The exonuclease Nibbler regulates age-associated traits and modulates piRNA length in Drosophila

Nibbler (Nbr) is a 3′‐to‐5′ exonuclease that trims the 3′end of microRNAs (miRNAs) to generate different length patterns of miRNAs in Drosophila. Despite its effect on miRNAs, we lack knowledge of its biological significance and whether Nbr affects other classes of small RNAs such as piRNAs and endo‐siRNAs. Here, we characterized the in vivo function of nbr by defining the Nbr protein expression pattern and loss‐of‐function effects. Nbr protein is enriched in the ovary and head. Analysis of nbr null animals reveals adult‐stage defects that progress with age, including held‐up wings, decreased locomotion, and brain vacuoles, indicative of accelerated age‐associated processes upon nbr loss. Importantly, these effects depend on catalytic residues in the Nbr exonuclease domain, indicating that the catalytic activity is responsible for these effects. Given the impact of nbr on miRNAs, we also analyzed the effect of nbr on piRNA and endo‐siRNA lengths by deep‐sequence analysis of libraries from ovaries. As with miRNAs, nbr mutation led to longer length piRNAs – an effect that was dependent on the catalytic residues of the exonuclease domain. These analyses indicate a role of nbr on age‐associated processes and to modulate length of multiple classes of small RNAs including miRNAs and piRNAs in Drosophila.

[1]  Gert-Jan Hendriks,et al.  Impact of age-associated increase in 2′-O-methylation of miRNAs on aging and neurodegeneration in Drosophila , 2014, Genes & development.

[2]  V. Ambros.  MicroRNAs and developmental timing. , 2011, Current opinion in genetics & development.

[3]  F. Slack,et al.  Ageing and the small, non-coding RNA world , 2013, Ageing Research Reviews.

[4]  Ammar S Naqvi,et al.  The Exoribonuclease Nibbler Controls 3′ End Processing of MicroRNAs in Drosophila , 2011, Current Biology.

[5]  Haifan Lin,et al.  A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. , 1998, Genes & development.

[6]  Zhiping Weng,et al.  Dicer Partner Proteins Tune the Length of Mature miRNAs in Flies and Mammals , 2012, Cell.

[7]  Nahum Sonenberg,et al.  The mechanics of miRNA-mediated gene silencing: a look under the hood of miRISC , 2012, Nature Structural &Molecular Biology.

[8]  Manolis Kellis,et al.  Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs. , 2007, Genome research.

[9]  Gene W Yeo,et al.  RNA sequence analysis defines Dicer's role in mouse embryonic stem cells , 2007, Proceedings of the National Academy of Sciences.

[10]  Yi Li,et al.  PCR-based accurate synthesis of long DNA sequences , 2006, Nature Protocols.

[11]  A. Spradling,et al.  A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. , 1997, Development.

[12]  J. Doudna,et al.  TRBP alters human precursor microRNA processing in vitro. , 2012, RNA.

[13]  Brian P. Brunk,et al.  Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq unified mapper (RUM) , 2011, Bioinform..

[14]  C. Sander,et al.  A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing , 2007, Cell.

[15]  L. Young,et al.  Two-step total gene synthesis method. , 2004, Nucleic acids research.

[16]  Julius Brennecke,et al.  Specialized piRNA Pathways Act in Germline and Somatic Tissues of the Drosophila Ovary , 2009, Cell.

[17]  Ryan D. Morin,et al.  Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. , 2008, Genome research.

[18]  Dominik Handler,et al.  The Genetic Makeup of the Drosophila piRNA Pathway , 2013, Molecular cell.

[19]  C. Bracken,et al.  IsomiRs--the overlooked repertoire in the dynamic microRNAome. , 2012, Trends in genetics : TIG.

[20]  RNA sequence analysis. , 1970, Annual review of biochemistry.

[21]  Y. Lacasse,et al.  From the authors , 2005, European Respiratory Journal.

[22]  S. Wyman,et al.  Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity. , 2011, Genome research.

[23]  G. Hannon,et al.  Small RNA sorting: matchmaking for Argonautes , 2011, Nature Reviews Genetics.

[24]  G. Rubin,et al.  Computational identification of Drosophila microRNA genes , 2003, Genome Biology.

[25]  N. Perrimon,et al.  An endogenous small interfering RNA pathway in Drosophila , 2008, Nature.

[26]  G. Hannon,et al.  The piRNA pathway in flies: highlights and future directions. , 2013, Current opinion in genetics & development.

[27]  J. Neilson,et al.  Zcchc11-dependent uridylation of microRNA directs cytokine expression , 2009, Nature Cell Biology.

[28]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[29]  S. Kawaoka,et al.  3' end formation of PIWI-interacting RNAs in vitro. , 2011, Molecular cell.

[30]  B. Langmead,et al.  Aligning Short Sequencing Reads with Bowtie , 2010, Current protocols in bioinformatics.

[31]  E. Lai,et al.  Common and distinct patterns of terminal modifications to mirtrons and canonical microRNAs. , 2012, RNA.

[32]  Caifu Chen,et al.  A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila. , 2013, Molecular cell.

[33]  Manolis Kellis,et al.  Discrete Small RNA-Generating Loci as Master Regulators of Transposon Activity in Drosophila , 2007, Cell.

[34]  Li-San Wang,et al.  The microRNA miR-34 modulates aging and neurodegeneration in Drosophila , 2012, Nature.

[35]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[36]  Z. Weng,et al.  Transposition-Driven Genomic Heterogeneity in the Drosophila Brain , 2013, Science.

[37]  Christopher M. Player,et al.  Large-Scale Sequencing Reveals 21U-RNAs and Additional MicroRNAs and Endogenous siRNAs in C. elegans , 2006, Cell.

[38]  Kuniaki Saito,et al.  Structure and function of Zucchini endoribonuclease in piRNA biogenesis , 2012, Nature.

[39]  T. Katoh,et al.  Selective stabilization of mammalian microRNAs by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2. , 2009, Genes & development.

[40]  G. Hannon,et al.  A transcriptome-wide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway. , 2013, Molecular cell.

[41]  Y. Hayashizaki,et al.  A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness. , 2010, Genome research.

[42]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[43]  P. Macdonald,et al.  Aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIF2C. , 2001, Development.

[44]  Jacob Cohen.  Statistical Power Analysis for the Behavioral Sciences , 1969, The SAGE Encyclopedia of Research Design.

[45]  J. Dubnau,et al.  Activation of transposable elements during aging and neuronal decline in Drosophila , 2013, Nature Neuroscience.

[46]  G. Hannon,et al.  The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis , 2012, Nature.

[47]  Bo W. Han,et al.  The 3′-to-5′ Exoribonuclease Nibbler Shapes the 3′ Ends of MicroRNAs Bound to Drosophila Argonaute1 , 2011, Current Biology.