A POLR3B-variant reveals a Pol III transcriptome response dependent on La protein/SSB

RNA polymerase III (POLR3) synthesizes tRNAs and other non-coding transcripts. POLR3 pathogenic variants cause neurodevelopmental disorders. General effects of POLR3-deficiency on Pol III transcripts are unknown. We show that a POLR3B:c.1625A>G;p.(Asn542Ser) disease variant exhibits mis-splicing with decreased POLR3B mRNA. Genome-edited POLR3B1625A>G HEK293 cells acquired this mis-splicing with decreases in multiple POLR3 subunits and TFIIIB and auto-upregulation of recycling subunit POLR3E. La protein was increased relative to its abundant pre-tRNA ligands which bind via their U(n)U-3p termini. Assays for cellular transcription revealed greater decreases for tRNA genes bearing 3p-terminators of 4Ts than >5Ts. La-knockdown decreased apparent transcription by Pol III unlinked to RNA stability. Consistent with La-mediated effects, small-RNAseq showed that POLR3B1625A>G and patient cells express more tRNA fragments (tRFs) from pre-tRNA 3p-trailers (tRF-1) than mature-tRFs relative to control cells. The data illustrate a tRF1-specific response to POLR3B-deficiency. Analysis of patient small-RNAseq data uncovered multiple tRNA gene-specific tRF-1/tRF-3 products as candidates for POLR3-deficiency biomarkers.