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PTRM Class 2008 Dan Wang 9-30-2008

PTRM Class 2008 Dan Wang 9-30-2008. Inefficient mRNA/mRNP reactions are challenged by nuclear surveillance at various points in mRNP biogenesis. Saguez, Olesen & Jensen, Curr Opin in Cell Biol, 2005. Co-transcriptional model for the recruitment of mRNA export factors in yeast.

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PTRM Class 2008 Dan Wang 9-30-2008

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  1. PTRM Class 2008 Dan Wang 9-30-2008

  2. Inefficient mRNA/mRNP reactions are challenged by nuclear surveillance at various points in mRNP biogenesis Saguez, Olesen & Jensen, Curr Opin in Cell Biol, 2005

  3. Co-transcriptional model for the recruitment of mRNA export factors in yeast Stutz&Izaurralde, Trends in Cell Biol, 2003

  4. THO/sub2 mutants are good models to study mRNA nuclear surveillance • Nuclear retention of mRNA • Decrease in steady-state levels of several mRNAs • Both can be restored upon RRP6 deletion

  5. Trf4p participates in HSP104 RNA nuclear surveillance TRAMP (Trf4p, Air2p, Mtr4p) THO (Hpr1p, Mft1p, Tho2p, Thp1p) trf4-236: polyadenylation- defective mutant HSP104 mRNA is still degraded without the polyadenylation activity of Trf4p

  6. Trf4p participates in HSP104 RNA nuclear surveillance TRAMP (Trf4p, Air2p, Mtr4p) THO (Hpr1p, Mft1p, Tho2p, Thp1p) TREX (THO, Sub2p, Yra1p) sub2-201: temperature-sensitive allele

  7. Nuclear retention of mRNA in THO/sub2 mutants in the absence of TRF4 TREX (THO, Sub2p, Yra1p) THO (Hpr1p, Mft1p, Tho2p, Thp1p) TRAMP (Trf4p, Air2p, Mtr4p) Rrp6p: exosome component exonuclease DAPI stains DNA

  8. Nuclear retention of mRNA in THO/sub2 mutants in the absence of TRF4 THO (Hpr1p, Mft1p, Tho2p, Thp1p) TRAMP (Trf4p, Air2p, Mtr4p)

  9. Nuclear retention of mRNA in THO/sub2 mutants in the absence of TRF4

  10. The entire TRAMP complex operates in mRNA surveillance TET-Off THO (Hpr1p, Mft1p, Tho2p, Thp1p) TRAMP (Trf4p, Air2p, Mtr4p) Rrp47p: exosome component interacts with Rrp6p

  11. The entire TRAMP complex operates in mRNA surveillance THO (Hpr1p, Mft1p, Tho2p, Thp1p) TRAMP (Trf4p, Air2p, Mtr4p) Rrp47p: exosome component interacts with Rrp6p

  12. Deletion of TRF4 does not restore mRNA retention in ∆hpr1/rrp6∆ cells Hypothesis: Large amounts of Trf4p-dependent polyadenylated rRNAs and sno/snRNAs accumulate in rrp6∆ cells and interfere with the retention process. Rrp6p: exosome component exonuclease Nop1p and Nsr1p: Nucleolar antigens THO (Hpr1p, Mft1p, Tho2p, Thp1p) TRAMP (Trf4p, Air2p, Mtr4p)

  13. Persistence of nuclear HSP104 RNAs in the sub2-201 mutant in the absence of transcription TREX (THO, Sub2p, Yra1p)

  14. Model of transcription site-associated mRNA surveillance in THO/sub2 mutants WT conditions: cotranscriptional mRNA packaging and 3’end formation occur efficiently and the mRNP is released for export. THO/sub2 mutant:a less efficient assembly/processing pathway leads to Rrp6p/exosome intervention and rapid RNA decay. A minor fraction of mRNA escape nuclear degradation and retained

  15. Premature transcription termination in THO/sub2 mutants is linked to p(A) site position WT: transcription terminated gradually over the 200-300nt following the p(A) site mft1∆: transcription terminated abruptly in close proximity to the p(A) site Ppr2p: transcription elongation factor component THO (Hpr1p, Mft1p, Tho2p, Thp1p)

  16. Premature transcription termination in THO/sub2 mutants is linked to p(A) site position TREX (THO, Sub2p, Yra1p) THO (Hpr1p, Mft1p, Tho2p, Thp1p) THO/sub2 mutants are distinct from the other TREX mutants

  17. Premature transcription termination in THO/sub2 mutants is linked to p(A) site position THO (Hpr1p, Mft1p, Tho2p, Thp1p) Rrp6: Exosome component exonuclease

  18. Premature transcription termination in THO/sub2 mutants is linked to p(A) site position A subset of RNAPII complexes were able to transcribe beyond the distance of the previous p(A) site position to prematurely terminate at the newly generated p(A) site around NRO probes L3 and 6.

  19. Genetic link between the THO complex and pre-mRNA 3’ end formation factors

  20. RNA 3’ end processing is impaired in THO/sub2-dificient extracts Pcf11p: Cleavage factor IA (CFIA) component TREX (THO, Sub2p, Yra1p, Gbp2p, Hrb1p) THO (Hpr1p, Mft1p, Tho2p, Thp2p)

  21. RNAs processed in tho2∆ mutant extract are polyadenylation impaired and unstable THO (Hpr1p, Mft1p, Tho2p, Thp2p) cyc1-512: lacks polyadenylation signal

  22. RNAs processed in tho2∆ mutant extract are polyadenylation impaired and unstable Cleavage only: CTP replaced ATP

  23. The processing defect in THO mutant extract is due to a modified CPF complex TREX (THO, Sub2p, Yra1p, Gbp2p, Hrb1p) THO (Hpr1p, Mft1p, Tho2p, Thp2p) Pap1 and Fip1p: Cleavage and polyadenylation factor components

  24. The polyadenylatoin cofactor Fip1p is downregulated in THO/sub2-deficient extracts in a ubiquitin/proteasome-dependent manner TREX (THO, Sub2p, Yra1p, Gbp2p, Hrb1p) THO (Hpr1p, Mft1p, Tho2p, Thp2p) Pti1p (CPF factor component) is posttranslationally modified in a THOmutant-dependent manner

  25. The polyadenylatoin cofactor Fip1p is downregulated in THO/sub2-deficient extracts in a ubiquitin/proteasome-dependent manner TREX (THO, Sub2p, Yra1p, Gbp2p, Hrb1p) THO (Hpr1p, Mft1p, Tho2p, Thp2p)

  26. The polyadenylatoin cofactor Fip1p is downregulated in THO/sub2-deficient extracts in a ubiquitin/proteasome-dependent manner MG132: proteasome inhibitor erg6∆: allows for efficient uptake of MG132

  27. Polyadenylation deficiency, low Fip1p levels, and RNA instability depend on mRNA surveillance factors

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