Publications (15)236.87 Total impact
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Article: CapSeq and CIP-TAP Identify Pol II Start Sites and Reveal Capped Small RNAs as C. elegans piRNA Precursors.
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ABSTRACT: Piwi-interacting (pi) RNAs are germline-expressed small RNAs linked to epigenetic programming. C. elegans piRNAs are thought to be transcribed as independent gene-like loci. To test this idea and to identify potential transcription start (TS) sites for piRNA precursors, we developed CapSeq, an efficient enzymatic method for 5' anchored RNA profiling. Using CapSeq, we identify candidate TS sites, defined by 70-90 nt sequence tags, for >50% of annotated Pol II loci. Surprisingly, however, these CapSeq tags failed to identify the overwhelming majority of piRNA loci. Instead, we show that the likely piRNA precursors are ∼26 nt capped small (cs) RNAs that initiate precisely 2 nt upstream of mature piRNAs and that piRNA processing or stability requires a U at the csRNA +3 position. Finally, we identify a heretofore unrecognized class of piRNAs processed from csRNAs that are expressed at promoters genome wide, nearly doubling the number of piRNAs available for genome surveillance.Cell 12/2012; 151(7):1488-500. · 32.40 Impact Factor -
Article: Specific miRNA Stabilization by Gld2-Catalyzed Monoadenylation.
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ABSTRACT: MicroRNAs (miRNAs) are small, noncoding RNAs that inhibit translation and promote mRNA decay. The levels of mature miRNAs are the result of different rates of transcription, processing, and turnover. The noncanonical polymerase Gld2 has been implicated in the stabilization of miR-122, possibly through catalyzing 3' monoadenylation; however, there is little evidence that this relationship is one of cause and effect. Here, we biochemically characterize Gld2's involvement in miRNA monoadenylation and its effect on miRNA stability. We find that Gld2 directly monoadenylates and stabilizes specific miRNA populations in human fibroblasts and that sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3' end. These results establish a mechanism of miRNA stability and resulting posttranscriptional gene regulation.Cell reports. 11/2012; -
Article: The translin-TRAX complex (C3PO) is a ribonuclease in tRNA processing.
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ABSTRACT: Conserved translin-TRAX complexes, also known as C3POs, have been implicated in many biological processes, but how they function remains unclear. Recently, C3PO was shown to be an endoRNase that promotes RNA interference (RNAi) in animal cells. Here, we show that C3PO does not play a significant role in RNAi in the filamentous fungus Neurospora crassa. Instead, the Neurospora C3PO functions as an RNase that removes the 5' pre-tRNA fragments after the processing of pre-tRNAs by RNase P. In addition, translin and trax mutants have elevated levels of tRNA and protein translation and are more resistant to a cell death-inducing agent. Finally, we show that C3PO is also involved in tRNA processing in mouse embryonic fibroblast cells. This study identifies the endogenous RNA substrates of C3PO and provides a potential explanation for its roles in apparently diverse biological processes.Nature Structural & Molecular Biology 07/2012; 19(8):824-30. · 12.71 Impact Factor -
Article: piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline.
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ABSTRACT: Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish "self" from "nonself" nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of "self" and "nonself" RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression.Cell 06/2012; 150(1):65-77. · 32.40 Impact Factor -
Article: C. elegans piRNAs mediate the genome-wide surveillance of germline transcripts.
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ABSTRACT: Piwi Argonautes and Piwi-interacting RNAs (piRNAs) mediate genome defense by targeting transposons. However, many piRNA species lack obvious sequence complementarity to transposons or other loci; only one C. elegans transposon is a known piRNA target. Here, we show that, in mutants lacking the Piwi Argonaute PRG-1 (and consequently its associated piRNAs/21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression with a concomitant depletion of RNA-dependent RNA polymerase (RdRP)-derived secondary small RNAs termed 22G-RNAs. Sequences depleted of 22G-RNAs are proximal to potential target sites that base pair imperfectly but extensively to 21U-RNAs. We show that PRG-1 is required to initiate, but not to maintain, silencing of transgenes engineered to contain complementarity to endogenous 21U-RNAs. Our findings support a model in which C. elegans piRNAs utilize their enormous repertoire of targeting capacity to scan the germline transcriptome for foreign sequences, while endogenous germline-expressed genes are actively protected from piRNA-induced silencing.Cell 06/2012; 150(1):78-87. · 32.40 Impact Factor -
Article: Cloning Argonaute-associated small RNAs from Caenorhabditis elegans.
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ABSTRACT: Small RNA pathways fulfill a plethora of gene-regulatory functions in a variety of organisms. In the nematode worm, Caenorhabditis elegans, a number of endogenous small RNA pathways have been described, including the microRNA pathway, the 21U/piRNA pathway, the 26G-RNA pathways, and the 22G-RNA pathways. Argonaute proteins are key effector molecules of each pathway that, together with their small RNA cofactors regulate various processes including developmental timing, fertility, transposon silencing, and chromosome segregation. Although several of the 26 Argonautes in the worm have been studied to date, a number have yet to be fully characterized or their small RNA binding complement defined. The identification of small RNAs that copurify with an Argonaute family member is central to understanding the targets and assessing the function of that Argonaute. Here we discuss the rationale for generating reagents to immunoprecipitate Argonaute complexes and provide a cohesive protocol for the cloning and Illumina deep-sequencing of Argonaute-associated small RNAs in C. elegans.Methods in molecular biology (Clifton, N.J.) 01/2011; 725:251-80. -
Article: Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi.
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ABSTRACT: A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNAs associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP, and an RNase III domain-containing protein, MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.Molecular cell 06/2010; 38(6):803-14. · 14.61 Impact Factor -
Article: Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans.
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ABSTRACT: Gametogenesis is a thermosensitive process in numerous metazoans, ranging from worms to man. In Caenorhabditis elegans, a variety of RNA-binding proteins that associate with germ-line nuage (P granules), including the Piwi-clade argonaute PRG-1, have been implicated in maintaining fertility at elevated temperature. Here we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3), in promoting thermotolerant male fertility. A rescuing GFP::alg-3 transgene is localized to P granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, the 26G-RNAs which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for thermotolerant fertility in C. elegans and support a model in which AGOs and their small RNA cofactors function to promote robustness in gene-expression networks.Proceedings of the National Academy of Sciences 02/2010; 107(8):3588-93. · 9.68 Impact Factor -
Article: Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway.
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ABSTRACT: Argonaute (AGO) proteins interact with distinct classes of small RNAs to direct multiple regulatory outcomes. In many organisms, including plants, fungi, and nematodes, cellular RNA-dependent RNA polymerases (RdRPs) use AGO targets as templates for amplification of silencing signals. Here, we show that distinct RdRPs function sequentially to produce small RNAs that target endogenous loci in Caenorhabditis elegans. We show that DCR-1, the RdRP RRF-3, and the dsRNA-binding protein RDE-4 are required for the biogenesis of 26-nt small RNAs with a 5' guanine (26G-RNAs) and that 26G-RNAs engage the Piwi-clade AGO, ERGO-1. Our findings support a model in which targeting by ERGO-1 recruits a second RdRP (RRF-1 or EGO-1), which in turn transcribes 22G-RNAs that interact with worm-specific AGOs (WAGOs) to direct gene silencing. ERGO-1 targets exhibit a nonrandom distribution in the genome and appear to include many gene duplications, suggesting that this pathway may control overexpression resulting from gene expansion.Proceedings of the National Academy of Sciences 02/2010; 107(8):3582-7. · 9.68 Impact Factor -
Article: Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.
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ABSTRACT: Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs.Molecular cell 10/2009; 36(2):231-44. · 14.61 Impact Factor -
Article: The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.
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ABSTRACT: RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.Cell 10/2009; 139(1):123-34. · 32.40 Impact Factor -
Article: PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans.
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ABSTRACT: In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.Molecular cell 08/2008; 31(1):67-78. · 14.61 Impact Factor -
Article: Rapid tRNA decay can result from lack of nonessential modifications.
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ABSTRACT: The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.Molecular Cell 02/2006; 21(1):87-96. · 14.18 Impact Factor -
Article: Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C.
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ABSTRACT: The essential Saccharomyces cerevisiae tRNA(His) guanylyltransferase (Thg1p) is responsible for the unusual G(-1) addition to the 5' end of cytoplasmic tRNA(His). We report here that tRNA(His) from Thg1p-depleted cells is uncharged, although histidyl tRNA synthetase is active and the 3' end of the tRNA is intact, suggesting that G(-1) is a critical determinant for aminoacylation of tRNA(His) in vivo. Thg1p depletion leads to activation of the GCN4 pathway, most, but not all, of which is Gcn2p dependent, and to the accumulation of tRNA(His) in the nucleus. Surprisingly, tRNA(His) in Thg1p-depleted cells accumulates additional m(5)C modifications, which are delayed relative to the loss of G(-1) and aminoacylation. The additional modification is likely due to tRNA m(5)C methyltransferase Trm4p. We developed a new method to map m(5)C residues in RNA and localized the additional m(5)C to positions 48 and 50. This is the first documented example of the accumulation of additional modifications in a eukaryotic tRNA species.Molecular and Cellular Biology 10/2005; 25(18):8191-201. · 5.53 Impact Factor -
Article: tRNAHis maturation: an essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNAHis.
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ABSTRACT: All tRNAHis molecules are unusual in having an extra 5' GMP residue (G(-1)) that, in eukaryotes, is added after transcription and RNase P cleavage. Incorporation of this G(-1) residue is a rare example of nucleotide addition occurring at an RNA 5' end in a normal phosphodiester linkage. We show here that the essential Saccharomyces cerevisiae ORF YGR024c (THG1) is responsible for this guanylyltransferase reaction. Thg1p was identified by survey of a genomic collection of yeast GST-ORF fusion proteins for addition of [alpha-32P]GTP to tRNAHis. End analysis confirms the presence of G(-1). Thg1p is required for tRNAHis guanylylation in vivo, because cells depleted of Thg1p lack G(-1) in their tRNAHis. His6-Thg1p purified from Escherichia coli catalyzes the guanylyltransferase step of G(-1) addition using a ppp-tRNAHis substrate, and appears to catalyze the activation step using p-tRNAHis and ATP. Thg1p is highlye conserved in eukaryotes, where G(-1) addition is necessary, and is not found in eubacteria, where G(-1) is genome-encoded. Thus, Thg1p is the first member of a new family of enzymes that can catalyze phosphodiester bond formation at the 5' end of RNAs, formally in a 3'-5' direction. Surprisingly, despite its varied activities, Thg1p contains no recognizable catalytic or functional domains.Genes & Development 01/2004; 17(23):2889-901. · 11.66 Impact Factor
Top co-authors
Top Journals
Institutions
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2008–2012
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University of Massachusetts Medical School
- Program in Molecular Medicine
Worcester, MA, USA
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2010
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Howard Hughes Medical Institute
Chevy Chase, MD, USA
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2004–2006
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University of Rochester
- Department of Biochemistry and Biophysics
Rochester, NY, USA
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