AAA+ Proteins RUVBL1 and RUVBL2 Coordinate PIKK Activity and Function in Nonsense-Mediated mRNA Decay

Abstract

Phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins play essential roles in DNA-based and RNA-based processes, such as the response to DNA damage, messenger RNA (mRNA) quality control, transcription, and translation, where they contribute to the maintenance of genome integrity and accurate gene expression. The adenosine triphosphatases associated with diverse cellular activities (AAA+) family proteins RuvB-like 1 (RUVBL1) and RUVBL2 are involved in various cellular processes, including transcription, RNA modification, DNA repair, and telomere maintenance. We show that RUVBL1 and RUVBL2 associate with each PIKK family member. We also show that RUVBL1 and RUVBL2 control PIKK abundance at least at the mRNA level. Knockdown of RUVBL1 or RUVBL2 decreased PIKK abundance and impaired PIKK-mediated signaling. Analysis of SMG-1, a PIKK family member involved in nonsense-mediated mRNA decay (NMD), revealed an essential role for RUVBL1 and RUVBL2 in NMD. RUVBL1 and RUVBL2 associated with SMG-1 and the messenger ribonucleoproteins in the cytoplasm and promoted the formation of mRNA surveillance complexes during NMD. Thus, RUVBL1 and RUVBL2 regulate PIKK functions on two different levels: They control the abundance of PIKKs, and they stimulate the formation of PIKK-containing molecular complexes, such as those involved in NMD.

The ATPase Activity of Reptin Is Required for Its Effects on Tumor Cell Growth and Viability in Hepatocellular Carcinoma

“…Reptin mutants retained the same subcellular distribution as WT Reptin, and also their ability to partner with Pontin, suggesting that they had no gross defects besides the loss of ATPase activity that could explain their phenotypic effects. We and others have previously shown that Reptin silencing induced the codepletion of Pontin, via a posttranslational mechanism (4,7,20). However, we show here that cells reconstituted with the Reptin Walker B mutants expressed normal levels of Pontin such as those reconstituted with WT siRNA-resistant Reptin (20), thus ruling out that some of the effects seen with the mutants might be due to the loss of Pontin.…”

“…Indeed, together with Pontin, Reptin is involved in the assembly of telomerase and their silencing leads to a decreased telomerase activity (4,9). It also controls the levels of all members of the phosphatidylinositol 3-kinase-related protein kinases family, notably mTOR, with Reptin silencing leading to decreased mTOR level and a consequent defect in signaling through S6 kinase (7). Our data also lead us to conclude accordingly that antagonists of Reptin ATPase activity may be of benefit for the therapy of hepatocellular carcinoma.…”

“…Overexpression of the Walker B mutant Pontin D302N in the presence of endogenous Pontin, thus, inhibited cell transformation by several oncogenes such as E1A (15), c-myc (16), or b-catenin (17). With regard to Reptin, the Walker B mutant D299N was not able to complement the effect of the loss of endogenous Reptin on the amount of PIKK proteins in HEK293 cells (7), suggesting that the ATPase activity of Reptin is indeed required for the regulation of PIKK levels. However, the same mutant was as efficient as wild-type (WT) Reptin for the repression of the influenza A virus polymerase (18) or of the transcriptional activity of ATF2 (19).…”

“…They are involved in the remodeling of chromatin, the regulation of transcription, and in DNA-damage sensing and repair (2,3). They function as chaperones and are required for the assembly of ribonucleoprotein complexes such as telomerase (4), snoRNPs (5), or, as part of the R2TP complex, for the assembly and/or stabilization of the RNA polymerase II complex (6) and of all members of the PIKK family including ATM, ATR, DNA-PKcs, SMG-1, TRRAP, and mTOR (7).…”

Tti1 and Tel2 Are Critical Factors in Mammalian Target of Rapamycin Complex Assembly

“…Finally, we showed that Tti1 could interact with all other PIKK family proteins. During revision of this report, Tti1 was also reported as an SMG-1-interacting protein with the name of SMG-10, which further interacts with RUVBL1 and RUVBL2 (37).…”