Chapter 1 14 In line with molecular disruptions of the affected subunits, several studies have documented reduced Pol III transcript levels in various patient-derived samples. Studies have shown that Pol III products such as tRNAs and BC200, which are involved in translation, are dysregulated in patient-derived fibroblasts and cellular models of 4H (Choquet, Forget, et al., 2019). Similar reductions have been observed in fibroblasts from patients with POLR3K variants, where tRNAs, H1 RNA, 5S rRNA, and 7SL RNA were significantly downregulated (Dorboz et al., 2018). Reduced levels of POLR3 transcripts have also been detected in blood RNA samples from patients with POLR3A variants (Azmanov et al., 2016). A mouse model with POLR3A variants further supports this, showing reduced tRNA levels (Moir et al., 2024). It is also shown in yeast that mutations associated with 4H decrease tRNA transcription (Arimbasseri et al., 2015). Tissue-specific vulnerability to Pol III mutations may also contribute to 4H syndrome’s pathology. For instance, it is hypothesized that oligodendrocytes, which require substantial protein synthesis during myelination, might be particularly affected due to the role of Pol III transcripts in translation (Perrier, Michell-Robinson, et al., 2020). Considering that Pol III products like tRNAs, have tissue specific expression profiles makes this hypothesis even more plausible (Dittmar et al., 2006). Additionally, the introduction of a POLR3A variant in MO3.13 cells (human oligodendrocytes) reduced myelin basic protein (MBP) levels in these oligodendrocytes (Choquet, Forget, et al., 2019), further pointing towards oligodendrocyte vulnerability. Additional evidence is found in the fact that OPCs with decreased RNA Pol III subunit expression showed altered oligodendrocyte differentiation, maturation and myelination capacity (Macintosh, Michell-Robinson, et al., 2023). Similarly, a mouse model with POLR3B exon 10 deletion exhibited defects in oligodendrocyte proliferation and differentiation, supporting the role of Pol III in myelination (Michell-Robinson et al., 2023). However, these mechanisms focus mainly on oligodendrocytes, this does not provide an explanation for 4H patients presenting mainly with neurological symptoms. An alternative hypothesis involves cell-type-specific effects mediated by alternative splicing. For instance, the intronic variant c.1771-7C>G in POLR3A leads to altered transcripts, including the skipping of exon 14, resulting in a premature stop codon or an in-frame deletion of exons 13 and 14 (Perrier, Gauquelin, et al., 2020) Similarly, POLR3A variant c.1771-6C>G is reported to result in exon 14 skip (Azmanov et al., 2016; Yoon Han et al., 2022). And also for POLR3B variants such as c.1625A>G and c.2084-6A>G, mis-splicing has been reported (Daoud et al.,
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