PMID:35277511

[manulera]

PMID:35277511

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

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.

Intro

• The LARP7 complex in higher eukaryotes

  • It works with 7SK snRNP, composed of:
  • 7SKsnRNA: transcribed by polIII
  • MePCE: Methyl Phosphatase Capping Enzyme (Bmc1 in Pombe, the gene of study in the paper)
  • HEXIM1/2: nothing said on this one
  • 7SK inhibits transcription by polII by sequestering P-TEFb (transcription elongation factors) in some nuclear structures. Don't really know what is the cellular context of this inhibition, perhaps DNA replication?
  • LARP7 binds to 7SK at the poly-u trailer and promotes the recruitment of MePCE.
  • MePCE methyl 5' capping prevents degradation of 7sk rna, but it's binding also contributes to its stabilisation.
  • MePCE also adds the methyl cap to u6 RNA (a component of the spliceosome)
  • Not much is known about MePCR outside drosophila and human.
  • It's overexpressed in certain cancers.

• Summary of findings:

  • Bmc1 binds to u6 as expected
  • Bmc1 is a component of telomerase
  • No cap addition to TER1
  • Bmc1 + Pof8 (LARP7) association promote telomerase assembly and TER1 accumulation.

Results

Results

• [x] IP + seq
  • [x] Bmc1 physically interacts with U6 RNA (spliced form)
• [x] Physical interactions: (RNA)
  • [x] Bmc1-Ter1
  • [x] Bmc1 interacts with the full-length, 3’ end- matured TER1 associated with the active telomerase holoenzyme
  • [x] Bmc1 interacts with the primary cohort of TMG-capped TER1 transcripts
  • [x] Lost in pof8D
  • [x] Bmc1-U6
  • [x] RNA gene
  • [x] Lost in pof8D
  • [x] pof8-u6
  • [x] pof8-ter1
  • [x] Lost in bmc1D
• [x] Physical interaction: Mass spec
  • [x] Bmc1-Pof8
  • [x] Bmc1-Lsm2-8 complex
  • [x] Bmc1-Trt1
  • [x] BMc1-Pop100
  • [x] NOT with the Tgs1 enzyme, meaning that it interacts with the mature form of the TER1 RNA.
  • [x] Interactions lost in presence of benzonase except for Pof8
• [x] CO-IP
  • [x] Bmc1-Pof8
  • [x] -> not lost with benzonase or ter1D
  • [x] Bmc1-Trt1
  • [x] -> lost with benzonase or ter1D
• [x] Bmc1 is required for telomerase activity in vitro, lost in a Pof8 background.
• [x] pof8D Bmc1OE, still reduced telomere elongation activity (no rescue)
• [x] Correct the fact that bmc1 can be deleted.

Fig. 5

• [x] bmc1D -> reduced TER1 levels (qRT pCR)
• [x] pof8D -> reduced TER1 levels (qRT pCR)
• [x] rrp6D -> increased TER1 levels (qRT pCR)
• [x] bmc1D rrp6D -> increased TER1 levels
• [x] pof8D rrp6D -> normal TER1 levels
• [x] bmc1D -> shorter telomeres
• [x] As expected, a strain lacking TER1 showed a complete loss of telomeric DNA, whereas deletion of pof8 and bmc1 only resulted in shorter telomeres, suggesting that while the two proteins are important for telomere maintenance, they are not strictly required like TER1

  FYPO

GO

• [x] Bmc1 is required for telomerase activity in vitro
• [x] telomerase RNA binding](https://www.pombase.org/term/GO:0070034)
• [x] Bmc1 is part of the telomerase holoenzyme
• [x] Bmc1 promotes TER1 accumulation by preventing 3' decay by the exosome

Existing annotations.

• [x] RNA modifications:
  • [x] addition of a 5’ trimethylguanosine (TMG) cap by the methyl- transferase Tgs1

Gene structure

• [x] It seems that the ter1 rna has an unusual way of being processed, which uses spliceosome machinery. If I understand correctly, the processing involves cutting the transcript at the 3' site without ligation, and some further processing that produces an RNA molecule that keeps the sequence from 1-1213. Should this be annotated to the sequence in some way?

Ask the authors:

• [x] If such annotations would be required for this RNA, are there other genes that would require similar annotations?
• [x] Why did they attempt the deletion?

Discuss with Val

• [x] Do we put links to transcriptomics datasets like the one in Fig. 1A?
• [x] Interaction data, can it be directly submitted to BioGRID? Raw data.
  • [x] Loss of interactions after treatment with benzonase, which digests nucleic acids (test that they are not mediated by a nucleic acid intermediate)
• [x] Should Bmc1 be anotated to telomerase activity? I guess not, since it does not have the enzymatic activity in itself, but it's rather part of the holoenzyme. However, in GO there is Telomeric repeat-binding factor contributes_to telomerase activity
• [x] Is the annotation to G0 ok?

Stuff

• snRNPs (pronounced "snurps"), or small nuclear ribonucleoproteins, are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs.