Project description:Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on human skeletal muscle transcripts from a patient with Becker muscular dystrophy. Methods: Tissue homogenates were prepared from frozen sections of a muscle biopsy obtained from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control. Ribosome-protected fragments and total RNA were prepared from a single homogenate, so starting RNA populations for both libraries were closely matched. Homogenates were not clarified before RNase digestion to avoid loss of ribosomes associated with large molecular weight complexes and RNA-Seq libraries were prepared after rRNA subtraction to avoid positional loss of 5’ reads. RPF-Seq libraries were built using the TruSeq Small RNA Sample Preparation Kit (Illumina) and RNA-Seq libraries were built using the TruSeq RNA Sample Preparation v2 Kit (Illumina). RPF-Seq and RNA-Seq libraries were subjected to 50 cycles of single-end sequencing on an Illumina HiSeq 2000 instrument. Trimmed and filtered RPF- and RNA-Seq reads were mapped to RefSeq fasta sequences downloaded from the UCSC genome browser (hg19 assembly). Results: Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that results in the expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosomal profiling. Conclusions: Our results provide a molecular explanation for the rescue of 5’ truncating mutations via a heretofore undescribed mechanism of post-transcriptional regulation of dystrophin expression. The presence of a glucocorticoid-inducible IRES within a highly conserved region of the DMD sequence strongly suggests a programmed role for alternate translation initiation, and ongoing efforts to understand the relevant cell lineage-specific and/or conditional activation signals will shed light on underlying mechanisms of IRES control and elucidate potentially novel functions of dystrophin. Skeletal muscle ribosome-protected fragment and RNA-Seq profiles from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control were generated by deep sequencing using the Illumina HiSeq 2000.
Project description:Sequencing of the most 3' end of mRNAs (3'-Tag-Seq) of 82 wild type strains from the Drosophila Genetic reference Panel (DGRP) during embryogenesis was performed. Three different time points (2-4hrs, 6-8hrs and 10 to 12rs after laying) were assayed. 14 biological replicates were performed from independent embryo collections.
Project description:modENCODE_submission_40 This submission comes from a modENCODE project of Susan Celniker. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We will generate over 600 RNA samples in biological triplicate and use them to generate expression profile maps detailing the sites of transcription across the fly genome using whole genome tiling arrays at 38 bp resolution as a broad survey of the transcriptome and 7 bp arrays resolution to identify at high resolution transcripts ends, splice sites, and small RNAs. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Keywords: tiling array analysis EXPERIMENT TYPE: Transcript tiling array analysis BIOLOGICAL SOURCE: Cell Line: 1182-4H TISSUE: embryo-derived cell-line GENOTYPE: mh SEX: Unknown NUMBER OF REPLICATES: 3; Replicate 1 applied to 1 array(s), Replicate 2 applied to 1 array(s), Replicate 3 applied to 1 array(s), No dye swap. EXPERIMENTAL FACTORS: Cell Line 1182-4H