Analyses of the stepwise maturation of the yeast telomerase

• Applicant: Professorin Dr. Heike Krebber
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 271459301

Telomerases are enzymatic ribonucleoprotein complexes that antagonize the constant loss of the eukaryotic chromosome ends upon replication by the synthesis of repetitive DNA sequences. They are composed of an RNA (TLC1 in S. cerevisiae) and several proteins. TLC1 undergoes nuclear maturation before it is exported into the cytoplasm to recruit additional proteins for complete assembly. The mature telomerase is subsequently re-imported into the nucleus where it fulfills its function.We have recently shown that TLC1 requires the mRNA export machinery (e.g. Mex67) in addition to the Ran-dependent karyopherin Crm1/Xpo1 for its export from the nucleus (Wu et al. 2014, Cell Reports). Moreover, our unpublished results further reveal that the three SR-proteins Npl3, Gbp2 and Hrb1, which interact with Mex67, also interact with TLC1. In fact, their gene deletions result in severe telomere shortening defects. Strikingly, none of their gene deletions shows TLC1 export defects; Deletion of NPL3 rather shows nuclear re-import defects. These findings indicate crucial functions for these genes in the live cycle of TLC1 and underline the necessity for its nucleo-cytoplasmic shuttling. In this project we will analyze the functions of the SR-proteins on TLC1 maturation and transport with genetic, biochemical and cellular localization studies. Moreover, we will purify TLC1 from wild type cells and from strains that trap this RNA in the nucleus (e.g. mex67-5) or in the cytoplasm (Deletion of MTR10) to enrich the ribonucleoparticle at different states of its maturation. The associated proteins will then be purified and identified in mass spectrometry.

• DFG Programme: Research Grants