The mammalian TRIM-NHL protein TRIM71/LIN-41 is a repressor of mRNA function

TRIM-NHL proteins are conserved regulators of development and differentiation but their molecular function has remained largely elusive. Here, we report an as yet unrecognized activity for the mammalian TRIM-NHL protein TRIM71 as a repressor of mRNAs. We show that TRIM71 is associated with mRNAs and that it promotes translational repression and mRNA decay. We have identified Rbl1 and Rbl2, two transcription factors whose down-regulation is important for stem cell function, as TRIM71 targets in mouse embryonic stem cells. Furthermore, one of the defining features of TRIM-NHL proteins, the NHL domain, is necessary and sufficient to target TRIM71 to RNA, while the RING domain that confers ubiquitin ligase activity is dispensable for repression. Our results reveal strong similarities between TRIM71 and Drosophila BRAT, the best-studied TRIM-NHL protein and a well-documented translational repressor, suggesting that BRAT and TRIM71 are part of a family of mRNA repressors regulating proliferation and differentiation.

[1]  G. Meister,et al.  Experimental identification of microRNA targets by immunoprecipitation of Argonaute protein complexes. , 2011, Methods in molecular biology.

[2]  A. Ballabio,et al.  Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties , 2008, BMC Evolutionary Biology.

[3]  David M. Umulis,et al.  Brat Promotes Stem Cell Differentiation via Control of a Bistable Switch that Restricts BMP Signaling , 2011, Developmental cell.

[4]  F. Slack,et al.  The let-7 microRNA target gene, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure , 2008, Cell cycle.

[5]  U. Baumann,et al.  An efficient one-step site-directed and site-saturation mutagenesis protocol. , 2004, Nucleic acids research.

[6]  F. Slack,et al.  Reciprocal expression of lin‐41 and the microRNAs let‐7 and mir‐125 during mouse embryogenesis , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[7]  F. Slack,et al.  The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. , 2000, Molecular cell.

[8]  C. Doe,et al.  Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. , 2006, Developmental cell.

[9]  Scott B. Dewell,et al.  Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP , 2010, Cell.

[10]  Mikael Bodén,et al.  MEME Suite: tools for motif discovery and searching , 2009, Nucleic Acids Res..

[11]  D. Blake,et al.  TRIM32 is an E3 ubiquitin ligase for dysbindin , 2009, Human molecular genetics.

[12]  A. Shearn,et al.  Mutations in the β-propeller domain of the Drosophila brain tumor (brat) protein induce neoplasm in the larval brain , 2000, Oncogene.

[13]  G. Hutvagner,et al.  HSP90 Protein Stabilizes Unloaded Argonaute Complexes and Microscopic P-bodies in Human Cells , 2010, Molecular biology of the cell.

[14]  N. Sonenberg,et al.  Cap-Dependent Translational Inhibition Establishes Two Opposing Morphogen Gradients in Drosophila Embryos , 2006, Current Biology.

[15]  Robert L. Judson,et al.  Opposing microRNA families regulate self-renewal in mouse embryonic stem cells , 2010, Nature.

[16]  M. Buszczak,et al.  Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling , 2012, Development.

[17]  M. Zavolan,et al.  MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells , 2008, Nature Structural &Molecular Biology.

[18]  Karl Mechtler,et al.  Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage , 2008, Nature.

[19]  K. Patel,et al.  TRIM32 Regulates Skeletal Muscle Stem Cell Differentiation and Is Necessary for Normal Adult Muscle Regeneration , 2012, PloS one.

[20]  Norman E. Davey,et al.  Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins , 2012, Cell.

[21]  R. Gregory,et al.  Trim71 cooperates with microRNAs to repress Cdkn1a expression and promote embryonic stem cell proliferation , 2012, Nature Communications.

[22]  G. Meister,et al.  Proteomic and functional analysis of Argonaute‐containing mRNA–protein complexes in human cells , 2007, EMBO reports.

[23]  A. Pasquinelli,et al.  Analysis of the regulation of lin‐41 during chick and mouse limb development , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[24]  Jennifer L. Bachorik,et al.  Gemin5-snRNA interaction reveals an RNA binding function for WD repeat domains , 2009, Nature Structural &Molecular Biology.

[25]  Robert Blelloch,et al.  Embryonic Stem Cell Specific MicroRNAs Regulate the G1/S Transition and Promote Rapid Proliferation , 2008, Nature Genetics.

[26]  F. Slack,et al.  let-7 microRNAs in development, stem cells and cancer. , 2008, Trends in molecular medicine.

[27]  M. Zavolan,et al.  Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. , 2008, RNA.

[28]  V. Ambros,et al.  nhl-2 Modulates MicroRNA Activity in Caenorhabditis elegans , 2009, Cell.

[29]  Mihaela Zavolan,et al.  Comparative Analysis of mRNA Targets for Human PUF-Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System , 2008, PloS one.

[30]  John Yu,et al.  Human TRIM71 and its nematode homologue are targets of let-7 microRNA and its zebrafish orthologue is essential for development. , 2007, Molecular biology and evolution.

[31]  Gordon K. Smyth,et al.  limma: Linear Models for Microarray Data , 2005 .

[32]  G. Hannon,et al.  A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases , 2008, Nature Structural &Molecular Biology.

[33]  K. Mechtler,et al.  Asymmetric Segregation of the Tumor Suppressor Brat Regulates Self-Renewal in Drosophila Neural Stem Cells , 2006, Cell.

[34]  T. Tuschl,et al.  Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. , 2004, Molecular cell.

[35]  W. Filipowicz,et al.  Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis. , 2004, RNA.

[36]  Eugene Berezikov,et al.  The TRIM-NHL Protein TRIM32 Activates MicroRNAs and Prevents Self-Renewal in Mouse Neural Progenitors , 2009, Cell.

[37]  T. Kanamoto,et al.  Cloning and regulation of the vertebrate homologue of lin‐41 that functions as a heterochronic gene in Caenorhabditis elegans , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[38]  Edward L. Huttlin,et al.  Systematic and quantitative assessment of the ubiquitin-modified proteome. , 2011, Molecular cell.

[39]  W. Filipowicz,et al.  Inhibition of Translational Initiation by Let-7 MicroRNA in Human Cells , 2005, Science.

[40]  R. Wharton,et al.  Drosophila Brain Tumor is a translational repressor. , 2001, Genes & development.

[41]  L. Smirnova,et al.  The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2 , 2009, Nature Cell Biology.

[42]  Shiva Seyedoleslami Esfahani,et al.  Regulation of the Drosophila lin‐41 homologue dappled by let‐7 reveals conservation of a regulatory mechanism within the LIN‐41 subclade , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[43]  Nicholas S M Putz,et al.  Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects. , 2011, RNA: A publication of the RNA Society.

[44]  F. Slack,et al.  A novel repeat domain that is often associated with RING finger and B-box motifs. , 1998, Trends in biochemical sciences.

[45]  Jian-Fu Chen,et al.  The ubiquitin ligase mLin41 temporally promotes neural progenitor cell maintenance through FGF signaling. , 2012, Genes & development.

[46]  W. Filipowicz,et al.  Kinetic analysis reveals successive steps leading to miRNA‐mediated silencing in mammalian cells , 2012, EMBO reports.

[47]  R. Jove,et al.  miR-302b maintains "stemness" of human embryonal carcinoma cells by post-transcriptional regulation of Cyclin D2 expression. , 2008, Biochemical and biophysical research communications.

[48]  R. Baines,et al.  Pumilio Binds para mRNA and Requires Nanos and Brat to Regulate Sodium Current in Drosophila Motoneurons , 2008, The Journal of Neuroscience.

[49]  A. Aggarwal,et al.  Model of the brain tumor-Pumilio translation repressor complex. , 2003, Genes & development.

[50]  R. Agami,et al.  MicroRNA regulation by RNA-binding proteins and its implications for cancer , 2011, Nature Reviews Cancer.