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Effect of Nbr on the length of piRNAs and endo-siRNAs in Drosophila

ABSTRACT: nbr/CG9247 gene regulates the length of a subset of miRNAs. It is not clear whether Nbr affects the length of other classes of small RNAs, such as piRNAs and endo-siRNAs. To address this, we compared small RNA population in wild-type, Df(2L)BSC312/+, nbr null (nbrf02257/Df(2L)BSC312), (nbr null; pCaSper-nbr (WT)), and (nbr null; pCaSper-nbr (D435A,E437A)). This approach revealed that, in addition to miRNAs, piRNAs and endo-siRNAs were also affected in their length in nbr null and nbr null; pCaSper-nbr (D435A,E437A). 4-7d ovaries were dissected in PBS, and 40ug total RNA was prepared from wild-type, Df(2L)BSC312/+, nbr null (nbrf02257/Df(2L)BSC312), (nbr null; pCaSper-nbr (WT)), and (nbr null; pCaSper-nbr (D435A,E437A)), using Trizol Reagent (#15596-018, Life Technologies, Carlsbad, CA) following the manufacturer's protocol. The small RNAs between ~16 to ~29 nt in size were purified from 15% TBE-urea gel (#EC6885BOX, Life Technologies, Carlsbad, CA). Small RNA libraries were prepared using Illumina's TruSeq small RNA sample preparation kit (#RS-200-0012, Illumina, Inc. San Diego, CA), following the manufacturer's protocol The libraries were sequenced on HiSeq2000 platform (Illumina).

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Stephen Fisher

PROVIDER: E-GEOD-50055 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Effect of Nbr on the length of piRNAs and endo-siRNAs in Drosophila

Project description:nbr/CG9247 gene regulates the length of a subset of miRNAs. It is not clear whether Nbr affects the length of other classes of small RNAs, such as piRNAs and endo-siRNAs. To address this, we compared small RNA population in wild-type, Df(2L)BSC312/+, nbr null (nbrf02257/Df(2L)BSC312), (nbr null; pCaSper-nbr (WT)), and (nbr null; pCaSper-nbr (D435A,E437A)). This approach revealed that, in addition to miRNAs, piRNAs and endo-siRNAs were also affected in their length in nbr null and nbr null; pCaSper-nbr (D435A,E437A).

2015-02-02 | GSE50055 | GEO

Drosophila melanogaster

Project description:Effect of Nbr on the length of piRNAs and endo-siRNAs in Drosophila

| PRJNA215892 | ENA

RNA-seq of Drosophila melanogaster testis with or without tbrd-1 knock out or tplus3a/b knock out

Project description:Many protein complexes are involved in gene regulation during Drosophila spermatogenesis. tplus3a and tplus3b code for Plus3 domain proteins and are enriched in spermatocytes. Male flies ∆tplus3a/b carrying deletions of both genes in trans to deficiency Df(2L)BSC151 show severely reduced fertility. tbrd-1 is known to regulate hundreds of genes during spermatogenesis in cooperation with tTAFs. To gain more insight into the regulatory mechanisms during spermatogenesis we used RNA from testes of ∆tplus3a/b/Df(2L)BSC151 and tbrd-11 mutant flies for RNAseq in comparison to w1118 wild-typic control. RNA was isolated with Trizol from 200 Testes dissected from w1118, homozygous tbrd-11 mutants or ∆tplus3a/b/Df(2L)BSC151. After DNAse I digestion RNA was purified and RNAseq was performed in three replicates. Genotype Description: w1118: wild-typic control tbrd-11: homozygous tbrd-11 mutants (knock-out through P{EPgy2}tbrd-11, as described in Leser et al., 2012) ∆tplus3a/b/Df(2L)BSC151: CRISPR/Cas9 deletion mutants, representing knock-out of both tplus3 and tplus3b. Male flies y1cho2v1;∆tplus3a/b/CyO were crossed with virgin females w1118;Df(2L)BSC151/CyO. Male offspring with w1118;∆tplus3a/b/Df(2L)BSC151 were used for RNAseq.

2019-11-04 | E-MTAB-7013 | biostudies-arrayexpress

Small RNA profiling in Drosophila wild-type and nbr[f02257] mutants

Project description:nbr/CG9247 gene regulates 3'end heterogeneity of a subset of miRNAs. It is not clear how broad this effect is on small RNA population. To address this, we compared small RNA population in wild-type and tmr[f02257] mutants. This approach identified more miRNAs whose 3'end heterogeneity was affected in nbr[f02257] mutants. 2-3 day old control (w homogeneous strain Bloomington stock center 5905) and nbr[f02257] null mutant flies were collected. nbr[f02257] line was in the homogenous (Bloomington stock center stock 5905) background through a minimum of 5 backcrosses. Total RNA from whole flies was extracted using TRIzol reagent (Invitrogen). 40ug of total RNA from each genotype was used for small RNA library preparation with Small RNA Sample Prep kit (v1.5) (Illumina).

2011-11-01 | E-GEOD-32684 | biostudies-arrayexpress

Buffering and proteolysis are induced upon segmental haploidy in Drosophila melanogaster.

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2012-04-04 | E-GEOD-34400 | biostudies-arrayexpress

Genes affected upon dsRNA knockdown treatment for nbr/CG9247 in Drosophila DL1 cells

Project description:nbr/CG9247 gene function regulates the length of the 3'end of miRNAs. Such 3'end heterogeneity of miRNAs may impact miRNA target gene silencing. To address this, we performed microarray analysis for genes altered upon nbr knockdown in Drosophila DL1 cells. This approach revealed a subset of genes upregulated upon nbr knockdown. Five independent replicates of DL1 cells treated with control (dsRNA to renilla) or nbr (dsRNA RNA to nbr/CG9247) were collected 5 days after dsRNA bathing. Total RNA from from each replicate was extracted using TRIzol reagent (Invitrogen). A portion of total RNA for each replicate was used to confirm the effect of nbr knockdown on one of the nbr-dependent miRNAs, miR-34, by small RNA northern.

2011-11-01 | E-GEOD-32683 | biostudies-arrayexpress

Characterization and parental contribution of piRNAs and endo-siRNAs to mouse zygotes.

Project description:Transposable elements (TEs) are widely represented in eukaryotic genomes. Recently, a set of small RNAs known as rasRNAs (repeat-associated small RNAs) have been related to the down-regulation of TEs conferring a means to safeguard genome integrity. Two key members of the rasRNAs group are piRNAs and endo-siRNAs. In this study, we have performed a comparative analysis of piRNAs and endo-siRNAs present in mouse oocytes, spermatozoa and zygotes, identified by deep sequencing and bioinformatic analysis. Both piRNAs and endo-siRNAs regulate TEs in addition to other repetitive elements such as tRNAs and rRNAs, suggesting an alternative role of rasRNAs with regard to translation regulation. The detection of piRNAs and endo-siRNAs in sperm cells and revealed also in zygotes, hints to their potential delivery to oocytes during fertilization. However, a comparative assessment of the three cell types indicates that both piRNAs and endo-siRNAs are mainly maternally inherited. Finally, we have assessed the role of the different rasRNA molecules in connection with amplification processes by way of the M-bM-^@M-^\ping-pong cycleM-bM-^@M-^]. Our results suggest that the ping-pong cycle can act on other rasRNAs, such as tRNA- and rRNA-derived fragments, thus not only being restricted to TEs during gametogenesis, as was evidenced in spermatozoa, oocytes and zygotes. Comparative analysis from deep sequencing of piRNAs and endo-siRNAs in mouse oocytes, spermatozoa and zygotes

2014-05-20 | E-GEOD-45983 | biostudies-arrayexpress

Characterization and parental contribution of piRNAs and endo-siRNAs to mouse zygotes.

Project description:Transposable elements (TEs) are widely represented in eukaryotic genomes. Recently, a set of small RNAs known as rasRNAs (repeat-associated small RNAs) have been related to the down-regulation of TEs conferring a means to safeguard genome integrity. Two key members of the rasRNAs group are piRNAs and endo-siRNAs. In this study, we have performed a comparative analysis of piRNAs and endo-siRNAs present in mouse oocytes, spermatozoa and zygotes, identified by deep sequencing and bioinformatic analysis. Both piRNAs and endo-siRNAs regulate TEs in addition to other repetitive elements such as tRNAs and rRNAs, suggesting an alternative role of rasRNAs with regard to translation regulation. The detection of piRNAs and endo-siRNAs in sperm cells and revealed also in zygotes, hints to their potential delivery to oocytes during fertilization. However, a comparative assessment of the three cell types indicates that both piRNAs and endo-siRNAs are mainly maternally inherited. Finally, we have assessed the role of the different rasRNA molecules in connection with amplification processes by way of the “ping-pong cycle”. Our results suggest that the ping-pong cycle can act on other rasRNAs, such as tRNA- and rRNA-derived fragments, thus not only being restricted to TEs during gametogenesis, as was evidenced in spermatozoa, oocytes and zygotes.

2014-05-20 | GSE45983 | GEO

Expression profiling pooled Drosophila melanogaster heterozygous for deletions on Chromosome 2L

Project description:We performed mRNA transcriptional profiling on 99 hemizygotic lines (Df/+) from the DrosDel project covering 68% of chromosome 2L, in order to understand how changes in gene copy number affect overall transcriptome.

2016-01-31 | GSE61509 | GEO

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Project description:PIWI-interacting RNAs (piRNAs) are germline-enriched small RNAs that control transposons to maintain genome integrity1,2,3. To achieve this, piRNAs bind PIWI proteins upon being processed from piRNA precursors1,2,3. Bioinformatic studies of piRNA biogenesis in Drosophila showed that the piRNA 5′ end is formed by PIWI-Slicer or Zucchini (Zuc) endonucleolytic cleavage, while the 3′ end is formed by Zuc or Nibbler (Nbr) 3′-to-5′ exonucleolytic activity4,5,6. piRNA 3′-end formation in Bombyx was shown to be mediated by PNLDC1/Trimmer (Trim) 3′-to-5′ exonuclease7, while piRNA intermediates are bound with PIWI anchored onto mitochondrial protein PAPI8. However, the requirement for Zuc and Nbr in piRNA biogenesis in Bombyx has not been elucidated. Here, we applied biochemical approaches to understand their involvement in piRNA biogenesis and revealed that Zuc endonuclease, but not Trim and Nbr exonucleases, plays a crucial role in Bombyx piRNA 3′-end formation. Loss of Zuc had little effect on the levels of Trim and Nbr, but led to the aberrant accumulation of piRNA intermediates within the PAPI complex, which were processed to mature piRNAs by recombinant Zuc. Zuc copurified with PAPI, and PAPI exerted RNA-binding activity only when Siwi coexisted with it and PAPI was phosphorylated, suggesting that complex assembly proceeds via a hierarchical process. Bioinformatic analyses of piRNA intermediates within the PAPI complex revealed that both the 5′ and the 3′ ends showed the hallmark of PIWI-Slicer, yet no phasing pattern was observed in mature piRNAs. These findings strongly support the notion that, in Bombyx piRNA, the 5′ end is formed by PIWI-Slicer, but independently of Zuc, while the 3′ end is formed by Zuc endonuclease. The Bombyx piRNA biogenesis is simpler than that of Drosophila, which is reasonable considering that Bombyx has no transcriptional silencing machinery relying on phased piRNAs.

2018-03-01 | GSE107371 | GEO

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