ZC3HC1 is a structural element of the nuclear basket effecting interlinkage of TPR polypeptides

Abstract

The nuclear basket (NB) is a fibrillar structure commonly looked upon as solely built of polypeptides of the large coiled-coil-forming protein TPR. Here, protein ZC3HC1 is reported as a novel second structural element of the NB functioning in the stepwise recruitment and stable interconnection of TPR subpopulations at the NB.

Nuclear basket protein ZC3HC1 and its yeast homolog Pml39p feature an evolutionary conserved bimodular construction essential for initial binding to NPC-anchored homologs of scaffold protein TPR

“…Altogether, such a reduction in the NE-associated amounts of Mlp1p, accompanied by a nucleoplasmic pool of Mlp1p due to Pml39p deficiency, resembled the effects we had by then noted in human cells, in which knockdown of ZC3HC1 by RNAi had caused a reduction in the NE-associated amounts of TPR, accompanied by a nucleoplasmic pool of TPR. Later, such findings also turned out in line with very similar results in ZC3HC1 KO cells (Gunkel et al, 2021) and eventually upon degron-mediated ZC3HC1 degradation too (Gunkel & Cordes, 2022), with the latter findings now also resembling the outcome of a study in which degronmediated degradation of Pml39p is shown affecting the NE-association of Mlp1p (Bensidoun et al, 2021b).…”

“…We corroborated this conclusion experimentally by demonstrating that two other species’ NuBaID-signature-containing proteins are true homologs of Hs ZC3HC1, namely a NuBaID-containing protein of Dictyostelium discoideum and the Pml39 protein of Saccharomyces cerevisiae , with these proteins together with Hs ZC3HC1 markedly exemplifying the diversity between the NuBaID proteins of different phyla. While Sc Pml39p had already been found to be an Mlp1/2-binding protein (Palancade et al , 2005) and recently stated to be the yeast homolog of Hs ZC3HC1 (Gunkel et al , 2021; Gunkel & Cordes, 2022), we here present the experimental evidence unveiling that Sc Pml39p just like Hs ZC3HC1 possesses a bimodular NuBaID and requires integrity of its NuBaID’s two modules for NB- and Mlp-interaction. Moreover, we demonstrate that NB-association of Pml39p is a prerequisite for the positioning of additional amounts of Mlp1p at the yeast’s NBs and that Pml39p even enables interconnections between Mlp1 polypeptides at sites remote from the NB.…”

“…Alternatively, we can also conceive of such ZC3HC1 loops as playing a role as spacer elements, defining distances between the NB and neighboring chromatin. Furthermore, while dispensable for the initial binding of ZC3HC1 to the NB, we can also imagine a scenario in which such loops might play a role as lariats in the regulatable formation of higher-order cylindrical arrangements between TPR and ZC3HC1 of the type discussed recently (Gunkel et al , 2021; Gunkel & Cordes, 2022).…”

“…However, at least concerning the Mlp1p foci, present in nup60Δ cells while absent in nup60Δ pml39Δ cells, we rated these foci as equivalent to the TPR foci in the NUP153-deficient but ZC3HC1-positive human cells while absent in the NUP153- and ZC3HC1-deficient cells. Moreover, since we later could prove via degron-mediated ZC3HC1 degradation that the TPR foci are indeed held together by ZC3HC1 (Gunkel & Cordes, 2022), we eventually also considered it legitimate to interpret our yeast data accordingly, namely by also regarding Pml39p as a protein with a direct role as a linker element between Mlp1 polypeptides even at sites remote from the NB.…”

“…However, differing from the other TPR-associated NB proteins, we found the NB-attached ZC3HC1 relevant for the NB-positioning of about half the total amount of NE-associated TPR in different cell types (Gunkel et al , 2021), with ZC3HC1 itself eventually found functioning as a structural NB component. Following its initial binding to the NPC-anchored TPR polypeptides, ZC3HC1 enables the subsequent recruitment of additional TPR polypeptides and their linkage to TPR already present at the NB (Gunkel & Cordes, 2022). However, it remained to be determined how such a reciprocally dependent positioning of ZC3HC1 and subpopulations of TPR at the NB can come about and which parts of ZC3HC1 enable it to engage in possibly different types of interactions with TPR.…”