Unknown,Transcriptomics,Genomics,Proteomics

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TDRD6 instructs correct assembly of mRNPs in the chromatoid body to potentiate UPF1 dependent mRNA decay.

ABSTRACT: RNA sequencing of heterozygote or Tudor domain contian protein 6 (TDRD6) knockout round spermatid cells. Chromatoid bodies (CBs) are germ cell-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family such as TDRD6. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay machinery such as UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 interaction, and for assembly of UPFs and other RNA binding proteins into super-complexes. In absence of TDRD6, the association of some mRNAs with UPF1 is impaired, and the long 3â UTR-stimulated but not the exon junction complex-stimulated pathway of NMD is distorted. Reduced association of mRNAs with UPF1 correlated with increased stability and presence in polysome fractions, i.e. enhanced translational activity. Thus, we define CBs as sites of UPF1-dependent mRNA degradation and provide evidence for the requirement for NMD in spermiogenesis. This function of CBs depends on TDRD6-promoted assembly of mRNA decay enzymes within mRNPs. RNA was extracted from quadruplicate samples and libraries generated for sequencing using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) at the Deep Sequencing Group SFB 655, Biotechnology Center of Technische UniversitÃ¤t Dresden. After enrichment and XP bead (Agencourt AMPure Kit; Beckman Coulter, Inc.) purification, quality control was done using Fragment AnalyzerTM (Advanced Analytical). The bar-coded libraries were equimolarly pooled and subjected to 76 bp single-end sequencing on Illumina HiSeq 2000, resulting in an average of 33 million reads per sample.

ORGANISM(S): Mus musculus

SUBMITTER: Grigorios Fanourgakis

PROVIDER: E-GEOD-63948 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on mouse heart and skeletal muscle transcripts. Methods: Tissue was rapidly harvested and snap-frozen to minimize bias to the pool of translating ribosomes. RNA was prepared from a single homogenate for each tissue so that 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. Trimmed reads from 50 cycles of Illumina single-end sequencing were mapped onto a non-redundant set of 18,499 mouse protein-coding RefSeq transcripts from the nuclear genome. Results: Mapped sequence reads to myosin, actin and the giant protein titin together account for ~20% of the total mRNA-derived ribosome protected fragments (RPFs). We observed large-scale uniformity in the distribution of RPFs on the >30,000 codon titin open reading frame, from which we inferred an in vivo ribosome elongation error rate of ≤10-5. Ribosome footprints on Ttn mRNA also uncovered a novel 5’ UTR within a phylogenetically conserved intronic element that would produce ~2.35 mDa titin isoform that corresponds to the titin 'T2' band frequently described as a proteolytic artifact. Local translation efficiency across several >10 kb muscle mRNAs was also uniform, while their global translation efficiencies varied by ~20-fold suggesting initiation rate plays a major role in the translation efficiency of large mRNAs. Evidence for RPFs on 5’ UTRs was widespread with particular enrichment for ribosomes positioned at CUG codons. Comparison of global translation efficiency in cardiac and skeletal muscle revealed novel examples of tissue-specific translational control including synthesis of the myogenic factor Mef2c, and the titin-binding stress response protein Ankrd23. Conclusions: Our study represents the first detailed analysis of translation in an adult mammalian tissue generated by ribosome profiling technology. Current limitations to using ribosomal profiling in tissues include unknown perturbations to the dynamic state of translation despite rapidly harvested and snap-frozen samples. The uniform 5’ to 3’ coverage observed on individual large mRNAs and the ability to observe footprints on the extremely small phospholamban coding sequence, suggests that initiation and elongation were halted on similar time scales. More detailed examination of the positional information within CDS region requires further understanding of the bias introduced during the library preparation steps for both RPF-and RNA-Seq, as well as local biases induced as translation is arrested. Despite these qualifications, this initial view of active translation in muscle tissue highlights the potential for ribosome profiling to monitor the dynamic translation response to exercise, injury or disease pathology in animal models at a level of resolution not easily attainable with other quantitative approaches. Heart and skeletal muscle ribosome-protected fragment and RNA-Seq profiles of 10-week old C57BL/6J male mice were generated by deep sequencing using the Illumina HiSeq 2000.

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TDRD6 instructs correct assembly of mRNPs in the chromatoid body to potentiate UPF1 dependent mRNA decay.

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