Marsupial phyloSNPs

Introduction to Marsupial phyloSNPs

In this project, new genomic data from the Tasmanian devil (Sarcophilus harrisii), Tasmanian tiger (Thylacinus cynocephalus), and echidna (Tachyglossus aculeatus) are analyzed for significant changes at the protein coding level. The goal is to find single amino acid changes in one of these species at a highly invariant residue in a well-conserved exon in a gene with known or predictable tertiary structure. Such changes are thought to enrich for genetic changes with significant, adaptive biochemical or phenotypic consequences (1,2,3,4), in contrast to ordinary SNPs at positions of low conservation. Thus phyloSNPs are informative to the distinctive biology of the species carrying them and suggest a focus for subsequent experiment.

Marsupial genomic and cDNA data to date has been quite limited compared to placental mammal. Yet as outgroup, metatheran animals provide important context to placentals and represent important context in understanding human protein evolution. The monotheres are inevitably limited by the paucity of extant species (basically platypus and echidna) and dim prospects for fossil DNA. Consequently echidna provides an important adjunct to the existing but incomplete platypus assembly. While extant birds and reptiles -- the preceding divergence node -- are abundant it must be remembered that a very considerable time elapsed (from 310 mry to 175 mry) prior to divergence of mammals with living representatives. This gap of 135 myr is comparable to the whole evolutionary record of theran mammals.

Assumed vertebrate phylogenetic tree

Marsupial relationships taken from 2009 paper establishing the mitochondrial genome sequence of the Tasmanian tiger (Thylacinus cynocephalus):

Newick tree that generates vertebrate phylogenetic tree used in the analysis here:

((((((((((((((((((homSap,panTro),gorGor),ponPyg),macMul),calJac),tarSyr),(micMur,otoGar)),tupBel),
(((((musMus,ratNor),dipOrd),cavPor),speTri),(oryCun,ochPri))),
(((((vicPac,susScr),turTru),bosTau),((equCab,(felCat,canFam)),(myoLuc,pteVam))),(eriEur,sorAra))),
(((loxAfr,proCap),echTel),(dasNov,choHof))),
(monDom,((macEug,triVul),(sarHar,thyCyn)))),
(ornAna,tacAcu)),
((galGal,taeGut),anoCar)),
xenTro),
(((tetNig,takRub),(gasAcu,oryLap)),danRer)),
calMil),
petMar);

Phylo-sorting data

-	-	-	-	-	-	-	((((((((((((((((((	-	-	-	-
10	26	10	>	27	gene	homSap	,	Homo	sapiens	(human)	hg181
11	38	11	>	40	gene	panTro	),	Pan	troglodytes	(chimp)	panTro
12	25	12	>	26	gene	gorGor	),	Gorilla	gorilla	(gorilla)	gorGor
13	40	13	>	42	gene	ponPyg	),	Pongo	pygmaeus	(orang)	ponAbe
14	28	14	>	30	gene	macMul	),	Macaca	mulatta	(rhesus)	rheMac
15	12	15	>	12	gene	calJac	),	Callithrix	jacchus	(marmoset)	calJac
16	48	16	>	53	gene	tarSyr	),(	Tarsius	syrichta	(tarsier)	tarSyr
17	29	17	>	31	gene	micMur	,	Microcebus	murinus	(mouse_lemur)	micMur
18	37	18	>	39	gene	otoGar	)),	Otolemur	garnettii	(bushbaby)	otoGar
19	50	19	>	57	gene	tupBel	),(((((	Tupaia	belangeri	(tree_shrew)	tupBel
20	31	20	>	33	gene	musMus	,	Mus	musculus	(mouse)	mm91
21	43	21	>	45	gene	ratNor	),	Rattus	norvegicus	(rat)	rn41
22	18	22	>	19	gene	dipOrd	),	Dipodomys	ordii	(kangaroo_rat)	dipOrd
23	14	23	>	15	gene	cavPor	),	Cavia	porcellus	(guinea_pig)	cavPor
24	45	24	>	48	gene	speTri	),(	Spermophilus	tridecemlineatus	(squirrel)	speTri
25	35	25	>	37	gene	oryCun	,	Oryctolagus	cuniculus	(rabbit)	oryCun
26	33	26	>	35	gene	ochPri	))),(((((	Ochotona	princeps	(pika)	ochPri
27	52	27	>	59	gene	vicPac	,	Vicugna	pacos	(lama)	vicPac
54	57	28	>	49	gene	susScr	),	Sus	scrofa	(pig)	
28	51	29	>	58	gene	turTru	),	Tursiops	truncatus	(dolphin)	turTru
29	11	30	>	11	gene	bosTau	),((	Bos	taurus	(cow)	bosTau
30	20	31	>	21	gene	equCab	,(	Equus	caballus	(horse)	equCab
31	22	32	>	23	gene	felCat	,	Felis	catus	(cat)	felCat
32	13	33	>	14	gene	canFam	)),(	Canis	familiaris	(dog)	canFam
33	32	34	>	34	gene	myoLuc	,	Myotis	lucifugus	(microbat)	myoLuc
34	42	35	>	44	gene	pteVam	))),(	Pteropus	vampyrus	(macrobat)	pteVam
35	21	36	>	22	gene	eriEur	,	Erinaceus	europaeus	(hedgehog)	eriEur
36	44	37	>	47	gene	sorAra	))),(((	Sorex	araneus	(shrew)	sorAra
37	27	38	>	28	gene	loxAfr	,	Loxodonta	africana	(elephant)	loxAfr
38	41	39	>	43	gene	proCap	),	Procavia	capensis	(hyrax)	proCap
39	19	40	>	20	gene	echTel	),(	Echinops	telfairi	(tenrec)	echTel
40	17	41	>	18	gene	dasNov	,	Dasypus	novemcinctus	(armadillo)	dasNov
41	15	42	>	16	gene	choHof	))),(	Choloepus	hoffmanni	(sloth)	choHof
42	30	43	>	32	gene	monDom	,((	Monodelphis	domestica	(opossum)	monDom
55	55	44	>	29	gene	macEug	,	Macropus	eugenii	(wallaby)	
56	56	45	>	46	gene	sarHar	),(	Sarcophilus	harrisii	(tasmanian_devil)	
57	60	46	>	56	gene	triVul	,	Trichosurus	vulpecula	(bushytail_possum)	
58	59	47	>	55	gene	thyCyn	)))),(	Thylacinus	cynocephalus	(tasmanian_tiger)	
43	34	48	>	36	gene	ornAna	,	Ornithorhynchus	anatinus	(platypus)	ornAna
59	58	49	>	50	gene	tacAcu	)),((	Tachyglossus	aculeatus	(echidna)	
44	23	50	>	24	gene	galGal	,	Gallus	gallus	(chicken)	galGal
45	46	51	>	51	gene	taeGut	),	Taeniopygia	guttata	(finch)	taeGut
46	10	52	>	10	gene	anoCar	)),	Anolis	carolinensis	(lizard)	anoCar
47	53	53	>	60	gene	xenTro	),(((	Xenopus	tropicalis	(frog)	xenTro
48	49	54	>	54	gene	tetNig	,	Tetraodon	nigroviridis	(pufferfish)	tetNig
49	47	55	>	52	gene	takRub	),(	Takifugu	rubripes	(fugu)	fr21
50	24	56	>	25	gene	gasAcu	,	Gasterosteus	aculeatus	(stickleback)	gasAcu
51	36	57	>	38	gene	oryLap	)),	Oryzias	latipes	(medaka)	oryLat
52	16	58	>	17	gene	danRer	)),	Danio	rerio	(zebrafish)	danRer
60	54	59	>	13	gene	calMil	),	Callorhinchus	milii	(elephantfish)	
53	39	60	>	41	gene	petMar	)	Petromyzon	marinus	(lamprey)	petMar
											
44	44	51	f	51	gene	fasta	tree_syntax	genus	species	common	ucsc
phy	alp	phy		alp

Candidate analysis

(methods explained here shortly)

Case of ERN2

chr6_5971 ERN2 4
contig00001  length=355   numreads=5
KLPFTIPELVHASPCRSSDGVLYT
.....................F..
               ^        
15      R=3(75) H=2(50

Read data format: the top row gives project gene name, HGNC gene name and exon number from ENSEMBL monDom5
and human orthology predictions, then Monodelphis amino-acid segment, then sequence differences in
tasmanian  devil (in this case, both individuals differ from Monodelphis by L->F), then differences between the two thylacines
(here one individual has R at position 15, the other has H), and finally the number of experimental reads that confirm the nucleotide
difference and the sum of the quality scores. The sequences were assembled by Newbler (the official 454 assembler) which uses 
lower-case letters for less confident calls.

Paralog and pseudogene issues: ERN2 has not generated potentially confusing recent pseudogenes (lack of human or opossum genome Blat matches to ERN2 query). GeneSorter shows a single remote full-length paralog ERN1. However this particular exon is a good match (3 differences out of 23), so there is potential for experimental difficulties in distinguishing them in short reads. However at positions 15 and 20, ERN1 is identical at the amino acid level to ERN2.

Homoplasy (recurrent mutation) issues: This exon is very conserved and does not exhibit repetitive sequence, compositional simplicity, or indels in any species in either paralog that could foster experimental error or alignment ambiguity. At position 15, the ancestral value is arginine in both paralogs. The G--> A transition to histidine in one individual is conservative under most circumstances (still basic) and arises from an arginine codon CpG hotspot conserved back to lamprey in 30 of 32 species with available data, yet histidine is not observed part of a reduced alphabet (ie R/H) at this position over many billions of years of branch length. Consequently R-->H is a significant change in this individual tasmanian devil.

Side issues: a very ancient conserved leucine at position 21 appears to be transitioning to phenylalanine at marsupial node but has not been fixed, so settles out as L or F depending on lineage-sorting on each terminal marsupial leaf whereas placentals are all changed to phenylalanine (a phyloSNP caught in mid-air). While L and F might seem about the 'same' as amino acids, the branch length conservation totals say both are important but for different reasons: this is not a waffle codon nor reduced alphabet situation. This raises the question -- given the extreme conservation of this exon otherwise -- of whether the L-->F change at position 21 in both individuals has 'enabled' (made neutral or adaptive) an otherwise unfavorable R-->H change at position 15 in one individual.

Structural significance: By good fortune, the crystal structure of ERN1 (alternately called IRE1) has been published. The PDB 2HZ6 structure has good coverage of this particular exon. Consequently the marsupial ERN2 could be very accurately modelled and the structural effects of L-->F with or without R-->H computed by submission to online SwissProt modelling service.

Alignment of Monodelphis ERN2 (key exon replaced by that of sarHar2) with crytallograph human ERN1 alpha luminal domain 
               Expect = 5.8e-65 Identities = 109/180 (60%), Positives = 141/180 (78%)

ERN2_monDom   1  PESLLFISTLDGSLHAVSKKTGDIQWTLKDDPIIQGPVYATEPAFLPDPSDGSLYILGEE  60
                 PE+LLF+STLDGSLHAVSK+TG I+WTLK+DP++Q P +  EPAFLPDP+DGSLY LG +
ERN1_homSap   8  PETLLFVSTLDGSLHAVSKRTGSIKWTLKEDPVLQVPTHVEEPAFLPDPNDGSLYTLGSK  67

ERN2_monDom  61  SKQGLMKLPFTIPELVHASPCHSSDGVFYTGRKQDTWFMVDPKSGKKQTMLSTETWDGLY  120
                 + +GL KLPFTIPELV ASPCRSSDG+LY G+KQD W+++D  +G+KQ  LS+   D L 
ERN1_homSap  68  NNEGLTKLPFTIPELVQASPCRSSDGILYMGKKQDIWYVIDLLTGEKQQTLSSAFADSLS  127

ERN2_monDom  121 PSAPLLYIGRTQYTVTMYDPRSQALRWNTTYRGYSAPLLDHLPGYQVGHFTCSGEGLVVT  180
                 PS  LLY+GRT+YT+TMYD +++ LRWN TY  Y+A L +    Y++ HF  +G+GLVVT
ERN1_homSap  128 PSTSLLYLGRTEYTITMYDTKTRELRWNATYFDYAASLPEDDVDYKMSHFVSNGDGLVVT  187

Functional significance: A considerable amount is known about the paralog ERN1. Annotation transfer may be applicable to ERN2.

"The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells adapt to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring kinase 1 (IRE1) and PKR-related ER kinase (PERK) are two type I transmembrane ER-localized protein kinase receptors that signal the UPR through a process that involves homodimerization and autophosphorylation. To elucidate the molecular basis of the ER transmembrane signaling event, we determined the x-ray crystal structure of the luminal domain of human IRE1alpha. The monomer of the luminal domain comprises a unique fold of a triangular assembly of beta-sheet clusters. Structural analysis identified an extensive dimerization interface stabilized by hydrogen bonds and hydrophobic interactions. Dimerization creates an MHC-like groove at the interface. However, because this groove is too narrow for peptide binding and the purified luminal domain forms high-affinity dimers in vitro, peptide binding to this groove is not required for dimerization. Consistent with our structural observations, mutations that disrupt the dimerization interface produced IRE1alpha molecules that failed to either dimerize or activate the UPR upon ER stress. In addition, mutations in a structurally homologous region within PERK also prevented dimerization. Our structural, biochemical, and functional studies in vivo altogether demonstrate that IRE1 and PERK have conserved a common molecular interface necessary and sufficient for dimerization and UPR signaling."

                            ^     *
ERN2_homSap  KLPFTIPELVHASPCRSSDGVFYT
ERN2_panTro  KLPFTIPELVHASPCRSSDGVFYT
ERN2_ponAbe  KLPFTIPELVHASPCRSSDGVFYT
ERN2_rheMac  KLPFTIPELVHASPCRSSDGVFYT
ERN2_calJac  KLPFTIPELVHASPCRSSDGVFYT
ERN2_tarSyr  KLPFTIPELVHASPCRSSDGVFYT
ERN2_micMur  KLPFTIPELVHASPCRSSDGVFYT
ERN2_tupBel  KLPFTIPELVHASPCRSSDGVFYT
ERN2_musMus  KLPFTIPELVHASPCRSSDGVFYT
ERN2_ratNor  KLPFTIPELVHASPCRSSDGVFYT
ERN2_cavPor  KLPFTIPELVHTSPCRSSDGVFYT
ERN2_speTri  KLPFTIPELVHASPCRSSDGVFYT
ERN2_oryCun  KLPFTIPELVHASPCRSSDGVFYT
ERN2_ochPri  KLPFSIPELVHASPCRSSDGVFYT
ERN2_turTru  RLPFTIPELVHASPCRSSDGVFYT
ERN2_bosTau  RLPFTIPELVHASPCRSSDGVFYT
ERN2_equCab  KLPFTIPELVHASPCRSSDGVFYT
ERN2_felCat  RLPFTIPELVHASPCRSSDGVFYT
ERN2_canFam  KLPFTIPELVHASPCRSSDGVFYT
ERN2_myoLuc  KLPFTIPELVHASPCRSSDGVFYT
ERN2_eriEur  KLPFTVPELVHTSPCRSSDGVFYT
ERN2_sorAra  KLPFTIPELVHASPCRSSDGVFYT
ERN2_loxAfr  KLPFTIPELVHAS-----------
ERN2_proCap  ---------------------FYT
ERN2_echTel  KLPFTIPELVLASPCRSSDGVFYT
ERN2_dasNov  KLPFTIPELVHTSPCRSSDGIFYT
ERN2_monDom  KLPFTIPELVHASPCRSSDGVLYT
ERN2_macEug  KLPFTIPELVQASPCRSSDGILYM
ERN2_sarHar1 KLPFTIPELVQASPCRSSDGIFYM
ERN2_sarHar2 KLPFTIPELVQASPCHSSDGIFYM
ERN2_ornAna  KLPFTIPELVQSSPCRSSDGILYT
ERN2_anoCar  KLPFTIPELVQSSPCRSSDGIIYT
ERN2_taeGut  KLPFTIPELVQSSPCRSSDGVLYT
ERN2_galGal  KLPFTIPELVQASPCRSSDGILYM
ERN2_xenTro  KLPFTIPELVQSSPCRSSDGILYT
ERN2_xenLae  KLPFTIPELVQSSPCRSSDGILYT
ERN2_tetNig  KLPFTIPELVQASPCRSSDGVLYM
ERN2_takRub  KLPFTIPELVQASPCRSSDGVLYM
ERN2_gasAcu  KLPFTIPDLVQSAPCRSSDGILYT
ERN2_oryLat  KLPFTIPELVQSAPCRSSDGILYT
ERN2_petMar  KLPFTIPELVHASPCRTSDGVLYT

ERN1_homSap KLPFTIPELVQASPCRSSDGILYM
ERN1_panTro KLPFTIPELVQASPCRSSDGILYM
ERN1_ponAbe KLPFTIPELVQASPCRSSDGILYM
ERN1_rheMac KLPFTIPELVQASPCRSSDGILYM
ERN1_calJac KLPFTIPELVQASPCRSSDGILYM
ERN1_tarSyr KLPFTIPELVQASPCRSSDGILYM
ERN1_micMur KLPFTIPELVQASPCRSTDGILYM
ERN1_otoGar KLPFTIPELVQASPCRSSDGILYM
ERN1_tupBel KLPFTIPELVQASPCRSSDGILYM
ERN1_musMus KLPFTIPELVQASPCRSSDGILYM
ERN1_ratNor KLPFTIPELVQASPCRSSDGILYM
ERN1_dipOrd KLPFTIPELVQASPCRSSDGILYM
ERN1_cavPor KLPFTIPELVQASPCRSSDGILYM
ERN1_speTri KLPFTIPELVQASPCRSSDGILYM
ERN1_oryCun KLPFTIPELVQASPCRSSDGILYM
ERN1_vicPac KLPFTIPELVQASPCRSSDGILYM
ERN1_turTru KLPFTIPELVQASPCRSSDGILYM
ERN1_bosTau KLPFTIPELVQASPCRSSDGILYM
ERN1_equCab KLPFTIPELVQASPCRSSDGILYM
ERN1_canFam KLPFTIPELVQASPCRSSDGILYM
ERN1_myoLuc KLPFTIPELVQASPCRSSDGILYM
ERN1_pteVam KLPFTIPELVQASPCRSSDGILYM
ERN1_eriEur KLPFTIPELVQASPCRSSDGILYM
ERN1_sorAra KLPFTIPELVQASPCRSSDGILYM
ERN1_loxAfr KLPFTIPELVQASPCRSSDGILYM
ERN1_proCap KLPFTIPELVQASPCRSSDGILYM
ERN1_echTel KLPFTIPELVQASPCRSSDGILYM
ERN1_dasNov KLPFTIPELVQASPCRSSDGILYM
ERN1_choHof KLPFTIPELVQASPCRSSDGILYM
ERN1_monDom KLPFTIPELVQASPCRSSDGILYM
ERN1_ornAna KLPFTIPELVHASPCRSSDGILYM
ERN1_galGal KLPFTIPELVQASPCRSSDGILYM
ERN1_taeGut KLPFTIPELVQASPCRSSDGILYM
ERN1_anoCar KLPFTIPELVQASPCRSSDGILYM
ERN1_xenTro KLPFTIPELVQSSPCRSSDGILYT
ERN1_tetNig KLPFTIPELVQASPCRSSDGVLYM
ERN1_takRub KLPFTIPELVQASPCRSSDGVLYM
ERN1_gasAcu KLPFTIPELVQASPCRSSDGVLYM
ERN1_oryLat KLPFTIPELVQASPCRSSDGVLYM
ERN1_danRer KLPFTIPELVQASPCRSSDGILYM

Ancient CpG in ERN2 homSap chr16:23625855-23625856
       Human  CG
       Chimp  CG
     Gorilla  --
   Orangutan  CG
      Rhesus  CG
    Marmoset  CG
     Tarsier  CG
 Mouse lemur  CG
    Bushbaby  --
   TreeShrew  CG
       Mouse  CG
         Rat  CG
Kangaroo rat  --
  Guinea Pig  CG
    Squirrel  CG
      Rabbit  CG
        Pika  CG
      Alpaca  --
     Dolphin  CG
         Cow  CG
       Horse  CG
         Cat  CG
         Dog  CG
    Microbat  CG
     Megabat  --
    Hedgehog  CG
       Shrew  CG
    Elephant  --
  Rock hyrax  --
      Tenrec  CG
   Armadillo  CG
     Opossum  CG
    Platypus  CG
      Lizard  CG
   Tetraodon  CG
        Fugu  CG
 Stickleback  CT
      Medaka  CT
     Lamprey  CG

Case of XXXX

(more shortly)

Case of YYYY

(more shortly)

Case of ZZZZ

(more shortly)