Proteomics

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SERRATE interacts with the nuclear exosome targeting complex to degrade primary miRNA precursors in Arabidopsis


ABSTRACT: 0.3g of flowers were ground in liquid nitrogen. The powder was extracted for 30 min [H2] in 1.5 ml of lysis buffer (50mM Tris HCl pH 8.0, 50mM NaCl, 1% Triton x-100 and 1 x cOmplete™ EDTA-free protease inhibitor (Roche)). After removal of cell debris by centrifugation (2 times 10 min, 16000 x g, 4 °C) the cleared supernatants were incubated for 30 min with GFP or FLAG antibodies coupled to magnetic microbeads (μMACS GFP and DYKDDDDK isolation Kits, Miltenyi). Beads were loaded on magnetized MACS separation columns equilibrated with lysis buffer and washed 4 times with 300 µl washing buffer (50mM Tris HCL pH 7.5, 25 or 50 mM NaCl, 0,1 % Triton). Samples were eluted in 50 µl of pre-warmed elution buffer (Milteny). Control IPS were performed in Col-O using either GFP or FLAG antibodies.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Flower

SUBMITTER: Lauriane Kuhn  

LAB HEAD: Heike Lange

PROVIDER: PXD014447 | Pride | 2020-04-15

REPOSITORIES: Pride

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Publications

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis.

Bajczyk Mateusz M   Lange Heike H   Bielewicz Dawid D   Szewc Lukasz L   Bhat Susheel S SS   Dolata Jakub J   Kuhn Lauriane L   Szweykowska-Kulinska Zofia Z   Gagliardi Dominique D   Jarmolowski Artur A  

Nucleic acids research 20200701 12


SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the id  ...[more]

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