Effects of HLD-associated POLR1C mutant proteins on cellular localization and differentiation.

Hypomyelinating leukodystrophis (HLDs) are composed of a group of congenital central nervous system (CNS) neuropathies, which resemble prototypic Pelizaeus-Merzbacher disease (PMD, also called HLD1). They display myelin dysfunction by causing repeated demyelination and remyelination, leading to severe demyelination [[1], [2], [3], [4], [5]]. CNS myelin is derived from morphologically differentiated oligodendrocyte plasma membranes. It plays an essential role in propagation of saltatory conduction and in protecting neuronal axons from physical and physiological stresses [[6], [7], [8]].

The polr1c gene encodes one subunit of nuclear RNA-catabolizing enzymes. RNA polymerase I and III subunit C (POLR1C) contributes to transcription of ribosomal RNA (rRNA), transfer RNA (tRNA), and other small RNAs. Two missense mutations Asn-32-to-Ile (N32I) and Asn-74-to-Ser (N74S) of the polr1c gene are associated with HLD11 (OMIN No. 616494) [9]; however, it is still unknown whether their mutations indeed affect intracellular localization of POLR1C proteins and/or cell morphological differentiation.

The plasmid encoding each human POLR1C construct or the wild type was introduced into oligodendrocyte cell line FBD-102b [10,11]. In the Fig. 1, the wild type was localized in the nuclei. In contrast, the N32I mutant proteins were primarily localized in punctate structures of the extranuclear region. Similar results were observed in the case of the N74S mutant (Fig. S1). The structures partially corresponded to the ones stained with an antibody against the lysosome marker LAMP1 (Fig. S2). Cells harboring the N32I or N74S mutant failed to exhibit differentiated phenotypes with myelin web-like structures along inner processes (Fig. S3).

Fig. 1

The Asn-32-to-Ile missense mutant proteins of POLR1C exhibit extracellular localization with punctate structures. (A) FBD-102b cells were transfected with the plasmid encoding the wild type or the Asn-32-to-Ile mutant of GFP-tagged POLR1C. Representative GFP-fluorescence (green) and DAPI-fluorescence (blue) images are shown. The white arrowheads indicate the representative aggregation-like punctate structures. (B) The line plot along a white line (from the left to right direction) of the respective images is shown. F.I. indicates fluorescent intensities. (C) Cells with punctate structures of GFP-tagged POLR1C are statistically shown (*, p < .01 of Student's t-test for the control; n = 3 fields). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. S1

The Asn-74-to-Ser missense mutant proteins of POLR1C exhibit extracellular localization with punctate structures. (A) FBD-102b cells were transfected with the plasmid encoding the Asn-74-to-Ser mutant of GFP-tagged POLR1C. Representative GFP-fluorescence (green) and DAPI-fluorescence (blue) images are shown. The white arrowhead indicates the representative aggregation-like punctate structure. (B) The line plot along a white line (from bottom to top) of the respective images is shown. F.I. indicates fluorescent intensities. (C) Cells with punctate structures of GFP-tagged POLR1C proteins are statistically shown.

Fig. S2

The POLR1C missense mutant proteins are localized in the lysosome. (A, B) FBD-102b cells were transfected with the plasmid encoding each of the respective mutants of GFP-tagged POLR1C (green) and stained with an anti-LAMP1 antibody (red). The line plots along a white line (from left to right) of the respective images are also shown. The white arrowheads indicate the representative aggregation-like punctate structures.

Fig. S3

The POLR1C missense mutants inhibit morphological differentiation. (A, B) FBD-102b cells were transfected with the plasmid encoding each of the respective mutants of GFP-tagged POLR1C. Neomycin-resistant gene-containing cells were selected. Cells stably harboring each of the respective POLR1C mutants, as well as their parental cells, were allowed to differentiate for 5 days. Cells with myelin-like web structures, along inner processes, are considered to be differentiated and are statistically shown (**, p < .01 of one-way ANOVA with post-hoc Fisher's test for the control; n = 3 fields). Representative small and large images of differentiated cells (indicated by surrounded, dotted lines) are shown.

The plasmid encoding each human POLR1C construct or the wild type was introduced into oligodendrocyte cell line FBD-102b [10,11]. In the Fig. 1, the wild type was localized in the nuclei. In contrast, the N32I mutant proteins were primarily localized in punctate structures of the extranuclear region. Similar results were observed in the case of the N74S mutant (Fig. S1). The structures partially corresponded to the ones stained with an antibody against the lysosome marker LAMP1 (Fig. S2). Cells harboring the N32I or N74S mutant failed to exhibit differentiated phenotypes with myelin web-like structures along inner processes (Fig. S3).

The N32 and N74 positions are closely placed either on an amino acid sequence (Fig. S4) or on a predicted three-dimensional structure (Fig. S5). But, they are different from missense mutation positions in Treacher-Collins syndrome 3 (OMIN No. 248390). Further analyses will allow us to promote our understanding of the relationship between POLR1C mutations and the cellular effects.

Fig. S4

Alignment among mammals in the POLR1C amino acid sequences. Human, mouse, and rat POLR1C amino acid sequences and some amino acid numbers are shown. Red letters indicate the position of missense mutations associated with HLD11. Blue letters indicate those associated with Treacher-Collins syndrome 3. Bold letters show common amino acids in human and rodents. Normal letters show different amino acids in human and rodents.

Fig. S5

Predicted, human POLR1C 3D structure. The human POLR1C 3D structure is predicted based on the X-ray structure of yeast POLR1C. A red letter indicates the position of a missense mutation associated with HLD11. Blue letters indicate those associated with Treacher-Collins syndrome 3. Mutated positions are shown as black space-filling spheres.

The N32 and N74 positions are closely placed either on an amino acid sequence (Fig. S4) or on a predicted three-dimensional structure (Fig. S5). But, they are different from missense mutation positions in Treacher-Collins syndrome 3 (OMIN No. 248390). Further analyses will allow us to promote our understanding of the relationship between POLR1C mutations and the cellular effects.

The following are the supplementary data related to this article.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgmr.2018.11.002.