Oral Presentation 43rd Lorne Genome Conference 2022

The DBHS family of RNA/DNA binding proteins regulate telomerase assembly and recruitment to the telomere (#54)

Alexander P Sobinoff 1 , Christopher B Nelson 1 , Scott B Cohen 2 , Tracy M Bryan 3 , Archa Fox 4 , Hilda A Pickett 1
  1. Telomere Length Regulation Unit, Children’s Medical Research Institute, University of Sydney, Sydney, NSW, Australia
  2. Children's Medical Research Institute, Westmead, NSW, Australia
  3. Cell Biology Unit, Children’s Medical Research Institute, University of Sydney, Westmead, NSW, Australia
  4. School of Human Sciences, The University of Western Australia, Perth, WA, Australia

As part of oncogenesis, cancer cells must activate a telomere maintenance mechanism to counteract telomere shortening and achieve cellular immortality. Approximately 85-90% of cancers do this by activating the enzyme telomerase, a ribonucleoprotein complex consisting of the catalytic reverse transcriptase hTERT and the RNA template component hTR. Telomere maintenance via telomerase requires the correct assembly of its protein and RNA components, as well as the organisation of co-factors involved in maturation, stability, and recruitment to the telomere. We have identified the Drosophila behaviour/human splicing (DBHS) family of RNA/DNA binding proteins as novel regulators of telomerase biology. Our data demonstrate all three members of the DBHS protein family (NONO, SFPQ and PSPC1) function to regulate telomerase activity via its recruitment to telomeres.

Immunoprecipitation revealed that the DBHS proteins associate with both hTR and catalytically active telomerase. Depletion of all three DBHS proteins decreased hTR co-localisations with telomeric DNA via IF-FISH, suggesting impaired telomerase trafficking to the telomere. Intriguingly, knockdown of NONO and SFPQ resulted in the sequestration of hTR in enlarged nuclear Cajal bodies, the hypothesised sites of hTR processing and maturation. In contrast, knockdown of PSPC1 had no effect on hTR localisation, but did reduce levels of coilin, an integral component of Cajal bodies. Rescue experiments using 3’-UTR siRNA and functional domain mutants revealed that NONO-mediated trafficking of active telomerase to telomeres requires a functional RNA recognition motif and the protein-protein interaction NONA/ParaSpeckle (NOPS) domain. Finally, continuous passage of NONO and PSPC1 CRISPR-Cas9 knockout clones over 60 population doublings demonstrated striking telomere shortening consistent with the absence of telomere maintenance, demonstrating inhibition of telomerase-mediated telomere extension.

Our results identify the DBHS proteins as novel and unexpected players in the regulation of telomerase recruitment/activity that have the potential to be exploited to prevent telomerase-mediated telomere extension in cancer.