Dataset Information

Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch

ABSTRACT: The maternal mRNA nanos is required for anterior-posterior patterning in the early Drosophila melanogaster development. The nanos-mRNA is translationally repressed and deadenylated during the first two hours of embryogenesis. A stem-loop structure in the 3'-UTR of this RNA (SRE - Smaug recogniiton element) is bound by the Smaug protein which recruits a protein complex to the RNA to sequester its translation and intiate transcript degradation via the ccr4/not-complex. To analyse the composition of this repressor complex, biotinylated RNAs carrying two SRE stem-loops were used for an affinity purification of bound proteins. As a negative control an RNA was used that contained two point mutations in the SRE loop, that abolish binding of Smaug. Seven subunits were identified as components of the repressor complex. The main deadenylase in Drosophila, the ccr4/not-complex was also significantly enriched.

INSTRUMENT(S):

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Embryo, Early Embryonic Cell

SUBMITTER: Michael Götze

LAB HEAD: Elmar Wahle

PROVIDER: PXD006596 | Pride | 2017-07-18

REPOSITORIES: Pride

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Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch.

Götze Michael M Dufourt Jérémy J Ihling Christian C Rammelt Christiane C Pierson Stephanie S Sambrani Nagraj N Temme Claudia C Sinz Andrea A Simonelig Martine M Wahle Elmar E

RNA (New York, N.Y.) 20170712 10

Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement ...[more]

PMID: 28701521

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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).

Project description:Integrated breeding strategies are used to increase both the yield potential and stability of crops. Most of these approaches have a direct genetic basis. The utility of epigenetics in breeding to improve complex traits such as yield and stress tolerance is not clear. A better understanding of the status of the epigenome and its contribution to the agronomic performance would help in developing strategies to incorporate the epigenetic component of complex traits in breeding,Starting from isogenic canola lines, epilines were generated by selecting recursively during three generations for lines with a higher energy use efficiency and drought tolerance. These epilines were more energy use efficient, drought tolerant, high nitrogen use efficient, and higher yielding under suboptimal conditions. Moreover, these characteristics were transgenerational inheritable. Transcriptome comparison with a line selected for energy use efficiency only revealed common differentially expressed genes related to the onset of signaling events regulating stress tolerance. Genes related to salt, osmotic, abscisic acid and drought were specifically differentially expressed in the drought tolerant epilines. The status of the epigenome, scored as differential trimethylation of lysine 4 of histone 3, supports the energy use efficient and drought tolerant phenotype by facilitating transcription of the genes that are found to be differentially expressed.From these results it can be concluded that the epigenome can be shaped by selection to increase yield and stress tolerance. This acquired knowledge will support further development of strategies to incorporate epigenetics in breeding. SRA study accession: SRP052946, Bioproject: PRJNA273932. Two canola (Brassica napus L. spp. oleifera (Metzg) Sinsk. f. biennis) epilines with low respiration and high NAD(P)H content were selected. Grinded leaf material from 26 day-old control and both epilines was collected in triplicate and served as input for deep sequencing on Illumina GAIIx.

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Dataset Information

Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch

Dataset's files

Publications

Translational repression of the <i>Drosophila nanos</i> mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch.

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