BRF1 mutations alter RNA polymerase III-dependent transcription and cause neurodevelopmental anomalies

Abstract

RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development.

Figure 1.

BRF1 mutations cause a cerebellar-dental-skeletal syndrome. (A) Patients 1 and 2 (family 1) at the ages of 12 and 10 yr, patients 3 and 4 (family 2) at the ages of 10 and 4 yr, and patients 5 and 6 (family 3). Note characteristic facial dysmorphism and dental anomalies. (B) Brain MRI (top, sagittal scans; bottom, coronal scans) of patients 1–4 (P1–P4) at the ages of 14 yr, 9 yr, 12 yr, and 18 mo, respectively, showing a thin corpus callosum (white filled arrows), flattened brainstem (white unfilled arrows), and cerebellar hypoplasia (black unfilled arrows). (C) Pedigrees of family 1 (left), family 2 (middle), and family 3 (right) with genotypes for BRF1 missense alterations. In family 2, the p.Pro292His mutation was likely transmitted by the unaffected father who did not participate in the study. In family 3, the two individuals denoted by an asterisk had Leber congenital amaurosis caused by a homozygous RDH12 mutation. (D) Multiple sequence alignment of BRF1 orthologs and human TFIIB showing evolutionary conservation of mutant BRF1 amino acid residues.

Figure 2.

Functional annotation of variants in isoforms 1 and 2 of BRF1 and its effects on the head size, optic tectum size, and cerebellar formation in zebrafish embryos. (A,E) Schematic representation of the location of BRF1 variants examined in zebrafish within each of the two isoforms evaluated: isoform 1 (NP_001229717.1; 650 aa) and isoform 2 (NP_001229715.1; 584 aa). In purple are alleles that when tested are shown to be null, and in brown are alleles that score as hypomorphs based on zebrafish assays. (B,B′) Dorsal views of control zebrafish embryos and embryos injected with brf1b MO, brf1b MO + WT human BRF1, and brf1bMO + variant (R223W, S226L, or T259M) human BRF1 RNA in the context of isoform 1, respectively, at 3 d.p.f., stained with anti-α acetylated tubulin. (B) Head size measurements were taken using brightfield images (highlighted with a yellow outline in panel F). (B′) The area of the optic tecta was measured in the fluorescent images (highlighted with a cyan oval in panel F′). (C,D) Bar graphs showing the relative head size (C) and the optic tecta area (D). Data are presented as mean ± SE. Two-tailed t-tests were performed to assess statistical significance. The embryos coinjected with the MO and each of the variants were statistically different from MO alone but not statistically different when compared to embryos injected with MO + WT human BRF1, therefore scoring as benign. (F–I′) Functional assessment of the BRF1 missense variants in the context of isoform 2. Three d.p.f. embryos injected with brf1b MO, brf1b MO + WT human BRF1, brf1b MO + variant human BRF1 RNA (P292H, R223W, S226L, or T259M), or variant human BRF1 RNA alone in the context of isoform 2 were observed following staining with anti-α acetylated tubulin for head size (F) and for optic tecta area (F′), as well as for cerebellar defects (F′; illustrated with a red dashed box where maximum disorganization is observed). (G–I′) Bar graphs showing average head size, optic tecta area, as well as the percentage of embryos with cerebellar defects evaluated among each condition. To assess statistical significance among the evaluated conditions, two-tailed t-tests were performed for head size and optic tecta, and χ² tests were performed for cerebellar disorganization to evaluate statistical significance across the conditions. In the context of isoform 2, functional analysis using zebrafish show that P292H and R223W are null, while S226L and T259M are hypomorphic alleles, showing an isoform-specific effect in which head size and optic tecta size are reduced and cerebellar disorganization occurs. No significant effects were observed for any of the phenotypes when the variants themselves were overexpressed. Each experiment was done at minimum in triplicate with at least 50 embryos per condition per replicate. (***) P-value ≤ 0.001 relative to MO + WT; (◊) P-value ≤ 0.001 relative to controls; (†) P-value ≤ 0.01 relative to MO.

Figure 3.

BRF1 mutations cause growth defects and reduced target promoter occupancy. (A) BRF1 mutations affect cell growth. Spot dilutions of the variants introduced into a BRF1 knockout strain grown at 30°C. Wild-type (WT) and variant Brf1 were encoded on plasmids. For the combination of two mutations, two plasmids were used, each harboring one mutation and a distinct marker. (5-FOA)5-fluoroorotic acid. (B) Three-dimensional modeling of human BRF1 missense alterations. The four identified amino acid substitutions were mapped to the structure of human TFIIB (green) in a complex with TBP (purple) and DNA (pdb code 1C9B) (Tsai and Sigler 2000). Amino acids affected by mutations in families 1 and 2 are shown in yellow and orange, respectively. (C–E) BRF1 variants show a decreased occupancy of tRNA promoters in yeast. Fold enrichments of ChIP experiments performed with tandem affinity purification (TAP)—tagged BRF1 variants in yeast. Data are presented as mean ± SD. (C) Mutations A226S to serine or S226L show no effect on fold enrichment compared to WT. (D) Mutation T259M leads to decreased fold enrichment on tRNA genes. (E) Combination of the two variants results in a much lower occupancy of the S226L and T259M variants. Summing up both signals results in less occupancy of the two mutated BRF1 than the WT BRF1.

Figure 4.

In vitro transcription defects caused by BRF1 mutations in yeast. (Top) A representative gel of the in vitro transcription reaction using a nuclear extract harboring a deficient Brf1 protein (p.Trp107Arg, W107R) that is impaired in Pol III–dependent transcription. Addition of WT Brf1 rescues activity whereas the different Brf1 variants and the combinations present in the affected children show a defect in transcription. Data are presented as mean ± SD. A quantification is shown in the middle panel. (*) P < 0.01; (**) P < 0.0001. The lower panel shows a twelve times excess of the Brf1 protein amount used for this assay separated on an SDS gel.