A protein-RNA interaction atlas of the ribosome biogenesis factor AATF

AATF is a central regulator of the cellular outcome upon p53 activation, a finding that has primarily been attributed to its function as a transcription factor. Recent data showed that AATF is essential for ribosome biogenesis and plays a role in rRNA maturation. AATF has been implicated to fulfil this role through direct interaction with rRNA and was identified in several RNA-interactome capture experiments. Here, we provide a first comprehensive analysis of the RNA bound by AATF using CLIP-sequencing. Interestingly, this approach shows predominant binding of the 45S pre-ribosomal RNA precursor molecules. Furthermore, AATF binds to mRNAs encoding for ribosome biogenesis factors as well as snoRNAs. These findings are complemented by an in-depth analysis of the protein interactome of AATF containing a large set of proteins known to play a role in rRNA maturation with an emphasis on the protein-RNA-complexes known to be required for the generation of the small ribosomal subunit (SSU). In line with this finding, the binding sites of AATF within the 45S rRNA precursor localize in close proximity to the SSU cleavage sites. Consequently, our multilayer analysis of the protein-RNA interactome of AATF reveals this protein to be an important hub for protein and RNA interactions involved in ribosome biogenesis.


Supplemental material and methods
Images were acquired using a Zeiss epifluorescence microscope (Zeiss Axiovert 200M).

RNA isolation and RT-qPCR
Whole RNA was isolated from all cells using TRIzol® and subsequent chloroform extraction or a commercial

Statistical analysis
Unless stated otherwise, a two-tailed, unpaired student's t-test was used for statistical analysis of e.g.

GO Term analysis
Gene ontology (GO) analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (https://david.ncifcrf.gov/) 3 . Online results were downloaded as .txt-files and depicted using GraphPad Prism v5 or Microsoft Excel.

LC-MS analysis (protein interactome)
All samples were analyzed on a Q Exactive Plus (Thermo Scientific) that was coupled to an EASY nLC 1200 Precursors were dynamically excluded for 20.0s.

RNA hydrolysis to nucleoside level
The RNA samples were digested into nucleosides as it was described before 6

Relative quantification of modified nucleosides via LC-MS/MS
For RNA analysis, an Agilent1260 series equipped with a diode array detector (DAD) and a Triple Quadrupole mass spectrometer (Agilent 6460) were utilized. In addition to that, a Synergy Fusion RP18 column (4 µm particle size, 80 Å pore size, 250 mm length, 2 mm inner diameter) from Phenomenex (Germany) was used at    Table M3. Resulting spectra were processed using Agilent MassHunter Qualitative Analysis Software: In a first step, the recorded UV chromatogram of the main nucleoside guanosine was extracted to receive the 'area under the curve' (AUC) and then, the UV-signal derived from the SIL-IS (see below) was subtracted. After that, calibration measurements of guanosine dilutions (5-500 pmol) were applied for exact quantification and the amount of injected guanosine (in pmol) of each RNA sample was calculated by using the resulting guanosine calibration factor.
Quantification of modified nucleosides was achieved by utilizing 13  was added and again the resulting ratio of modified nucleoside and its 13 C-labeled isotope was calculated.
Afterwards, the latter was divided by the corresponding modification response factor to receive the modification amount in fmol. Lastly, the ratios of modified nucleosides and the injected guanosine amounts were calculated.
Further and more detailed information can be found in Thüring et al. 7 and Kellner et al. 6 .  F Western blot related to RIP-qPCR analysis of 18S rRNA and 45S pre-rRNA (Fig. 1D). Western blot analysis (anti-FLAG antibody) shows efficient precipitation of FLAG-tagged proteins, which were expressed and precipitated at the same level. FLAG-RFP (red fluorescent protein) served as negative control.
G U2OS cells were transfected with either an siRNA pool targeting the AATF coding sequence or non-targeted control siRNA. After 48h, cells were harvested and RNA was isolated. qPCR for ACTB, AATF and 18S rRNA on RNA derived from these cells confirms the significant decrease of 18S rRNA after AATF depletion.
Nucleolar and non-nucleolar localization of RBPs was based on ENCODE I data (see methods section).

Suppl. Figure 3: Impact of AATF depletion on rRNA modifications
Analysis of rRNA modifications of whole RNA isolated from murine IMCD cells after transfection with AATFtargeted siRNA or a control using LC-MS/MS (see suppl. methods for details). Here, no differences between control cells and cells depleted of AATF were detected for the most common modifications.

Suppl. Figure 5: Original blots and images
Supplemental Table legends Suppl. Each dataset was filtered for significant peaks FC>=3 and p-value>=5, and pooled.
To define a list of bound RNAs, replicates ENSG identifiers were removed The ENSG IDs were then annotated for Gene Stable ID, Gene name and Gene Type using biomart on the "B_nucleolar" and "C_non nucleolar": subsets of RBPs were chosen according to their localization based mostly on immunofluorescence information from the website: http://rnabiology.ircm.qc.ca/RBPImage/.
All proteins for which insufficient or contradictory data were available to support either localization or biological function, were removed from this analysis.