Fibrillarin Ribonuclease Activity is Dependent on the GAR Domain and Modulated by Phospholipids

Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear shape, and cell cycle progression. We show that fibrillarin has an additional activity as a ribonuclease. The activity is affected by phosphoinositides and phosphatidic acid and insensitive to ribonuclease inhibitors. Furthermore, the presence of phosphatidic acid releases the fibrillarin-U3 snoRNA complex. We show that the ribonuclease activity localizes to the GAR (glycine/arginine-rich) domain conserved in a small group of RNA interacting proteins. The introduction of the GAR domain occurred in evolution in the transition from archaea to eukaryotic cells. The interaction of this domain with phospholipids may allow a phase separation of this protein in nucleoli.


Fibrillarin mutagenesis
Mutagenesis was performed with Phusion Hot Start II DNA Polymerase and the PCR product processed DpnI, kinase and ligase enzymes using specific primers for each mutation. The vector pGFP-Fibrillarin was used as DNA template for the first mutagenesis cycle and confirmed by sequencing. For the second mutation, the sequence confirmed of mutant K131E was used as template for the A250P mutation. Primers used for K131E mutant are the following: Fw 5'-AGGAGATGACGAAATTGAGTACC-3' and Rv 5'-TCCGAAATCGAGACTCTC-3'. Their transient transfection in Fibrillarin-SNAP stable cell line was performed at 80% confluence using polyethylenimine (PEI) with 10 µg of fibrillarin-mutant K131E, A250P-GFP plasmid 1 ml of DMEM (Dulbecco's Modified Eagle's Medium) without fetal calf serum. Transfection cocktail were vortexed and incubated 5 min at RT, added dropwise to the cell culture. The fluorescent substrate used for this work was SNAP-Cell® TMR-Star (555 nm) is a red fluorescent substrate added by dropwise in the culture media and washed after 5 min to stain the fibrillarin with SNAP tag.After 48 hr of incubation after transient transfection the TMR-Star (555 nm) was added and the cells were fixed in 4% with formaldehyde solution in PBS for 15 min and washed once in PBS and analyzed by microscopy. Images were acquired with a DM6000B fluorescent microscope, the illumination with Leica EL6000 with HXP 120W / 45C VIS Hg lamp for fluorescent lamp for transmitted light. Using a 555nm channel for SNAP tag, 488 for GFP tag.

Supplementary figure 7. Co-localization of immunolocalization fibrillarin and SNAP tagged
Fibrillarin. Stable cell culture expressing SNAP-Fibrillarin (red) and immunolocalize fibrillarin in green. Black and white slides only show a specific channel. 488 nm for anti-fibrillarin and 555 nm for the SNAP-Fibrillarin. The Inset shows a typical nucleolus of the cell

Supplementary figure 10. Flow diagram of sequences analyzed by bioinformatics tools.
Bioinformatic analysis of the different sequences and domains evaluated in the work are presented as a flow chart with the different strategies used.

S-12
Supplementary figure 11. GAR domain amino acid alignment. The sequence alignment of the basal GAR group, group A1, group A2 and group B showing two conserved arginine: R34 and R45 are represented in the figure. S-13

Supplementary figure 12. Phylogenetic analysis of the GAR domain branches founded in the proteomes of different species.
Diagram showing the two branches from the HMM analysis of GAR1 protein sequences indicated by RGG-box1 and RGG-box2.

List of fibrillarin protein sequeces retrieved from 36 complete genomes of vertebrate species and a yaest (S. cerevisiae).
*The FIB sequences were retrived from the listed genomes in S1