pike na ugm multiple expressed isoforms of tin2 stimulate …€¦ · pike na ugm multiple...
TRANSCRIPT
Multiple expressed isoforms of TIN2 stimulate human telomerase
Alexandra Mims PikePacBio UGM
September 26, 2019
Telomere length regulation is crucial in cells that divide many times
TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3’AATCCCAATCCCAATCCCAATCCCAATCCCAATCC-5’
Telo
mer
e le
ngth
Age (years)
Baerlocher & Lansdorp. Methods Cell Biol. (2004) Stanley & Armanios Cur. Opin. Genetics & Dev. (2015)
Telomere length is heterogeneous in the population
Short Telomere Syndromes • Dyskeratosis congenita• Pulmonary Fibrosis• Aplastic Anemia
Cancer • Rare telomere lengthening mutations• Upregulation of telomerase
Shelterin component TIN2 is mutated in short telomere syndromes
TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3’AATCCCAATCCCAATCCCAATCCCAATCCCAATCC-5’
TIN2TIN2
TRF1 TRF2
TPP1
POT1
TRF1 Mutations C-terminalExtension
Shelterin-interacting Region C-terminal Region
TPP1 TRF2
1 451354TIN2S TIN2L
conservation
Human TINF2 is alternatively spliced into TIN2S and TIN2L isoforms
3’5’ 1 2 3 4 5 6 7 8 9
~3kbTINF2
TIN2S3’5’ 1 2 3 4 5 6
TIN2L3’5’ 1 2 3 4 5 6 7 8 9
3’5’ 1 2 3 4 5 6 7 8 9
~3kbTINF2
With Jon Alder
293T CRISPR clones
+
51
39
1 2 3 4 5 6 7
anti myc
TIN2S
TINF2
TIN2L
myc
tubulin
51
39
myc
293T CRISPR clones
+
51
39
1 2 3 4 5 6 7
anti myc
TIN2S
TINF2
TIN2L
myc
tubulin
51
39
CRISPR knock-in of a myc epitope tag reveals an undescribed TIN2 isoform
PacBio sequencing identifies a new TIN2 isoform, “TIN2M”
Johns Hopkins Deep Sequencing & Microarray Core FacilityHaiping Hao, Jasmeet Sethi
Human • 293T• HeLa• RPE1• K562• LCL
Mouse • C57BL/6• CAST/EiJ
Filter, trim, align
Aligned Reads:
AlignedNot Covered (intronic)
3’5’ 1 2 3 4 5 6 7 8 9
~3kbTINF2PacBio sequencing identifies a new TIN2 isoform, “TIN2M”
Aligned Reads:
AlignedNot Covered (intronic)
3’5’ 1 2 3 4 5 6 7 8 9
~3kbTINF2
TIN2M3’5’ 1 2 3 4 5 6 7 8
TIN2S3’5’ 1 2 3 4 5 6
TIN2L3’5’ 1 2 3 4 5 6 7 8 9
PacBio sequencing identifies a new TIN2 isoform, “TIN2M”
Each TIN2 isoform localizes to telomeres
TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3’AATCCCAATCCCAATCCCAATCCCAATCCCAATCC-5’
TIN2 TPP1
POT1
Telomerase
TIN2 stimulates telomerase processivity
TIN2 isoforms may have functional differences in vivo
• The C-terminal extension may hold keys to TIN2 function in vivo
- Novel binding partner?
- Post-translational modifications1
TRF1 Mutations C-terminalExtension
Shelterin-interacting Region C-terminal Region
TPP1 TRF2
1 451354TIN2S
420TIN2M TIN2L
conservation
• CRISPR + PacBio Isoseq identified a new isoform that was missed in previous studies
• TIN2 isoforms:
- localize to telomeres
- stimulate telomerase processivity
- Differ only in the variable C-terminal extension1 Nelson et al. MCB 2018
Acknowledgements
Greider Lab, Johns Hopkins Carol Greider Margaret Strong Carla Connelly
Jonathan Alder, U Pitt John Paul Ouyang Sarah Wheelan
JHMI Deep Sequencing and Microarray Core Facility Haiping Hao Jasmeet Sethi
Bell Lab, MIT/HHMI Stephen Bell