Telomerase biogenesis requires a novel Mex67 function and a cytoplasmic association with the Sm7 complex

Abstract

The templating RNA is the core of the telomerase reverse transcriptase. In Saccharomyces cerevisiae, the complex life cycle and maturation of telomerase includes a cytoplasmic stage. However, timing and reason for this cytoplasmic passage are poorly understood. Here, we use inducible RNA tagging experiments to show that immediately after transcription, newly synthesized telomerase RNAs undergo one round of nucleo-cytoplasmic shuttling. Their export depends entirely on Crm1/Xpo1, whereas re-import is mediated by Kap122 plus redundant, kinetically less efficient import pathways. Strikingly, Mex67 is essential to stabilize newly transcribed RNA before Xpo1-mediated nuclear export. The results further show that the Sm7 complex associates with and stabilizes the telomerase RNA in the cytoplasm and promotes its nuclear re-import. Remarkably, after this cytoplasmic passage, the nuclear stability of telomerase RNA no longer depends on Mex67. These results underscore the utility of inducible RNA tagging and challenge current models of telomerase maturation.

Unraveling the stepwise maturation of the yeast telomerase

“…1E). These findings confirm that Sm-ring loading to TLC1 occurs in the cytoplasm, which was also shown earlier via an inducible TLC1-RNA tagging experiment and is very similar to the cytoplasmic Sm-ring loading onto the snRNAs 24,29 . Therefore, we investigated the interaction between the Sm-ring and TLC1 in the import receptor mutant mtr10∆.…”

“…Remarkably, although the RNA is less stable in mex67-5 mutants, the immature TLC1 still increased five fold in the double mutant rrp6∆ mex67-5, suggesting a) that its trimming occurs in the nucleus and b) that the immature form is still protected without Mex67. Furthermore it has to be noted, that in contrast to Vasianovich and colleagues, we find no rescue of the decreased TLC1 in the double mutant rrp6∆ mex67-5 as detected by northern blot 29 . Since we applied in our experiments the highly sensitive qPCR method, we were able to detect an increase of the immature TLC1 when its export is blocked in mex67-5, despite the overall decrease of the total TLC1 in both mutants (Fig.…”

“…Secondly, we assayed the TLC1-forms in the mex67-5 nuclear export mutant. We detected a decrease of total TLC1, which is in agreement with earlier findings in which Mex67 was suggested to protect TLC1 from its degradation 29 . However, in our experiments we still see a two-fold increase of the immature TLC1 in mex67-5 mutants, which suggests that immature TLC1 is not immediately degraded when Mex67 is missing (Fig.…”

“…Thus, the immediate cytoplasmic drain of these noncoding RNAs is crucial for cell survival and might also be similarly important for the transcribed immature TLC1. Interestingly, maturation events such as trimming and TMG-capping were suggested to be nuclear events that occur prior to TLC1 shuttling and current models suggest that only the Est-proteins and the Sm-ring are loaded in the cytoplasm 23,29,30 . Therefore, we investigated the maturation steps of TLC1 in detail and uncovered a stepwise process.…”

“…After loading of the Est-proteins and the Sm-ring in the cytoplasm, TLC1 is reimported into the nucleus via Mtr10 23,28,29 . Prevention of shuttling, either by mutations in the export factors or the import factor results in telomere shortening defects and reduced TLC1 levels 23,29,30 .…”