Metazoan E(y)2/ENY2 is a multifunctional protein important for transcription activation and mRNA export, being a component of SAGA/TFTC and the mRNA export complex AMEX. Here, we show that ENY2 in Drosophila is also stably associated with THO, the complex involved in mRNP biogenesis. The ENY2-THO complex is required for normal Drosophila development, functioning independently on SAGA and AMEX. ENY2 and THO arrive on the transcribed region of the hsp70 gene after its activation, and ENY2 plays an important role in THO recruitment. ENY2 and THO show no direct association with elongating RNA polymerase II. Recruitment of ENY2 and THO occurs by their loading onto nascent mRNA, apparently immediately after its synthesis, while the AMEX component Xmas-2 is loaded onto mRNA at a later stage. Knockdown of either ENY2 or THO, but not SAGA or AMEX, affects the processing of the transcript's 39 end. Thus, ENY2, as a shared subunit of several protein complexes governing the sequential steps of gene expression, plays an important role in the coordination of these steps.[Keywords: THO; mRNA export; mRNP formation; gene expression; protein complex; ENY2] Supplemental material is available at http://www.genesdev.org.
SAGA/TFTC is a histone acetyltransferase complex that has a second enzymatic activity because of the presence of a deubiquitination module (DUBm). Drosophila DUBm consists of Sgf11, ENY2 and Nonstop proteins. We show that Sgf11 has other DUBm-independent functions. It associates with Cbp80 component of the cap-binding complex and is thereby recruited onto growing messenger ribonucleic acid (mRNA); it also interacts with the AMEX mRNA export complex and is essential for hsp70 mRNA export, as well as for general mRNA export from the nucleus. Thus, Sgf11 functions as a component of both SAGA DUBm and the mRNA biogenesis machinery.
ENY2/Sus1, a protein involved in the coupling of transcription with mRNA export, is a component of SAGA/TFTC and TREX-2/AMEX complexes. Recently, we have described the association of ENY2 with the third protein complex, THO. Moreover, our data indicate that ENY2 is also associated with other factors, both in the nucleus and cytoplasm. Thus, being a shared components of several protein complexes, ENY2 appears to function as an adapter molecule involved in integration of cellular processes, in particular, subsequent stages of gene expression.
The general snRNA gene transcription apparatus has been extensively studied. However, the role of coactivators in this process is far from being clearly understood. Here, we have demonstrated that the Drosophila SAGA complex interacts with the PBP complex, the key component of the snRNA gene transcription apparatus, and is present at the promoter regions of the snRNA genes transcribed by both the RNA polymerase II and RNA polymerase III (U6 snRNA). We show that SAGA interacts with the Brf1 transcription factor, which is a part of the RNA polymerase III transcription apparatus and is present at promoters of a number of Pol III-transcribed genes. Mutations inactivating several SAGA subunit genes resulted in reduced snRNA levels in adult flies, indicating that SAGA is indeed the transcriptional coactivator for the snRNA genes. The transcription of the Pol II and Pol III-transcribed U genes was reduced by mutations in all tested SAGA complex subunits. Therefore, the transcription of the Pol II and Pol III-transcribed U genes was reduced by the mutations in the deubiquitinase module, as well as in the acetyltransferase module of the SAGA, indicating that the whole complex is essential for their transcription. Therefore, the SAGA complex activates snRNA genes suggesting its wide involvement in the regulation of gene transcription, and consequently, in the maintenance of cellular homeostasis.
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