Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity

ABSTRACT: Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a nontelomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons, and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release. RNA immunoprecipitated samples using either anti-rat TRF2-S or rabbit IgG antibodies were used to generate biotin-labeled RNA using the Illumina Total Prep RNA Amplification Kit (Ambion), which was hybridized to Illumina's rat Ref-12 Expression BeadChips (Illumina, San Diego, CA), containing 22,523 well-annotated RefSeq transcripts with ~30-fold redundancy. The arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using Illumina BeadStudio software, version 1.5, normalized by Z-score transformation and used to calculate differences in signal intensities. Significant values were calculated from two groups of independent experiments, using a two-tailed Z-test with P < 0.05, a false discovery rate <0.30, a z ratio absolute value not less than 1.5, and an average signal intensity not less than zero. The results also had to pass the filtering and one-way independent ANOVA test by sample groups less than 0.05, and detection p-value for any probe in the comparison group less than 0.02.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Kevin Becker

PROVIDER: E-GEOD-72887 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:FMRP loss-of-function causes Fragile X Syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here we use high throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins, and transcripts implicated in autism spectrum disorders Polyribosomes were prepared from UV254-crosslinked pooled littermate FVB mouse brains as described in detail in published paper PMID 21784246. Polyribosomes were dissociated under denaturing conditions using two different protocols to disrupt ribonucleoprotein complexes. In most experiments Fmr1-null littermates were used in parallel to confirm FMRP specificity. After mild RNAse treatment to reduce the size of RNA to 60-100 nucleotides, endogenous FMRP was immunoprecipitated with one of two antibody combinations, either mixed monoclonal antibodies 7G1-1 and 2F5, or polyclonal antibody ab17722 (Abcam). The ages of the mice were P11, P13, P14, P15, and P25. In addition, to assess a different neuronal polysome-associated protein, as another control, lysates prepared from two samples, the P11 and P13 mice, were split in half and one half IPed with a human anti-Hu antisera as the neuronal Hu proteins are polysome-associated in brain. In sum, 7 FMRP HITS-CLIP experiments were performed including (1) P14 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (2) P14 mouse polysomes prepared by Protocol 1 and IPed with ab17722 (3) P15 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (4) P25 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (5) P25 mouse polysomes prepared by Protocol 1 and IPed with ab17722 (6) P11 mouse polysomes prepared by Protocol 2 and IPed with ab17722 and (7) P13 mouse polysomes prepared by Protocol 2 and IPed with ab17722. FMRP-bound or Hu-bound RNA tags were cloned and submitted for high throughput sequencing on the Life Science 454 platform (samples 1-5) or the Illumina platform (samples 6 and 7 and the 2 Hu samples processed in parallel with samples 6 and 7). Full details are given in PMID 21784246.

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Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity

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