Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Translational activation of developmental mRNAs during neonatal mouse testis development

ABSTRACT: The sequence of gene regulatory events that drive neonatal germ cell development in the mammalian testis is not yet clear. We assessed changes in mRNA utilization in the neonatal testis at 1 and 4 dpp, times when the testis contains quiescent gonocytes (1 dpp) and proliferating spermatogonia (4 dpp). There are not thought to be major changes in the nature or number of somatic cells over that interval. We used microarrays to detail the global expression levels of mRNA distribution between non-translating mRNAs and efficiently translating mRNAs during testis development at 1 dpp and 4 dpp Extracts of mouse testes from 1 dpp and 4 dpp CD-1 mice were fractionated over a sucrose gradient in triplicate. Gradients were fractionated using an ISCO gradient fractionation system equipped with a UA-6 detector. The position of the 80S peak defined the boundary between translating and non-translating mRNAs. Pooled non-polysomal (non-translating; fractions 1-3) and heavy polysomal (efficiently translating, fractions 7-13) RNAs were isolated from sucrose gradient fractions for hybridization on Affymetrix 420 2.0 microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Christopher Geyer

PROVIDER: E-GEOD-49283 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Translational activation of developmental messenger RNAs during neonatal mouse testis development.

Chappell Vesna A VA Busada Jonathan T JT Keiper Brett D BD Geyer Christopher B CB

Biology of reproduction 20130919 3

The basic tenets of germ cell development are conserved among metazoans. Following lineage commitment in the embryo, germ cells proliferate, transition into meiosis, and then differentiate into gametes capable of fertilization. In lower organisms such as Drosophila and C. elegans, germline stem cells make the decision to proliferate or enter meiosis based in large part on the regulated expression of genes by translational control. This study undertakes a direct characterization of mRNAs that exp ...[more]

PMID: 23926285

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Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).

Dataset Information

Translational activation of developmental mRNAs during neonatal mouse testis development

Publications

Translational activation of developmental messenger RNAs during neonatal mouse testis development.

Similar Datasets

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