Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse polysome vs RNP fractions from testis obtained from prepuberttal and adult animals to investigate post transcriptional control mechanisms in gametes

ABSTRACT: Gametes rely heavily on post-transcriptional control mechanisms to regulate their differentiation. In eggs, the storage and selective temporal activation of maternal mRNAs is essential for normal development. In the male, transcription ceases during spermiogenesis necessitating the post-transcriptional regulation of many paternal mRNAs required for spermatid differentiation and spermatozoan function. Messenger RNAs that are being actively translated form polysomes. whereas translationally inactive mRNAs are often sequestered in ribonucleoproteins (RNPs). Here we combine polysome display and microarray analyses of RNP and polysome fractions of testes from prepubertal and adult mice to characterize the translation state of individual mRNAs as spermatogenesis proceeds.. Consistent with published reports, many post-meiotic mRNAs known to be translationally delayed shift from the RNPs into the polysomes, confirming the validity of this approach. In addition, based upon the criterion of movement from RNPs to polysomes, we detect another 742 mouse testicular genes showing dramatic shifts between RNPs and polysomes. One sub-group of 35 genes including the known translationally delayed Pgk2, are initially transcribed and translationally repressed in meiotic spermatocytes, and translated post-meiotically. This high-through-put approach defines the changing translation patterns of a large number of genes as male germ cells differentiate and identifies a new group of post-transcriptionally regulated meiotic transcripts for future study. Mouse testes from animals of 3 different ages were fractionated on a sucrose gradient and transcripts were quantified in the RNP and Polysome fractions. Transcripts were identified that changed their RNP/Polysome representation at the different developmental time points.

ORGANISM(S): Mus musculus

SUBMITTER: Norman Hecht

PROVIDER: E-GEOD-4711 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated.

Iguchi Naoko N Tobias John W JW Hecht Norman B NB

Proceedings of the National Academy of Sciences of the United States of America 20060508 20

Gametes rely heavily on posttranscriptional control mechanisms to regulate their differentiation. In eggs, maternal mRNAs are stored and selectively activated during development. In the male, transcription ceases during spermiogenesis, necessitating the posttranscriptional regulation of many paternal mRNAs required for spermatozoan assembly and function. To date, most of the testicular mRNAs known to be translationally regulated are initially transcribed in postmeiotic cells. Because protein syn ...[more]

PMID: 16682651

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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

Transcription profiling of mouse polysome vs RNP fractions from testis obtained from prepuberttal and adult animals to investigate post transcriptional control mechanisms in gametes

Publications

Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated.

Similar Datasets

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