Unknown,Transcriptomics,Genomics,Proteomics

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Comprehensive assembly and spatio-temporal analysis of the C. elegans 3' UTRome through isolation and sequencing of 3'UTRs in germline-dereived cell types and somatic tissues.

ABSTRACT: Our computational analyses of sequencing depth and the discovery rates of sequence elements in the 3M-bM-^@M-^YUTR strongly demonstrate that interrogation of the 3M-bM-^@M-^YUTRome in specific tissues and cell types across development will greatly expand the identification of new 3M-bM-^@M-^YUTR isoforms and the sequence elements therein. Therefore, we will generate and sequence the 3M-bM-^@M-^YUTRs from the cell types isolated from the developing germline, mature germ cells, and early embryogenesis. In addition, we will identify the 3M-bM-^@M-^YUTRs for the transcripts isolated from the major somatic tissues during late- and post-embryonic development. Based on our sequencing estimates, we anticipate that the tissue-specific interrogation of the 3M-bM-^@M-^YUTRome will reveal a far greater diversity of novel 3M-bM-^@M-^YUTR isoform expression that is masked in the whole-worm 3M-bM-^@M-^YUTRome sequence data. Using the transgenic myo-3::PABP strain, we have generated polyA-captured libraries for late embryos and across the major stages of post-embryonic development for the muscle transcriptome. We propose to generate these polyA-captured libraries for transcripts expressed in the other major tissues across development. PolyA-captured libraries will be generated for deep sequencing following the tissue-specific isolation of mRNAs by PABP pulldowns (the PABP immunoprecipitations will take advantage of a FLAG epitope which is fused to PABP in all of the transgenic strains). We propose to validate the expression of tissue- specific transcripts by qPCR for select transcripts known to be expressed in those particular tissues. In addition, following the strategy we have employed for the large-scale polyA-captured libraries from muscle, we will perform quality checks for 3M-JM-< end capture by manually sequencing ~60 clones isolated from each library. Once we have obtained the deep sequencing data, we will analyze all of the sequence reads using the bioinformatics pipeline that we established for the whole-worm polyA- captured sequences (Mangone et al., 2010). Four samples representing gravid, L2, L3, and L4 developmental stages were analyzed.

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Sang Chun

PROVIDER: E-GEOD-33431 | 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.

Dataset Information

Comprehensive assembly and spatio-temporal analysis of the C. elegans 3' UTRome through isolation and sequencing of 3'UTRs in germline-dereived cell types and somatic tissues.

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