How does genetic variation affect transcript and protein abundance? DNA RNA Protein DNA RNA Protein Transcrip)on Transla)on DNA RNA Protein Francis Crick 1956
Nature July 2013 “We estimate that approximately one-half of pQTLs are probably also eQTLs. However, many pQTLs do not correspond to eQTLs, even at a relaxed stringency.” Accumulating evidence of a disconnect between transcript and protein expression. September 2014 February 2015 “QTLs affecting mRNA levels are, on average, attenuated or buffered at the protein level…
50 40 30 20 10 Body weight (gm) 7/11/2014 7/31/2014 8/20/2014 date 50 40 30 20 10 Body weight (gm) 7/11/2014 7/31/2014 8/20/2014 date female DO mice male DO mice DO mice are genetically and phenotypically diverse Alan Attie & Mark Keller Female DO mice Male DO mice
192 DO Livers Transcripts Short Reads RNA-Seq eQTL pQTL eQTL Mapping pQTL Mapping Proteins Peptides MS/MS Compare ? Munger et al. 2014 Chick*, Munger* et al. Nature, 2016 How does genetic variation influence transcript and protein abundance?
The founder origin of each allele provides direct es)mates of allele specific expression. Only alleles derived from 129S1 express Gm12976 in the DO popula)on.
192 DO Livers Transcripts Short Reads RNA-Seq eQTL pQTL eQTL Mapping pQTL Mapping Proteins Peptides MS/MS Compare ? Munger et al. 2014 Chick*, Munger* et al. Nature 2016 How does genetic variation affect protein abundance?
Searching for protein and transcript mediators of distant pQTL –> Mediation Analysis RNA Protein QTL trans cis RNA Protein Target Causal Intermediates RNA Protein trans QTL cis Target Target Protein ~ pQTLdistant Target Protein ~ pQTLdistant + MediatorProtein x 8000 proteins Target Protein ~ pQTLdistant + MediatorRNA x 21000 Transcripts X
43,102 SNPs in region 3 Candidate SNPs 1 Short Deletion Nnt eQTL B6 alleles do not express Nnt Low abundance of NNT in C57BL/6J drives low abundance of TMEM68.
TMEM68-NNT Next Step: Valida)on Transcript Abundance Protein Abundance Tmem68 TMEM68 trans 13 cis 3 cis cis Nnt NNT Alex Stanton – Tums predoc Tmem68 TMEM68 3 cis Ques)on 1: Where does the disconnect between Tmem68 transcript and protein occur? At the level of transla)on, or by post-transla)onal mechanisms? ?
Stoichiometric buffering of CCT complex: Next steps • Observa)on: DO animals with NOD allele at Cct6a have lower protein abundance of all CCT members. • NOD muta)on affects both Cct6a transcript and protein levels. • NOD has a transversion subs)tu)on in conserved KLF4 binding domain in Cct6a promoter. • Luciferase assays to quan)fy effect of this subs)tu)on on promoter strength. • CRISPR to “fix” muta)on in NOD and introduce same muta)on into B6. • Test stoichiometric buffering hypothesis by introducing muta)on into other CCT member -> Can we transfer the regulatory role in the complex by knocking down the expression of another protein?
In Progress: Using genetic diversity to identify kinases for specific phosphorylation sites. Liver Phospho-Proteome Kinase <–> phospho site iden)fica)on by media)on
“Pulling the weeds” to iden)fy subtle gene)c interac)ons Step 2: Mediate all subthreshold peaks LOD > 5. Aka “Pull the weeds”. Step 3: Repeat process for all 8k proteins. Step 4: Find proteins that share same subthreshold peak and mediator
Conclusions • Most gene)c varia)on that affects transcript abundance does not affect protein abundance. – For local gene)c varia)on that does affect protein abundance, 80% act proximally on transcrip)on (standard model). • 99+% of distant pQTL act on the target protein’s abundance independent of the target’s transcript abundance. • Media)on analysis iden)fies 700 RNA/protein causal intermediates of distant pQTL and infers >5000 protein interac)ons. • Stoichiometric buffering is a common post-transla)onal mechanism governing protein abundance of binding partners and complex members. CCT2 CCT3 CCT5 CCT6A CCT4 CCT7 CCT8 TCP1
Searching for protein and transcript mediators of distant pQTL –> Mediation Analysis RNA Protein QTL trans cis RNA Protein Target Causal Intermediates RNA Protein trans QTL cis Target Target Protein ~ pQTLdistant Target Protein ~ pQTLdistant + MediatorProtein x 8000 proteins Target Protein ~ pQTLdistant + MediatorRNA x 21000 Transcripts X
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