A Short CEP135 Splice Isoform Controls Centriole Duplication

Abstract

Summary Centriole duplication is coordinated such that a single round of duplication occurs during each cell cycle. Disruption of this synchrony causes defects including supernumerary centrosomes in cancer and perturbed ciliary signaling [1–5]. To preserve the normal number of centrioles, the level, localization, and post-translational modification of centriole proteins is regulated so that when centriole protein expression and/or activity is increased, centrioles self-assemble. Assembly is initiated by the formation of the cartwheel structure that comprises the base of centrioles [6–11]. SAS-6 constitutes the cartwheel and SAS-6 levels remain low until centriole assembly is initiated at S-phase onset [3, 12, 13]. Cep135 physically links to SAS-6 near the site of microtubule nucleation and binds to CPAP for triplet microtubule formation [13, 14]. We identify two distinct protein isoforms of Cep135 that antagonize each other to modulate centriole duplication: full length Cep135 (Cep135full) promotes new assembly while a short isoform, Cep135mini, represses it. Cep135mini represses centriole duplication by limiting the centriolar localization of Cep135full binding proteins (SAS-6 and CPAP) and the pericentriolar localization of γ-tubulin. The Cep135 isoforms exhibit distinct and complementary centrosomal localization during the cell cycle. Cep135mini protein decreases from centrosomes upon anaphase onset. We suggest that the decrease in Cep135mini from centrosomes promotes centriole assembly. The repression of centriole duplication by a splice isoform of a protein that normally promotes it serves as a novel mechanism to limit centriole duplication.

The Human Centriolar Protein CEP135 Contains a Two-Stranded Coiled-Coil Domain Critical for Microtubule Binding

“…The present data do not reveal the function of this most N-terminal segment; due to its high conservation across evolution, we expect that this part of CEP135/Bld10p may represent a binding site for centriolar protein partner(s) that awaits identification. It has been recently reported that there is a short human CEP135/Bld10p splice isoform (dubbed CEP135mini) comprising the first 249 N-terminal residues of HsCEP135, plus 16 amino acids that differ in sequence from the full-length protein (Dahl et al, 2015). This shorter HsCEP135 variant negatively regulates centriole assembly by inhibiting the activity of the full-length protein, presumably by preventing it from interacting with centriolar binding partners such as HsSAS-6 (Dahl et al, 2015).…”

“…It has been recently reported that there is a short human CEP135/Bld10p splice isoform (dubbed CEP135mini) comprising the first 249 N-terminal residues of HsCEP135, plus 16 amino acids that differ in sequence from the full-length protein (Dahl et al, 2015). This shorter HsCEP135 variant negatively regulates centriole assembly by inhibiting the activity of the full-length protein, presumably by preventing it from interacting with centriolar binding partners such as HsSAS-6 (Dahl et al, 2015). Notably, our HsCEP135-N atomic model accounts for the first approximately two-thirds of CEP135mini.…”

“…By contrast, primary fibroblasts derived from patients suffering from microcephaly due to a premature termination codon in the human CEP135/ Bld10p gene display aberrations in centrosome numbers (Hussain et al, 2012). Furthermore, small interfering RNA-mediated depletion of CEP135/Bld10p in human cultured cells causes a loss of centriolar microtubules, as well as a reduction in centriole length (Lin et al, 2013;Dahl et al, 2015), reminiscent of the phenotype obtained in Drosophila. Together, these observations suggest that CEP135/Bld10p plays an important role in promoting centriole assembly and stability, although its exact mode of action appears to differ somewhat depending on the species.…”

Centriole assembly at a glance

“…For example, deletion of CEP135 from chicken DT40 cells does not prevent procentriole assembly, even though it reduces its efficiency (Inan et al, 2013). Likewise, RNAi-mediated depletion of CEP135 in human cells leads to partial impairment of the process (Lin et al, 2013), although the interpretation of these depletion experiments is complicated by the existence of two CEP135 transcripts with opposing roles (Dahl et al, 2015). Overall, it appears that either the pinhead is dispensable for procentriole assembly in some systems or else proteins other than CEP135 operate in a partially redundant manner in some cases.…”

WBP11 is required for splicing the TUBGCP6 pre-mRNA to promote centriole duplication

“…Expression of the centriole proteins PLK4 and CP110 is downregulated by the binding of miRNAs to the 39 UTR of the mRNA (Bao et al, 2018;Cao et al, 2012;Song et al, 2014). Moreover, two alternative isoforms of CEP135 have been shown to play opposing roles in centriole biogenesis, and dysregulation of isoform expression promotes centrosome amplification in breast cancer cells (Dahl et al, 2015;Ganapathi Sankaran et al, 2019). A genome-wide RNAi screen revealed a strong connection between splicing factors and centriole biology, with 14 of 38 genes required for centriole biogenesis playing established roles in mRNA splicing (Balestra et al, 2013).…”