Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs

Abstract

The DNA-dependent adenosine triphosphatase (ATPase) Plk1-interacting checkpoint helicase (PICH) has recently been implicated in spindle checkpoint (SAC) signaling (Baumann et al., Cell 128(1):101–114, 2007). Depletion of PICH by siRNA abolished the SAC and resulted in an apparently selective loss of Mad2 from kinetochores, suggesting a role for PICH in the regulation of the Mad1–Mad2 interaction. An apparent rescue of SAC functionality by overexpression of PICH in PICH-depleted cells initially seemed to confirm a role for PICH in the SAC. However, we have subsequently discovered that all PICH-directed siRNA oligonucleotides that abolish the SAC also reduce Mad2 mRNA and protein expression. This reduction is functionally significant, as PICH siRNA does not abolish SAC activity in a cell line that harbors a bacterial artificial chromosome driving the expression of murine Mad2. Moreover, we identified several siRNA duplexes that effectively deplete PICH but do not significantly affect SAC functionality or Mad2 abundance or localization. Finally, we discovered that the ability of overexpressed PICH to restore SAC activity in PICH-depleted cells depends on sequestration of the mitotic kinase Plk1 rather than ATPase activity of PICH, pointing to an underlying mechanism of “bypass suppression.” In support of this view, depletion or inhibition of Plk1 also rescued SAC activity in cells harboring low levels of Mad2. This observation suggests that a reduction of Plk1 activity partially compensates for reduced Mad2 levels and argues that Plk1 normally reduces the strength of SAC signaling. Collectively, our results question the role of PICH in the SAC and instead identify Mad2 as a sensitive off target for small RNA duplexes. In support of the latter conclusion, our evidence suggests that an off-target effect on Mad2 may also contribute to explain the apparent role of the Tao1 kinase in SAC signaling (Draviam et al., Nat Cell Biol 9(5):556–564, 2007).Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-009-0244-2) contains supplementary material, which is available to authorized users.

Centromere DNA decatenation depends on cohesin removal and is required for mammalian cell division

“…Originally, it had been speculated that centromere-associated PICH might function as a tension sensor in the SAC (Baumann et al, 2007). Although a role for PICH in the SAC appears unlikely in the light of recent results (Huebner et al, 2010), a persistent connection between sister kinetochores may indeed provide a convenient platform for the placement of a tension sensor. In line with this hypothesis, centromere-associated Aurora B has recently been proposed to contribute to the monitoring of tension through its spatial separation from kinetochore-associated substrates (Liu et al, 2009).…”

“…Its predicted enzymatic properties and specific association with centromeres originally suggested a potential role for this protein as a tension sensor in the SAC (Baumann et al, 2007). However, in the light of recent findings, this now appears unlikely (Huebner et al, 2010;Maresca and Salmon, 2009;Uchida et al, 2009). However, the surprising discovery that PICH persisted on centromere-derived DNA during anaphase renewed interest in the role and regulation of DNA decatenation during mitotic progression .…”

Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints

“…Clearly, there is no room in this scheme for a tension-sensing kinetochore/centromere-based checkpoint element. [The recent report that after a long search this element has finally been discovered (Baumann et al 2007) was erroneous (Hubner et al 2010)]. Instead, the checkpoint detects unattached kinetochores, and the checkpoint signal is shut down at a kinetochore as MTs progressively accumulate on its surface (Rieder et al 1994;Waters et al 1998); this is a quick process (McEwen et al 1997) promoted by tension which slows the turnover rate of kinetochore-associated MTs (King and Nicklas 2000).…”

Kinetochores accelerate centrosome separation to ensure faithful chromosome segregation

“…We had previously linked this difference to the absence of the spindle checkpoint protein Mad2 at kinetochores in siCENP-O-treated cells (McAinsh et al, 2006;McClelland et al, 2007). This difference seemed specific because it was based on two independent siRNAs for each depletion (McAinsh et al, 2006;McClelland et al, 2007); however, recent studies showed that the Mad2 mRNA is a frequent off-target for various siRNAs, raising the possibility that our siRNAs targeting CENP-O might also target Mad2 (Hubner et al, 2010;Westhorpe et al, 2010). To test for this possibility and to unequivocally confirm the specificity of our phenotypes, we generated stable HeLa cell lines expressing either siRNA-resistant EGFP-CENP-L or siRNA-resistant EGFP-CENP-O.…”