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Series GSE41246 Query DataSets for GSE41246
Status Public on Sep 01, 2015
Title Ribosome profiling in muscle reveals the translation landscape of the giant protein titin
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary 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.
Overall design 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.
Contributor(s) Weiss RB
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Submission date Oct 01, 2012
Last update date May 15, 2019
Contact name Robert B Weiss
Phone (801) 585-5606
Organization name University of Utah
Department Human Genetics
Lab EIHG, Rm 2260B
Street address 15 N 2030 E
City Salt Lake City
State/province UT
ZIP/Postal code 84112
Country USA
Platforms (1)
GPL13112 Illumina HiSeq 2000 (Mus musculus)
Samples (4)
GSM1012165 9071X2_Heart_RPF
GSM1012166 9071X2_Heart_RNA
GSM1012167 9071X4_TA_RPF
BioProject PRJNA176310
SRA SRP015946

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SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE41246_Heart_RPF-RNA_Abundance.txt.gz 401.8 Kb (ftp)(http) TXT
GSE41246_TA_RPF-RNA_Abundance.txt.gz 384.1 Kb (ftp)(http) TXT
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