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

Abstract

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.

Limited Proteolysis-Coupled Mass Spectrometry Identifies Phosphatidylinositol 4,5-Bisphosphate Effectors in Human Nuclear Proteome

“…However, the comparison of results from GO analysis provides a similar set of participating nuclear processes such as gene expression, RNA processing, and chromatin regulation [ 7 ]. This is in agreement with our previous findings that PIP2 regulates both Pol I and Pol II mediated transcription, epigenetic regulation and RNA processing [ 2 , 8 , 9 , 26 ]. We validated PIPsLiP-qMS approach by control pull-down experiments followed by WB detection of two chosen proteins MPRIP and CWC25 and showed that indeed both proteins have the binding capacity for PIP2 ( Supplementary Figure S2 ).…”

“…Moreover, the nucleolar protein nucleophosmin is involved in non-canonical autophagy when Pol I is inhibited [ 53 ]. PIP2 is a nucleolar factor affecting the activity of nucleolar proteins such as Pol I and fibrillarin [ 8 , 26 ]. When ubiquitin proteasome system is inhibited, the fibrillarin colocalizes in nucleolar aggregates with ubiquitin and LC3 [ 49 ].…”

Emerging Data on the Diversity of Molecular Mechanisms Involving C/D snoRNAs

“…Nevertheless, the mechanisms of sdRNA biogenesis and regulation are poorly understood. Interestingly enough, FBL has been recently shown to exhibit an RNase activity regulated by the presence of phosphatidic acid [137], which opens the possibility that snoRNA processing is an intrinsic property of C/D snoRNPs upon specific stimuli. The interaction of sdRNAs with proteins is largely unknown, except for a small subset of sdRNAs that associate with proteins of the Argonaute family and harbor microRNA-like [129,138] or piRNA-like [139] properties.…”

Fibrillarin evolution through the Tree of Life: Comparative genomics and microsynteny network analyses provide new insights into the evolutionary history of Fibrillarin

“…The GAR domain was not present in Archaea FIB proteins, suggesting acquisition during early eukaryotic evolution. Three different Hidden Markov Models (HMM) for RG-rich regions reported in [13] were used identify whether other RG-rich regions (like the GAR domain in FIB) were present in other proteins from Bacteria and Archaea, we had examined; no significant match was found against the proteomes of prokaryotes (S4 Table).…”

“…An early experiment in yeast identified impaired ribosomal processing, including impaired rRNA methylation, in thermos-sensitive FIB-deficient mutants [ 12 ]. Recently, a novel ribonuclease function was described for FIB; this activity is dependent on the GAR domain and is impaired by phospholipids [ 13 ]. Specific localization in the nucleoli and Cajal bodies of cells has made it possible to use it as a nucleolus marker.…”