AATF/Che-1 binds ribosome-associated RNAs and regulates ribosome biogenesis
AATF is a predominantly nuclear protein that has been shown to be an apoptosis-inhibiting factor essential for both embryonic development and tumor growth. Several studies have shown a role of AATF in the modulation of pivotal cellular signal transduction pathways such as p53-, mTOR- and HIF-signaling. However, the exact molecular functions underlying its essential nature to cell proliferation and survival have remained elusive. Interestingly, several lines of evidence point towards a pivotal role of this protein in ribosome biogenesis and the maturation of ribosomal RNA. The function of AATF in this process is not clear, especially since the identity of the RNA-binding protein that links the AATF-containing protein complex (ANN) to rRNA precursors is unknown. In this study, we identify AATF in a screen for RNA-binding proteins. Importantly, AATF does not only bind to polyA-tailed RNA but CLIP-experiments reveal its association with ribosomal RNA. The RNA-binding domain of AATF is essential to maintain its positive effect on cellular rRNA levels. Furthermore, AATF binds to both mRNAs encoding for ribosome biogenesis factors as well as proteins involved with this process indicating AATF to be a central hub for the coordination of ribosome maturation. Consequently, our data for the first time identify the molecular basis to the role of AATF in ribosome biosynthesis and corroborate the link between tumorigenesis and ribosome biology.
Sample Processing Protocol
AATF Co-IP samples were processed using standard protocols, digested using trypsin and purified via StageTips. Peptides were labeled using dimethyl triplex labeling. Labeled peptides were combined and measured on Q-Exactive Plus mass spectrometers that were coupled to EASY nLC 1000 UPLC. Peptides were loaded with solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm — 75 µm I.D., filled with 2.7 µm Poroshell EC120 C18, Agilent**). Peptides were chromatographically separated at a constant flow rate of 250 nL/min using the following gradient: 10-23% solvent B (0.1% formic acid in 80 % acetonitrile) within 75.0 min, 23-39% solvent B within 5.0 min, 39-95% solvent B within 5.0 min, followed by washing and column equilibration. The mass spectrometer was operated in data-dependent acquisition mode. The MS1 survey scan was acquired from 300-1750 m/z at a resolution of 70,000. The top 10 most abundant peptides were isolated within a 1.8 Th window and subjected to HCD fragmentation at a normalized collision energy of 27%. The AGC target was set to 5e5 charges, allowing a maximum injection time of 120 ms. Product ions were detected in the Orbitrap at a resolution of 35,000. Precursors were dynamically excluded for 20.0 s.
Data Processing Protocol
All mass spectrometric raw data were processed with Maxquant (version 220.127.116.11) using default parameters. Briefly, MS2 spectra were searched against the Uniprot HUMAN database, including a list of common contaminants. False discovery rates on protein and PSM level were estimated by the target-decoy approach to 1% (Protein FDR) and 1% (PSM FDR) respectively. The minimal peptide length was set to 7 amino acids and carbamidomethylation at cysteine residues was considered as a fixed modification. Oxidation (M) and Acetyl (Protein N-term) were included as variable modifications. The match-between runs option was enabled. Dimethyl triplex labeling quantification was used, and the re-quantify option was enabled. Further processing was performed in Perseus (v 18.104.22.168)
Kaiser RWJ, Ignarski M, Van Nostrand EL, Frese CK, Jain M, Cukoski S, Heinen H, Schaechter M, Seufert L, Bunte K, Frommolt P, Keller P, Helm M, Bohl K, Höhne M, Schermer B, Benzing T, Höpker K, Dieterich C, Yeo GW, Müller RU, Fabretti F. A protein-RNA interaction atlas of the ribosome biogenesis factor AATF. Sci Rep. 2019 9(1):11071 PubMed: 31363146