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

RNA polymerase III (Pol III, POLR3) synthesizes tRNAs and other small non-coding RNAs. Human POLR3 pathogenic variants cause a range of developmental disorders, recapitulated in part by mouse models, yet some aspects of POLR3 deficiency have not been explored. We characterized a human POLR3B:c.1625A>G;p.(Asn542Ser) disease variant that was found to cause mis-splicing of POLR3B. Genome-edited POLR3B(1625A>G) HEK293 cells acquired the mis-splicing with decreases in multiple POLR3 subunits and TFIIIB, although display auto-upregulation of the Pol III termination-reinitiation subunit POLR3E. La protein was increased relative to its abundant pre-tRNA ligands which bind via their U(n)U-3prime-termini. Assays for cellular transcription revealed greater deficiencies for tRNA genes bearing terminators comprised of 4Ts than of greater than or equal to 5Ts. La-knockdown decreased Pol III ncRNA expression unlinked to RNA stability. Consistent with these effects, small-RNAseq showed that POLR3B(1625A>G) and patient fibroblasts express more tRNA fragments (tRFs) derived from pre-tRNA 3prime-trailers (tRF-1) than from mature-tRFs, and higher levels of multiple miRNAs, relative to control cells. The data indicate that decreased levels of Pol III transcripts can lead to functional excess of La protein which reshapes small ncRNA profiles revealing new depth in the Pol III system. Finally, patient cell RNA analysis uncovered a strategy for specific Pol III-transcribed ncRNAs as POLR3-deficiency biomarkers.