2012; 99:1-30. · 6.91 Impact Factor
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    ABSTRACT: The highly conserved let-7 microRNA (miRNA) regulates developmental pathways across animal phyla. Mis-expression of let-7 causes lethality in C. elegans and has been associated with several human diseases. We show that timing of let-7 expression in developing worms is under complex transcriptional and post-transcriptional control. Expression of let-7 primary transcripts oscillates during each larval stage, but precursor and mature let-7 miRNAs do not accumulate until later in development after LIN-28 protein has diminished. We demonstrate that LIN-28 binds endogenous primary let-7 transcripts co-transcriptionally. We further show that LIN-28 binds endogenous primary let-7 transcripts in the nuclear compartment of human ES cells, suggesting that this LIN-28 activity is conserved across species. We conclude that co-transcriptional interaction of LIN-28 with let-7 primary transcripts blocks Drosha processing and, thus, precocious expression of mature let-7 during early development.
    Nature Structural & Molecular Biology 02/2011; 18(3):302-8. · 11.90 Impact Factor
  • Dimitrios G Zisoulis, Gene W Yeo, Amy E Pasquinelli
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    ABSTRACT: MicroRNAs (miRNAs) are small RNA molecules that posttranscriptionally regulate the expression of protein-coding genes. The mature miRNAs are loaded into Argonaute-containing protein complexes (miRISC, miRNA Induced S  ilencing Complex), and guide these complexes to the 3' UTR of targeted mRNA transcripts via base-pairing interactions. However, the imperfect complementarity that characterizes the interactions between animal miRNAs and target sites complicates the identification of direct target genes. We developed a biochemical method to identify on a large scale the target sequences recognized by miRISC in vivo. The mRNA sites bound by miRISC are stabilized by cross-linking and isolated by immunoprecipitation of Argonaute-containing complexes. The bound RNA molecules are trimmed to the regions protected by Argonaute, subjected to a series of isolation and linker ligation steps and identified by high-throughput sequencing methods.
    Methods in molecular biology (Clifton, N.J.) 01/2011; 732:169-85. · 1.29 Impact Factor
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    ABSTRACT: Originally discovered in C. elegans, microRNAs (miRNAs) are small RNAs that regulate fundamental cellular processes in diverse organisms. MiRNAs are encoded within the genome and are initially transcribed as primary transcripts that can be several kilobases in length. Primary transcripts are successively cleaved by two RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to produce ∼70 nucleotide (nt) long precursor miRNAs and 22 nt long mature miRNAs, respectively. Mature miRNAs regulate gene expression post-transcriptionally by imperfectly binding target mRNAs in association with the multiprotein RNA induced silencing complex (RISC). The conserved sequence, expression pattern, and function of some miRNAs across distinct species as well as the importance of specific miRNAs in many biological pathways have led to an explosion in the study of miRNA biogenesis, miRNA target identification, and miRNA target regulation. Many advances in our understanding of miRNA biology have come from studies in the powerful model organism C. elegans. This chapter reviews the current methods used in C. elegans to study miRNA biogenesis, small RNA populations, miRNA-protein complexes, and miRNA target regulation.
    Methods in cell biology 01/2011; 106:219-52. · 1.44 Impact Factor
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    ABSTRACT: Transcription and multiple processing steps are required to produce specific 22 nucleotide microRNAs (miRNAs) that can regulate the expression of target genes. In C. elegans, mature lin-4 miRNA accumulates at the end of the first larval stage to repress its direct targets lin-14 and lin-28, allowing the progression of several somatic cell types to later larval fates. In this study, we characterized the expression of endogenous lin-4 and found that temporally regulated independent transcripts, but not constitutive lin-4 containing RNAs derived from an overlapping gene, are processed to mature lin-4 miRNA. Through an RNAi screen, we identified a conserved RNA binding protein gene rbm-28 (R05H10.2), homologous to the human RBM28 and yeast Nop4p proteins, that is important for lin-4 expression in C. elegans. We also demonstrate that rbm-28 genetically interacts with the lin-4 developmental timing pathway and uncover a previously unrecognized role for lin-14 and lin-28 in coordinating organismal growth.
    Developmental Biology 10/2010; 348(2):210-21. · 3.87 Impact Factor
  • Amy E Pasquinelli
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    ABSTRACT: MicroRNAs (miRNAs) repress gene expression by forming base pairs with specific sequences in protein-coding messenger RNAs (mRNAs). The degree of complementarity between a miRNA and its target mRNA determines the mechanism of posttranscriptional regulation (1). Nearly perfect pairing induces cleavage of the target mRNA, whereas partial pairing results in translational repression and mRNA decay through deadenylation pathways. On pages 1534 and 1563 of this issue, Ameres et al. (2) and Cazalla et al. (3) reveal that, surprisingly, the extent of base-pairing also affects stability of the miRNA itself.
    Science 06/2010; 328(5985):1494-5. · 31.20 Impact Factor

Publication Stats

9k Citations
691.40 Total Impact Points

Institutions

  • 2003–2014
    • University of California, San Diego
      • • Department of Cellular and Molecular Medicine (CMM)
      • • Section of Molecular Biology
      San Diego, California, United States
  • 2012
    • University of Helsinki
      Helsinki, Southern Finland Province, Finland
  • 2010
    • California Stem Cell
      Irvine, California, United States
  • 2000–2005
    • Massachusetts General Hospital
      • • Department of Molecular Biology
      • • Molecular Biology Laboratory
      Boston, MA, United States
  • 2002
    • Harvard Medical School
      • Department of Genetics
      Boston, MA, United States