Protein Hit1, a novel box C/D snoRNP assembly factor, controls cellular concentration of the scaffolding protein Rsa1 by direct interaction

Abstract

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82–R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3′-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p317–352–Hit1p70–164 complex reveals a novel mode of protein–protein association explaining the strong stability of the Rsa1p–Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p–Rsa1p–Hit1p heterotrimer.

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

“…As mentioned previously, ZNHIT6 and ZNHIT3 are snoRNP biogenesis factors that interact with NUFIP1 and the AAA+ ATPases91415. ZNHIT1 and INO80B/ZNHIT4 are components of chromatin remodelling complexes SRCAP and INO80, respectively, both of which also harbour a RUVBL1 and RUVBL2 module7.…”

Structural Features of the Box C/D snoRNP Pre-assembly Process Are Conserved through Species

“…Regions 223-260 in NUFIP1 and 233-266 in Rsa1p are highly homologous and correspond to the conserved PEP motif, which directly contacts the helix a3 of the core protein SNU13/Snu13p (Rothe et al, 2014b). Point mutations of conserved residues in the PEP motif produced similar yeast two-hybrid (Y2H) effects in yeast and human.…”

Implication of the box C/D snoRNP assembly factor Rsa1p in U3 snoRNP assembly

“…This result demonstrated that association of Rsa1p, which occurs early on the nascent transcript (54), persists at least until the +12/+18 cleavage steps. Of note, both in the input and IP fractions no long PCR products corresponding to unspliced forms were detected, confirming the rapid elimination of the intron prior to 3΄-end processing.…”