The methyl phosphate capping enzyme Bmc1/Bin3 is a stable component of the fission yeast telomerase holoenzyme

Abstract

The telomerase holoenzyme is critical for maintaining eukaryotic genome integrity. In addition to a reverse transcriptase and an RNA template, telomerase contains additional proteins that protect the telomerase RNA and promote holoenzyme assembly. Here we report that the methyl phosphate capping enzyme (MePCE) Bmc1/Bin3 is a stable component of the S. pombe telomerase holoenzyme. Bmc1 associates with the telomerase holoenzyme and U6 snRNA through an interaction with the recently described LARP7 family member Pof8, and we demonstrate that these two factors are evolutionarily linked in fungi. Our data suggest that the association of Bmc1 with telomerase is independent of its methyltransferase activity, but rather that Bmc1 functions in telomerase holoenzyme assembly by promoting TER1 accumulation and Pof8 recruitment to TER1. Taken together, this work yields new insight into the composition, assembly, and regulation of the telomerase holoenzyme in fission yeast as well as the breadth of its evolutionary conservation.

The fission yeast methyl phosphate capping enzyme Bmc1 guides 2’-O-methylation of the U6 snRNA

“…Our previous work characterizing Bmc1 as a component of the telomerase holoenzyme also revealed interactions between Bmc1 and various other noncoding RNAs, including the U6 snRNA (Figure S1A) (20). We therefore tested whether Bmc1 has a role in the biogenesis, stability, or function of these transcripts, and if this function is linked to the Bmc1-interacting telomerase components Pof8 and Thc1.…”

“…Having already demonstrated that Pof8 is required to recruit Bmc1 to the telomerase RNA TER1 (20), we determined the protein binding requirements for U6. In contrast to what has been reported for TER1, for which reduced binding to Pof8 persists in the absence of Bmc1 (20,34), we found that all three proteins are necessary for an interaction with U6 (Figure 1A, S1B). Our results indicating an interaction between Pof8 and U6 are also consistent with previous work identifying mammalian LARP7 as a U6-interacting protein (26,27), suggesting that LARP7 family members have conserved functions related to U6, in addition to the established evolutionary conservation of LARP7 in telomerase (29)(30)(31).…”

“…qRT-PCR and semi-quantitative RT-PCR 1 µg TURBO DNase-treated RNA was reverse transcribed with the iScript cDNA reverse transcription kit (Biorad, 1708890) or 5 U AMV-RT (NEB, M0277S) and gene-specific reverse primers. qRT-PCR was performed with the SensiFAST SYBR No-Rox kit (Bioline, BIO-98005) and 1 µM of each primer, with settings outlined in (20). For semi-quantitative RT-PCR, cDNA was amplified with Taq polymerase (NEB, MO273L) using the following cycling conditions: 5 min initial denaturation at 94˚C, 26 (pud1, alp41) or 27 (rpl1603, bor1) cycles of 30 s at 94˚C, 30 s at 50˚C, and 1 minute at 72˚C, and a final 5 minute extension at 72˚C.…”

“…We therefore tested whether Bmc1 has a role in the biogenesis, stability, or function of these transcripts, and if this function is linked to the Bmc1-interacting telomerase components Pof8 and Thc1. Having already demonstrated that Pof8 is required to recruit Bmc1 to the telomerase RNA TER1 (20), we determined the protein binding requirements for U6. In contrast to what has been reported for TER1, for which reduced binding to Pof8 persists in the absence of Bmc1 (20,34), we found that all three proteins are necessary for an interaction with U6 (Figure 1A, S1B).…”

“…Conversely, ciliate and fission yeast LARP7 homologs have been well studied for their roles in telomerase biogenesis (28)(29)(30)(31)(32)(33). We and others have reported that the S. pombe LARP7 protein Pof8 associates with the Bin3/MePCE homolog Bmc1 in the telomerase holoenzyme, that this interaction is important for optimal telomerase activity, and that the link between these proteins is evolutionarily conserved across diverse fungal species (19,20,34). Thus, while much has been learned about MePCE/Bmc1 function in the 7SK snRNP and telomerase, its precise role in U6 biogenesis and function remains unknown.…”