Two stories Two stories 1) reconstruction the 1) reconstruction the evolution of a complex evolution of a complex 2) Adding qualitative 2) Adding qualitative labels to predicted labels to predicted interactions interactions Paulien Smits & Thijs Ettema Department of Paediatrics, NCMD
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Two stories 1) reconstruction the evolution of a complex 2) Adding qualitative labels to predicted interactions Paulien Smits & Thijs Ettema Department.
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Two storiesTwo stories1) reconstruction the evolution of 1) reconstruction the evolution of
a complexa complex2) Adding qualitative labels to 2) Adding qualitative labels to
predicted interactionspredicted interactions
Paulien Smits & Thijs Ettema
Department of Paediatrics, NCMD
Introduction – MRPs
• Human mitoribosome– 2 rRNAs, encoded by mtDNA
– 79 MRPs, encoded by nDNA
• Select candidate MRPs for genetic disease– Conservation
– Function
– Location
55S
28S
39S
12S
16S
31
48
Science at a Distance. http://www.brooklyn.cuny.edu/bc/ahp/BioInfo/TT/Tlatr.html, 2006
Objectives Detection of MRPs
• Orthology relations between MRPs from different species
• New human MRPs based on comparison with MRPs in other species
• Specific functions of MRPs based on comparison with MRPs in other species
• Extra domains in MRPs• Find MRP associated proteins
New orthology relations (profile-to-profile)
Human MRP Yeast MRP
MRPS25 Mrp49
MRPS33 Rsm27
MRPL9 Mrpl50
MRPL24 Mrpl40
MRPL40 Mrpl28
MRPL45 Mba1
MRPL53 Mrpl44
Human MRP Bacterial MRP
MRPS24 S3
MRPL47 L29
New mammalian MRPs: Rsm22
• Small subunit protein in yeast mitoribosome
• Orthologs in eukaryotes and prokaryotes
• Homologous to rRNA methylase
• S. pombe: fusion protein Rsm22+Cox11
Yeast: Cox11 attached to mitoribosomeRsm22 is novel mammal MRP with a rRNA
methylase function
New mammalian MRPs: Mrp10
• Small subunit protein in yeast mitoribosome
• Yeast mutant has mitochondrial translation defect
• Orthologs in eukaryotes
• Distant homology with Cox19Mrp10 orthologs in Mammals are novel
candidate MRPs
Proteome data available
Smits et al, NAR 2007
Origins of supernumerary subunits
• MRPL43, MRPS25 & complex I subunit
• MRPL43, MRPS25 & complex I subunit
• MRPL39 & threonyl-tRNA synthetase
Origins of supernumerary subunits
• MRPL43, MRPS25 & complex I subunit
• MRPL39 & threonyl-tRNA synthetase
• MRPL44, dsRNA-binding proteins
Origins of supernumerary subunits
Origins of supernumerary subunits
• MRPL43, MRPS25 & complex I subunit
• MRPL39 & threonyl-tRNA synthetase
• MRPL44, dsRNA-binding proteins
• Mrp1, Rsm26 & superoxide dismutase
Triplication of the S18 protein in the metazoa
Where do the supernumerary subunits come from?
One new, metazoa specific protein of the Large subunit (L48) has been obtained by duplication of a protein from the small subunit (S10)
Where do the supernumerary subunits come from?
Addition of « new » paralogous subunits in the large and the small subunit in the metazoa
Where do the supernumerary subunits come from?
Addition of a new subunit (L45 / MBA1) that is homologous to TIM44 (protein import) and bacterial proteins of unknown function
Homology between Mba1/MRPL45 and TIM44
Dolezal P, Likic V, Tachezy J, Lithgow T. Evolution of the molecular machines for protein import into mitochondria. Science 2006;313:314-8
MRPL45, Mba1 & Tim44
• Mba1 is physically associated with LSU• Transcription of Mba1 and MRPs is co-regulated• Function of MRPL45 unknown• COG4395 (MRPL45&Tim44) has similar
phylogenetic distribution as COG3175 (Cox11) Alpha-proteobacterial Tim44 is ancestor of
MRPL45 and yeast ortholog Mba1, losing the N-terminus and acquiring a function in translation and COX assembly as a constituent of the mitoribosome
• Comprehensive complex purification data (Krogan, Gavin)
• Shared Synthetic lethality
• Co-regulation (ChIP-on-chip)
• Co-expression
• Conserved co-expression (orthologous, paralogous, four species)
• Gene Neighborhood conservation (STRING pink)
• Gene CoOccurrence (STRING pink)
Complex purifications
• Fuse query protein with a hook• Pull down hook from in vivo extracts• Identify proteins that co-purify• Socio-Affinity score
Synthetic lethality
• One knock-out not lethal, second knock-out not lethal, knock-out both lethal
• Points to complementary pathways
• Shared synthetic lethality points to same pathway
Objective: distinguish physical from metabolic in omics data
• We integrate omics data sets for the budding yeast S.cerevisiae because of many high quality data sets as well as classical knowledge about protein functions
• We construct two separate reference sets: one for physical interactions and one for metabolic interactions.