SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription

Blank, Maximilian F.; Chen, Sifan; Poetz, Fabian; Schnölzer, Martina; Voit, Renate; Grummt, Ingrid

To examine which region of SIRT7 mediates RNAdependent interactions, we performed pull-down experiments using N-terminally truncated SIRT7 mutants. SIRT7 comprises an unstructured arginine-and dipeptide-rich region within the N-terminal 78 amino acids, a motif that is often present in RNA binding proteins (28) (Supplementary Figure S2A ). To investigate whether this region is important for RNA-dependent protein interactions, 5external spacer (5 -ETS) RNA was immobilized on streptavidin beads and incubated with lysates of cells expressing Flag-tagged wildtype SIRT7 or mutants lacking 32 ( N32) or 78 N-terminal amino acids ( N78). Both wildtype SIRT7 and the N32 mutant were efficiently pulleddown by immobilized RNA. Deletion of 78 amino acids, however, abolished RNA binding (Figure 2A , upper panels). Furthermore, a GST-fusion protein comprising amino acids 1-81 (GST-SIRT7/1-81) efficiently interacted with RNA, supporting that the arginine-rich N-terminal part of SIRT7 mediates the interaction of SIRT7 with RNA (Figure 2A bottom panel and Supplementary Figure S2B) .