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

Abstract

The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1-TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed.

Figure 1. Interactors of the R2TP/PFDL chaperone.

Representation of known interactions (left) and previously undescribed (right) interactions of the R2TP/PFDL complex described in this study. Associated chaperones are shown in red and known or putative cofactors for each client complex are identified inside arrows.

Figure 2. Affinity purification of R2TP/PFDL subunits reveals interactors.

(a) Diagram of the network of high-confidence interactions formed around the R2TP/PFD complex. Solid lines denote interactions with a FDR lower than 0.1 while dashed lines are interactions of relevance in this network with FDR scores higher than 0.1, but lower than 0.2 (Supplementary Data 1). Green-coloured nodes are tagged subunits used in this experiment. (b) CoImmunoprecipitation (CoIP) of FLAG-tagged subunits of the R2TP/PFD-like complex (RPAP3 and URI1) in HeLa S3 cells. Various Western blots were made to detect endogenous or recombinant proteins, as marked to the right. (c) Outline of the splicing cycle with emphasis on the recycling steps of the U5 snRNP and its reintegration within the U4/U6.U5 tri-snRNP (inner loop). (d) Regulation of the TORC1 kinase complex by TSC1–TSC2 (annotated here as ‘TSC'). Guanine nucleotides (GTP, GDP) and phosphate groups (P) are indicated in green or red, depending on whether they have an activating or inhibitory effect on their associated proteins, respectively.

Figure 3. R2TP/PFDL is a major interactor of the TSC1–TBC1D7 subcomplex.

Diagram of the network of high-confidence interactions formed around the TSC1–TSC2 complex. Solid lines denote interactions with a FDR lower than 0.1 while dashed lines are interactions of relevance in this network with FDR scores higher than 0.1, but lower than 0.2 (Supplementary Data 1). Red arrows are R2TP/PFD-like subunits that copurified in TSC1–TSC2 complex purifications. Coloured nodes are tagged subunits used in this experiment.

Figure 4. ZNHIT2 is a cofactor of R2TP/PFDL that targets the U5 snRNP.

(a) Diagram of the network of high-confidence interactions formed around ZNHIT2. Solid lines denote interactions with a FDR lower than 0.1 while dashed lines are interactions of relevance in this network with FDR scores higher than 0.1, but lower than 0.2 (Supplementary Data 1). Red arrows are target proteins identified in the ZNHIT2 purification. Coloured nodes are tagged subunits used in this experiment. White nodes are known protein complex subunits that were not detected. (b) CoImmunoprecipitation (CoIP) of FLAG-tagged subunits of the R2TP/PFD complex (RPAP3 and URI1) in HEK-293 cells following transfection with either non-specific (siCTRL) or ZNHIT2-targetting siRNAs (siZNHIT2). Various Western blots were made to detect endogenous or recombinant proteins, as indicated on the right side.

Figure 5. The zinc finger HIT domain mediates interaction with RUVBL2.

(a) Multiple sequence alignment of the zf-HIT domain in all six members of the human ZNHIT family in accordance with He et al. Red residues are small, hydrophobic, aromatic; blue are acidic; magenta are basic; and all other residues are green. Residue similarity and identity are represented by colon (:) and asterisk (*), respectively. Zinc coordinating cysteine and histidine residues (or corresponding positions) and region deleted in ΔHIT mutants are indicated overhead. (b) Linear representation of protein domain architecture within members of the human ZNHIT family. Residues delineating each domain are indicated below. HIT, zinc finger HIT domain; LXXLL, coactivator LXXLL nuclear receptor recognition motif; PAPA-1, PAPA-1 homology region; DEAD, DEAD-box RNA helicase domain. (c) In vitro GST pull-down assays of GST-ZNHIT2 (either full-length or lacking the zf-HIT domain) with RUVBL1-His and/or RUVBL2-His in presence of ATP, ADP or non-hydrolyzable ATP analogue, γ-S-ATP. (d) In vitro GST pull-down assays of various GST-tagged ZNHIT proteins (either full-length or lacking the zf-HIT domain) with RUVBL1-His and RUVBL2-His in presence of ADP.

Figure 6. Pre-mRNA splicing is affected by ZNHIT2 and RUVBL2 expression levels.

(a) Diagram of alternative splicing events monitored in this experiment. Primer pairs targeting alternative splicing events are shown. (b) Significant splicing shifts (P<0.05) following ZNHIT2, or RUVBL2 knockdown. PSI (Ψ) values for three biological replicates of six alternative splicing events following transfection of a non-targeting siRNA, ZNHIT2 knockdown and RUVBL2 knockdown are indicated.

Figure 7. ZNHIT2 and RUVBL2 regulate the composition of the U5 snRNP.

(a) Workflow of triple SILAC experiments. (b) Expression assessment of various U5 snRNP subunits following treatment with siZNHIT2 or siRUVBL2. (c–f) Interpretation of the SILAC data on scatterplots. Intensity ratio of medium-labelled proteins over unlabelled proteins (M/L) on the X axis denotes IP enrichment. Intensity ratio of medium-labelled proteins over heavy-labelled proteins (M/H) on the Y axis represents experimental variations. Marked proteins in the upper right quadrant (X>1, Y>1) are proteins enriched in the IP that were less abundant following siRNA treatment. Position of the protein used for IP is shown in all experiments.