Abstract
In order to discover novel proteins that promote the nuclear export of newly synthesized mRNAs in mammalian cells, we carried out a limited RNAi screen for proteins required for the proper cytoplasmic distribution of a model intronless mRNA. From this screen we obtained two hits, Ubc9 (SUMO-conjugating E2 enzyme) and GANP (germinal center-associated nuclear protein). Depletion of Ubc9 inhibited the proper cytoplasmic distribution of certain overexpressed intronless mRNAs, while depletion of GANP affected all tested mRNAs. Depletion of Sae1, which is also required for sumoylation, partially inhibited the cytoplasmic distribution of our model mRNA. Interestingly, the block in cytoplasmic accumulation in Ubc9-depleted cells could be overcome if an intron was incorporated into the mRNA. Surprisingly, Ubc9-depleted cells had normal nuclear export of newly synthesized intronless mRNAs, indicating that the observed accumulation of the model mRNA in the nuclei of transfected cells was likely due to some more general perturbation. Indeed, depletion of Ubc9, coupled with the overexpression of the intronless mRNAs, caused the redistribution of the nuclear speckle protein SC35 to cytoplasmic foci. Our results suggest that sumoylation may play a role in the proper assembly of mRNPs and/or the distribution of key RNA binding proteins, and may thus contribute to general protein expression patterns.
Highlights
Eukaryotic cells are divided into two compartments, the nucleoplasm, where mRNAs are synthesized, processed, and assembled into messenger ribonucleoprotein complexes, and the cytoplasm, where these mRNAs are translated into proteins
A Screen to Identify Genes Required for mRNA Nuclear Export
Many reports have surfaced in the literature claiming to identify novel components required for mRNA nuclear export in mammalian cells, few of these reports have been verified independently
Summary
Eukaryotic cells are divided into two compartments, the nucleoplasm, where mRNAs are synthesized, processed, and assembled into messenger ribonucleoprotein (mRNP) complexes, and the cytoplasm, where these mRNAs are translated into proteins. TREX-2 associates with the nuclear pore [10,11,12,13,14], and at least one component of this complex, germinal center-associated nuclear protein (GANP), has been implicated in the export of mRNAs in mammalian cells [12,13,14]. Other key nuclear export factors have undergone gene duplication to produce several proteins that act redundantly to promote export in mammals, a prime example being the RNA helicases UAP56 and URH49 [22,23]. Additional factors such as Aly and the Tho complex only produce effects when they are depleted in combination [24]. We conducted a screen for genes that are required for the efficient cytoplasmic steady-state distribution of a model intronless mRNA, MHC-ftz-i
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