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ʻO ka ulu ʻana o nā parasites microbial e pili ana i kahi kūʻē ma waena o ke koho kūlohelohe, kahi e hoʻomaikaʻi ai nā parasites, a me ka genetic drift, kahi e hoʻolilo ai i nā parasites i nā genes a hōʻiliʻili i nā mutations deleterious. Maanei, i mea e hoʻomaopopo ai pehea e hana ai kēia kūʻē ma ka pālākiō o hoʻokahi macromolecule, wehewehe mākou i ke ʻano cryo-EM o ka ribosome o Encephalitozoon cuniculi, kahi meaola eukaryotic me kekahi o nā genomes liʻiliʻi loa ma ke ʻano. ʻO ka hōʻemi nui ʻana o ka rRNA i loko o nā ribosomes E. cuniculi e hele pū ʻia me nā loli kūkulu i ʻike ʻole ʻia, e like me ka ulu ʻana o nā mea hoʻopili rRNA fused i ʻike ʻole ʻia ma mua a me rRNA me ka ʻole o nā bulges. Eia kekahi, ua ola ka ribosome E. cuniculi i ka nalowale ʻana o nā ʻāpana rRNA a me nā protein ma o ka hoʻomohala ʻana i ka hiki ke hoʻohana i nā mole liʻiliʻi ma ke ʻano he mimics kūkulu o nā ʻāpana rRNA i hoʻohaʻahaʻa ʻia a me nā protein. Ma keʻano holoʻokoʻa, hōʻike mākou he nui nā ʻano molekala i manaʻo ʻia ua hoʻemi ʻia, degenerate, a i loaʻa i nā mutations debilitating he nui nā ʻano hana compensatory e hoʻomau iā lākou i ka hana ʻoiai ʻo nā ʻūlū molekala koʻikoʻi.
No ka mea, ʻo ka hapa nui o nā hui o nā parasites microbial he mau mea hana molekala kūikawā e hoʻohana ai i kā lākou mau kikowaena, pono pinepine mākou e hoʻomohala i nā therapeutics like ʻole no nā hui like ʻole o nā parasites1,2. Eia nō naʻe, hōʻike nā hōʻike hou e hui pū ana kekahi mau ʻano o ka ulu ʻana o nā parasite a ua wānana nui ʻia, e hōʻike ana i kahi kumu kūpono no nā hana therapeutic ākea i nā parasites microbial3,4,5,6,7,8,9.
Ua ʻike mua ʻia kahi ʻano evolutionary maʻamau i nā parasites microbial i kapa ʻia ʻo ka hoʻemi ʻana o ka genome a i ʻole ka genome decay10,11,12,13. Hōʻike ka noiʻi o kēia manawa i ka wā e haʻalele ai nā microorganism i ko lākou nohona ola manuahi a lilo i mau parasites intracellular (a i ʻole endosymbionts), e loli mālie akā kupaianaha ko lākou genomes i nā miliona miliona o nā makahiki9,11. Ma kahi hana i ʻike ʻia ʻo ka genome decay, hōʻiliʻili nā parasites microbial i nā mutations deleterious e hoʻololi i nā genes koʻikoʻi he nui i pseudogenes, e alakaʻi ana i ka nalowale mālie o ka gene a me ka mutational collapse14,15. Hiki i kēia hiolo ʻana ke luku a hiki i ka 95% o nā genes i loko o nā meaola intracellular kahiko loa i hoʻohālikelike ʻia me nā ʻano ola manuahi pili loa. No laila, ʻo ka evolution o nā parasites intracellular he huki kaua ma waena o nā mana kūʻē ʻelua: ke koho kūlohelohe Darwinian, e alakaʻi ana i ka hoʻomaikaʻi ʻana o nā parasites, a me ka hiolo ʻana o ka genome, e hoʻolei ana i nā parasites i ka poina. Pehea i hoʻokele ai ka parasite e puka mai kēia huki kaua a mālama i ka hana o kona ʻano molekala ʻaʻole maopopo.
ʻOiai ʻaʻole i maopopo loa ke ʻano o ka palaho ʻana o ka genome, me he mea lā e kū pinepine ana ia ma muli o ka hele pinepine ʻana o ka genetic. No ka mea, noho nā parasites i nā heluna liʻiliʻi, asexual, a me nā heluna i kaupalena ʻia e ka genetics, ʻaʻole hiki iā lākou ke hoʻopau pono i nā mutations damage e kū pinepine ana i ka wā o ka hana hou ʻana o ka DNA. Ke alakaʻi nei kēia i ka hōʻiliʻili hou ʻana o nā mutations ʻino a me ka emi ʻana o ka genome parasite. ʻO ka hopena, ʻaʻole wale ka parasite e nalowale i nā genes i pono ʻole no kona ola ʻana i loko o ke kaiapuni intracellular. ʻO ka hiki ʻole o nā heluna parasite ke hoʻopau pono i nā mutations damage sporadic e hōʻiliʻili ai kēia mau mutations ma o ka genome, me kā lākou mau genes koʻikoʻi loa.
ʻO ka hapa nui o kā mākou ʻike o kēia manawa no ka hōʻemi ʻana o ka genome e pili ana i ka hoʻohālikelike ʻana o nā moʻo genome, me ka nānā ʻole ʻana i nā loli i nā molekala maoli e hana ana i nā hana mālama hale a lawelawe ma ke ʻano he mau pahuhopu lāʻau lapaʻau. Ua hōʻike ʻia nā haʻawina hoʻohālikelike e like me ke kaumaha o nā mutations microbial intracellular badges e hoʻoholo i nā protein a me nā waikawa nucleic e kuhihewa a hōʻuluʻulu, e hoʻolilo ana iā lākou i mea hilinaʻi chaperone a hypersensitive i ka wela19,20,21,22,23. Eia kekahi, ʻo nā parasites like ʻole - ka evolution kūʻokoʻa i kekahi manawa i hoʻokaʻawale ʻia e like me 2.5 biliona mau makahiki - ua ʻike i kahi nalowale like o nā kikowaena kaohi maikaʻi i kā lākou synthesis protein5,6 a me nā ʻano hana hoʻoponopono DNA24. Eia naʻe, ʻaʻole ʻike nui ʻia e pili ana i ka hopena o ke ʻano ola intracellular ma nā waiwai ʻē aʻe o nā macromolecules cellular, me ka hoʻololi ʻana o ka molekala i kahi kaumaha e hoʻonui nei o nā mutations badges.
Ma kēia hana, i mea e hoʻomaopopo maikaʻi ai i ka ulu ʻana o nā protein a me nā waikawa nucleic o nā microorganism intracellular, ua hoʻoholo mākou i ke ʻano o nā ribosomes o ka parasite intracellular ʻo Encephalitozoon cuniculi. ʻO E. cuniculi kahi meaola e like me ka fungus e pili ana i kahi hui o nā microsporidia parasitic nona nā genomes eukaryotic liʻiliʻi loa a no laila ua hoʻohana ʻia ma ke ʻano he mau meaola hoʻohālike e aʻo ai i ka palaho genome25,26,27,28,29,30. I kēia manawa, ua hoʻoholo ʻia ke ʻano o ka ribosome cryo-EM no nā genomes i hoʻemi iki ʻia o Microsporidia, Paranosema locustae, a me Vairimorpha necatrix31,32 (~3.2 Mb genome). Hōʻike kēia mau ʻano ua uku ʻia kekahi nalowale o ka hoʻonui ʻana o rRNA e ka hoʻomohala ʻana o nā pilina hou ma waena o nā protein ribosomal kokoke a i ʻole ka loaʻa ʻana o nā protein ribosomal msL131,32 hou. ʻO nā ʻano Encephalitozoon (genome ~2.5 miliona bp), me ko lākou hoahānau pili loa ʻo Ordospora, e hōʻike ana i ke kiʻekiʻe hope loa o ka hōʻemi ʻana o ka genome i nā eukaryotes - he emi iho ko lākou ma mua o 2000 mau genes protein-coding, a ua manaʻo ʻia ʻaʻole wale ko lākou ribosomes i nele i nā ʻāpana hoʻonui rRNA (nā ʻāpana rRNA e hoʻokaʻawale i nā ribosomes eukaryotic mai nā ribosomes bacteria) he ʻehā mau protein ribosomal ma muli o ko lākou nele i nā homologues i loko o ka genome E. cuniculi26,27,28. No laila, ua hoʻoholo mākou e hiki i ka ribosome E. cuniculi ke hōʻike i nā hoʻolālā i ʻike ʻole ʻia ma mua no ka hoʻololi ʻana i ka molekala i ka palaho genome.
Hōʻike kā mākou ʻano cryo-EM i ka ribosome cytoplasmic eukaryotic liʻiliʻi loa e wehewehe ʻia a hāʻawi i ka ʻike i ke ʻano o ka hopena o ka hōʻemi ʻana o ka genome i ke ʻano, ka hōʻuluʻulu ʻana, a me ka ulu ʻana o ka mīkini molekala i hoʻohui ʻia i ke kelepona. Ua ʻike mākou ua hōʻino ka ribosome E. cuniculi i nā kumumanaʻo i mālama nui ʻia o ka pelu ʻana o RNA a me ka hōʻuluʻulu ʻana o ribosome, a ua ʻike i kahi protein ribosomal hou, i ʻike ʻole ʻia ma mua. Me ka manaʻo ʻole, hōʻike mākou ua hoʻomohala nā ribosome microsporidia i ka hiki ke hoʻopaʻa i nā mole liʻiliʻi, a kuhi ʻia e hoʻoulu nā truncations i loko o rRNA a me nā protein i nā hana hou evolutionary e hāʻawi hope loa i nā ʻano pono ma ka ribosome.
No ka hoʻomaikaʻi ʻana i ko mākou ʻike i ka ulu ʻana o nā protein a me nā waikawa nucleic i loko o nā meaola intracellular, ua hoʻoholo mākou e hoʻokaʻawale i nā spores E. cuniculi mai nā moʻomeheu o nā cell mammalian i loaʻa i ka maʻi i mea e hoʻomaʻemaʻe ai i kā lākou ribosomes a hoʻoholo i ke ʻano o kēia mau ribosomes. He mea paʻakikī ke loaʻa ka nui o nā microsporidia parasitic no ka mea ʻaʻole hiki ke hoʻoulu ʻia ka microsporidia i loko o kahi medium nutrient. Akā, ulu lākou a hana hou i loko o ke cell host wale nō. No laila, no ka loaʻa ʻana o ka biomass E. cuniculi no ka hoʻomaʻemaʻe ʻana i ka ribosome, ua hoʻopili mākou i ka laina cell kidney mammalian RK13 me nā spores E. cuniculi a hoʻoulu i kēia mau cell i loaʻa i ka maʻi no kekahi mau pule e ʻae iā E. cuniculi e ulu a hoʻonui. Ma ka hoʻohana ʻana i kahi monolayer cell i loaʻa i ka maʻi ma kahi o ka hapalua mika square, ua hiki iā mākou ke hoʻomaʻemaʻe ma kahi o 300 mg o nā spores Microsporidia a hoʻohana iā lākou e hoʻokaʻawale i nā ribosomes. A laila ua hoʻopau mākou i nā spores i hoʻomaʻemaʻe ʻia me nā momi aniani a hoʻokaʻawale i nā ribosomes maka me ka hoʻohana ʻana i ka polyethylene glycol fractionation stepwise o nā lysates. Ua ʻae kēia iā mākou e loaʻa ma kahi o 300 µg o nā ribosomes E. cuniculi maka no ka nānā ʻana i ke ʻano.
A laila ua hōʻiliʻili mākou i nā kiʻi cryo-EM me ka hoʻohana ʻana i nā laʻana ribosome i loaʻa a hana i kēia mau kiʻi me ka hoʻohana ʻana i nā masks e pili ana i ka subunit ribosomal nui, ke poʻo subunit liʻiliʻi, a me ka subunit liʻiliʻi. I loko o kēia kaʻina hana, ua hōʻiliʻili mākou i nā kiʻi o kahi 108,000 mau ʻāpana ribosomal a ua helu ʻia nā kiʻi cryo-EM me ka hoʻonā o 2.7 Å (Nā Kiʻi Hoʻohui 1-3). A laila ua hoʻohana mākou i nā kiʻi cryoEM e hoʻohālike i ka rRNA, ka protein ribosomal, a me ka hibernation factor Mdf1 e pili ana me E. cuniculi ribosomes (Kiʻi 1a, b).
a Ke ʻano o ka ribosome E. cuniculi i ka hui pū ʻana me ka hibernation factor Mdf1 (pdb id 7QEP). b Palapala ʻāina o ka hibernation factor Mdf1 e pili ana me ka ribosome E. cuniculi. c Palapala ʻāina lua e hoʻohālikelike ana i ka rRNA i hoʻōla ʻia i nā ʻano Microsporidian i nā ʻano ribosomal i ʻike ʻia. Hōʻike nā panela i kahi o nā ʻāpana rRNA i hoʻonui ʻia (ES) a me nā wahi hana ribosome, me ke kahua decoding (DC), ka sarcinicin loop (SRL), a me ke kikowaena peptidyl transferase (PTC). d ʻO ka nui o ka electron e pili ana i ke kikowaena peptidyl transferase o ka ribosome E. cuniculi e hōʻike ana he ʻano like kēia wahi catalytic i loko o ka parasite E. cuniculi a me kona mau kikowaena, me H. sapiens. e, f ʻO ka nui o ka electron e pili ana o ke kikowaena decoding (e) a me ke ʻano schematic o ke kikowaena decoding (f) e hōʻike ana he mau koena U1491 ko E. cuniculi ma kahi o A1491 (E. coli numbering) i loko o nā eukaryotes ʻē aʻe he nui. Hōʻike kēia hoʻololi he maʻalahi paha ʻo E. cuniculi i nā lāʻau antibiotic e kuhikuhi ana i kēia wahi hana.
I ka hoʻohālikelike ʻana i nā ʻano i hoʻokumu mua ʻia o nā ribosomes V. necatrix a me P. locustae (ʻo nā ʻano ʻelua e hōʻike ana i ka ʻohana microsporidia like ʻo Nosematidae a ua like loa kekahi me kekahi), 31,32 E. cuniculi ribosomes e hana i nā kaʻina hana he nui o ka rRNA a me ka protein fragmentation. Denaturation hou aʻe (Nā Kiʻi Hoʻohui 4-6). I loko o ka rRNA, ʻo nā loli koʻikoʻi loa, ʻo ia ka nalowale piha ʻana o ka ʻāpana 25S rRNA i hoʻonui ʻia ʻo ES12L a me ka degeneration hapa o nā helices h39, h41, a me H18 (Kiʻi 1c, Kiʻi Hoʻohui 4). Ma waena o nā protein ribosomal, ʻo nā loli koʻikoʻi loa, ʻo ia ka nalowale piha ʻana o ka protein eS30 a me ka pōkole ʻana o nā protein eL8, eL13, eL18, eL22, eL29, eL40, uS3, uS9, uS14, uS17, a me eS7 (Nā Kiʻi Hoʻohui 4, 5).
No laila, ʻo ka emi nui ʻana o nā genomes o nā ʻano Encephalotozoon/Ordospora e hōʻike ʻia ana i ko lākou ʻano ribosome: ʻO E. cuniculi ribosomes ka mea e ʻike ai i ka nalowale nui loa o ka ʻike protein i loko o nā ribosome cytoplasmic eukaryotic i kau ʻia ma ke ʻano o ke ʻano, a ʻaʻohe o lākou mau ʻāpana rRNA a me nā protein i mālama nui ʻia ʻaʻole wale i loko o nā eukaryotes, akā i loko o nā ʻāpana ʻekolu o ke ola. Hāʻawi ke ʻano o ka ribosome E. cuniculi i ke kumu hoʻohālike molekala mua no kēia mau loli a hōʻike i nā hanana evolutionary i nānā ʻole ʻia e nā genomics hoʻohālikelike a me nā haʻawina o ke ʻano biomolecular intracellular (Kiʻi Hoʻohui 7). Ma lalo nei, wehewehe mākou i kēlā me kēia o kēia mau hanana me ko lākou kumu evolutionary a me ko lākou hopena hiki ke loaʻa i ka hana ribosome.
A laila ua ʻike mākou, ma waho aʻe o nā truncations rRNA nui, loaʻa i nā ribosomes E. cuniculi nā ʻokoʻa rRNA ma kekahi o kā lākou mau wahi hana. ʻOiai ʻo ke kikowaena peptidyl transferase o ka ribosome E. cuniculi he ʻano like me nā ribosomes eukaryotic ʻē aʻe (Kiʻi 1d), ʻokoʻa ke kikowaena decoding ma muli o ka ʻokoʻa o ke kaʻina ma ka nucleotide 1491 (E. coli numbering, Kiʻi 1e, f). He mea nui kēia nānā ʻana no ka mea ʻo ka wahi decoding o nā ribosomes eukaryotic e loaʻa pinepine nā koena G1408 a me A1491 i hoʻohālikelike ʻia me nā koena ʻano bacterial A1408 a me G1491. ʻO kēia ʻokoʻa ke kumu o ka ʻokoʻa o ka ʻike o nā ribosomes bacteria a me eukaryotic i ka ʻohana aminoglycoside o nā antibiotics ribosomal a me nā mole liʻiliʻi ʻē aʻe e kuhikuhi ana i ka wahi decoding. Ma ka wahi decoding o ka ribosome E. cuniculi, ua pani ʻia ke koena A1491 me U1491, e hana ana i kahi interface hoʻopaʻa kū hoʻokahi no nā mole liʻiliʻi e kuhikuhi ana i kēia wahi hana. Loaʻa pū ka ʻano like ʻo A14901 i nā microsporidia ʻē aʻe e like me P. locustae a me V. necatrix, e hōʻike ana ua laha nui ia i waena o nā ʻano microsporidia (Kiʻi 1f).
No ka mea, ua hoʻokaʻawale ʻia kā mākou mau laʻana ribosome E. cuniculi mai nā spores metabolically inactive, ua hoʻāʻo mākou i ka palapala ʻāina cryo-EM o E. cuniculi no ka hoʻopaʻa ʻana o ka ribosome i wehewehe mua ʻia ma lalo o ke kaumaha a i ʻole nā ​​kūlana pōloli. Nā kumu hibernation 31,32,36,37, 38. Ua hoʻohālikelike mākou i ke ʻano i hoʻokumu mua ʻia o ka ribosome hibernating me ka palapala ʻāina cryo-EM o ka ribosome E. cuniculi. No ka docking, ua hoʻohana ʻia nā ribosome S. cerevisiae me ka hibernation factor Stm138, nā ribosome locust me ka Lso232 factor, a me nā ribosome V. necatrix me nā kumu Mdf1 a me Mdf231. I ka manawa like, ua loaʻa iā mākou ka nui o ka cryo-EM e pili ana i ka rest factor Mdf1. E like me ka hoʻopaʻa ʻana o Mdf1 i ka ribosome V. necatrix, hoʻopaʻa pū ʻo Mdf1 i ka ribosome E. cuniculi, kahi e ālai ai i ka pūnaewele E o ka ribosome, e kōkua ana paha i ka loaʻa ʻana o nā ribosome i ka wā e lilo ai nā spores parasite i mea hana ʻole i ka metabolically ma hope o ka hoʻopau ʻia ʻana o ke kino (Kiʻi 2).
Hoʻopaʻa ʻo Mdf1 i ka pūnaewele E o ka ribosome, kahi mea e kōkua ai i ka hoʻopau ʻana i ka ribosome ke lilo nā spores parasite i mea hana ʻole i ka metabolism. I loko o ke ʻano o ka ribosome E. cuniculi, ua ʻike mākou ua hoʻokumu ʻo Mdf1 i kahi pilina i ʻike ʻole ʻia ma mua me ke kumu ribosome L1, ka ʻāpana o ka ribosome e kōkua ai i ka hoʻokuʻu ʻana o ka deacylated tRNA mai ka ribosome i ka wā o ka synthesis protein. Hōʻike kēia mau pilina e hoʻokaʻawale ʻo Mdf1 mai ka ribosome me ka hoʻohana ʻana i ke ʻano like me ka deacetylated tRNA, e hāʻawi ana i kahi wehewehe hiki no ke ʻano o ka wehe ʻana o ka ribosome iā Mdf1 e hoʻāla hou i ka synthesis protein.
Eia nō naʻe, ua hōʻike kā mākou ʻano i kahi pilina ʻike ʻole ʻia ma waena o Mdf1 a me ka wāwae ribosome L1 (ka ʻāpana o ka ribosome e kōkua i ka hoʻokuʻu ʻana i ka tRNA deacylated mai ka ribosome i ka wā o ka synthesis protein). Ma ke ʻano kūikawā, hoʻohana ʻo Mdf1 i nā pilina like me ka ʻāpana kuʻekuʻe o ka molekala tRNA deacylated (Kiʻi 2). Ua hōʻike kēia hoʻohālike molekala i ʻike ʻole ʻia ma mua ua hoʻokaʻawale ʻo Mdf1 mai ka ribosome me ka hoʻohana ʻana i ke ʻano like me ka deacetylated tRNA, e wehewehe ana pehea e wehe ai ka ribosome i kēia kumu hibernation e hoʻāla hou i ka synthesis protein.
I ke kūkulu ʻana i ke kumu hoʻohālike rRNA, ua ʻike mākou ua pelu ʻia nā ʻāpana rRNA o ka ribosome E. cuniculi, a mākou i kapa aku ai he rRNA i hoʻohui ʻia (Kiʻi 3). I loko o nā ribosome e uhi ana i nā ʻāpana ʻekolu o ke ola, pelu ʻo rRNA i loko o nā ʻano kahi e hui pū ai ka hapa nui o nā kumu rRNA a pelu pū kekahi me kekahi a i ʻole e launa pū me nā protein ribosomal38,39,40. Eia nō naʻe, i loko o nā ribosome E. cuniculi, me he mea lā ua uhaki nā rRNA i kēia kumumanaʻo pelu ma ka hoʻololi ʻana i kekahi o kā lākou helices i nā ʻāpana rRNA i wehe ʻia.
ʻO ke ʻano o ka helix H18 25S rRNA ma S. cerevisiae, V. necatrix, a me E. cuniculi. ʻO ka maʻamau, i loko o nā ribosomes e uhi ana i nā ʻāpana ola ʻekolu, ʻōwili kēia mea hoʻopili i loko o kahi helix RNA nona nā koena 24 a 34. I Microsporidia, i ka hoʻohālikelike ʻana, ua hoʻemi mālie ʻia kēia mea hoʻopili rRNA i ʻelua mau mea hoʻopili waiwai uridine hoʻokahi kaula i loaʻa wale nō 12 mau koena. Hoʻopili ʻia ka hapa nui o kēia mau koena i nā mea hoʻoheheʻe. Hōʻike ke kiʻi e hōʻike ana ua hōʻino nā microsporidia parasitic i nā loina laulā o ka pelu ʻana o ka rRNA, kahi e hoʻopili pinepine ʻia ai nā kumu rRNA i nā kumu ʻē aʻe a i ʻole e pili ana i nā pilina rRNA-protein. I ka microsporidia, lawe kekahi mau ʻāpana rRNA i kahi pelu maikaʻi ʻole, kahi e lilo ai ka helix rRNA mua i ʻāpana kaula hoʻokahi i hoʻolōʻihi ʻia kokoke i kahi laina pololei. ʻO ke alo o kēia mau wahi ʻokoʻa e hiki ai i ka microsporidia rRNA ke hoʻopaʻa i nā ʻāpana rRNA mamao me ka hoʻohana ʻana i kahi helu liʻiliʻi o nā kumu RNA.
Hiki ke ʻike ʻia ka laʻana koʻikoʻi loa o kēia hoʻololi evolutionary ma ka H18 25S rRNA helix (Kiʻi 3). I nā ʻano mai E. coli a i kānaka, aia nā kumu o kēia rRNA helix i loko o 24-32 nucleotides, e hana ana i kahi helix ʻano ʻē iki. I nā ʻano ribosomal i ʻike mua ʻia mai V. necatrix lāua ʻo P. locustae,31,32 ua wehe hapa ʻia nā kumu o ka H18 helix, akā ua mālama ʻia ka hui ʻana o ke kumu nucleotide. Eia nō naʻe, ma E. cuniculi, lilo kēia ʻāpana rRNA i nā mea hoʻopili pōkole loa 228UUUUGU232 a me 301UUUUUUUUUU307. ʻAʻole e like me nā ʻāpana rRNA maʻamau, ʻaʻole hoʻopili kēia mau mea hoʻopili waiwai uridine a hana paha i kahi pilina nui me nā protein ribosomal. Akā, hoʻohana lākou i nā ʻano wehe ʻia a wehe piha ʻia kahi e hoʻolōʻihi ʻia ai nā kaula rRNA me ka kokoke pololei. Wehewehe kēia ʻano hoʻohālikelike i ke ʻano o ka hoʻohana ʻana o E. cuniculi i 12 mau kumu RNA wale nō e hoʻopiha i ka hakahaka 33 Å ma waena o nā helices H16 a me H18 rRNA, ʻoiai ʻo nā ʻano ʻē aʻe e koi ana i ka liʻiliʻi he pālua ka nui o nā kumu rRNA e hoʻopiha i ka hakahaka.
No laila, hiki iā mākou ke hōʻike, ma o ka pelu ʻana i ka ikehu ʻole, ua hoʻomohala nā microsporidia parasitic i kahi hoʻolālā e hoʻopaʻa i kēlā mau ʻāpana rRNA i mālama nui ʻia ma waena o nā ʻano ma nā ʻano ʻekolu o ke ola. ʻIke ʻia, ma ka hōʻiliʻili ʻana i nā mutations e hoʻololi i nā helices rRNA i nā loulou poly-U pōkole, hiki iā E. cuniculi ke hana i nā ʻāpana rRNA maʻamau ʻole e loaʻa ana nā nucleotides liʻiliʻi e like me ka hiki no ka ligation o nā ʻāpana rRNA distal. Kōkua kēia i ka wehewehe ʻana pehea i hoʻokō ai ʻo microsporidia i kahi hōʻemi nui i ko lākou ʻano molekala kumu me ka nalowale ʻole o ko lākou kūpaʻa kūkulu a me ka hana.
ʻO kekahi hiʻohiʻona ʻē aʻe o ka E. cuniculi rRNA ʻo ia ke ʻano o ka rRNA me ka ʻole o nā mānoanoa (Kiʻi 4). ʻO nā bulges he mau nucleotides me ka ʻole o nā hui kumu e wili ana i waho o ka RNA helix ma kahi o ka peʻe ʻana i loko. ʻO ka hapa nui o nā protrusions rRNA e hana ma ke ʻano he mau mea hoʻopili molekala, e kōkua ana i ka hoʻopaʻa ʻana i nā protein ribosomal e pili ana a i ʻole nā ​​​​ʻāpana rRNA ʻē aʻe. Hana kekahi o nā bulges ma ke ʻano he hinges, e ʻae ana i ka rRNA helix e flex a pelu maikaʻi no ka synthesis protein productive 41.
ʻAʻohe puka rRNA (S. cerevisiae numbering) mai ka ʻōnaehana ribosome E. cuniculi, akā aia i ka hapa nui o nā eukaryotes ʻē aʻe b E. coli, S. cerevisiae, H. sapiens, a me E. cuniculi internal ribosomes. ʻAʻohe o nā parasites i nā puʻupuʻu rRNA kahiko a mālama nui ʻia. Hoʻopaʻa kēia mau mānoanoa i ka ʻōnaehana ribosome; no laila, ʻo ko lākou nele i ka microsporidia e hōʻike ana i ka emi ʻana o ke kūpaʻa o ka pelu ʻana o ka rRNA i nā parasites microsporidia. Hōʻike ka hoʻohālikelike ʻana me nā kumu P (nā kumu L7/L12 i loko o ka bacteria) i ka nalowale ʻana o nā puʻupuʻu rRNA i kekahi manawa e kūlike me ka ʻike ʻia ʻana o nā puʻupuʻu hou ma ka ʻaoʻao o nā puʻupuʻu i nalowale. Loaʻa i ka helix H42 i loko o ka 23S/28S rRNA kahi puʻupuʻu kahiko (U1206 ma Saccharomyces cerevisiae) i manaʻo ʻia he 3.5 biliona mau makahiki ma muli o kona pale ʻana ma ʻekolu mau wahi o ke ola. I ka microsporidia, ua hoʻopau ʻia kēia puʻupuʻu. Eia naʻe, ua ʻike ʻia kahi puʻupuʻu hou ma ka ʻaoʻao o ka puʻupuʻu i nalowale (A1306 ma E. cuniculi).
ʻO ka mea kupanaha, ua ʻike mākou ʻaʻole i loaʻa i nā ribosomes E. cuniculi ka hapa nui o nā puʻupuʻu rRNA i loaʻa i nā ʻano ʻē aʻe, me ka ʻoi aku o 30 mau puʻupuʻu i mālama ʻia i nā eukaryotes ʻē aʻe (Kiʻi 4a). Hoʻopau kēia pohō i nā pilina he nui ma waena o nā subunits ribosomal a me nā helices rRNA e pili ana, i kekahi manawa e hana ana i nā hakahaka nui i loko o ka ribosome, e hoʻolilo ana i ka ribosome E. cuniculi i porous i hoʻohālikelike ʻia me nā ribosomes kuʻuna (Kiʻi 4b). ʻO ka mea nui, ua ʻike mākou ua nalowale hoʻi ka hapa nui o kēia mau puʻupuʻu i nā ʻano ribosome V. necatrix a me P. locustae i ʻike mua ʻia, kahi i nānā ʻole ʻia e nā loiloi kūkulu mua31,32.
I kekahi manawa, hele pū ka nalowale ʻana o nā puʻupuʻu rRNA me ka hoʻomohala ʻana o nā puʻupuʻu hou ma ka ʻaoʻao o ka puʻupuʻu i nalowale. No ka laʻana, aia i loko o ke kumu ribosomal P kahi puʻupuʻu U1208 (ma Saccharomyces cerevisiae) i ola mai E. coli a i kānaka a no laila ua manaʻo ʻia he 3.5 biliona mau makahiki. I ka wā o ka synthesis protein, kōkua kēia puʻupuʻu i ke kumu P e neʻe ma waena o nā conformations hāmama a pani ʻia i hiki i ka ribosome ke hoʻopaʻa i nā mea unuhi a hāʻawi iā lākou i kahi hana. Ma nā ribosomes E. cuniculi, ʻaʻohe kēia mānoanoa; akā naʻe, hiki i kahi mānoanoa hou (G883) i loaʻa wale i loko o ʻekolu mau hui kumu ke kōkua i ka hoʻihoʻi ʻana i ka maʻalahi kūpono o ke kumu P (Kiʻi 4c).
Hōʻike kā mākou ʻikepili ma ka rRNA me ka ʻole o nā puʻupuʻu ʻaʻole i kaupalena ʻia ka hoʻēmi ʻana o ka rRNA i ka nalowale ʻana o nā mea rRNA ma ka ʻili o ka ribosome, akā hiki ke pili pū i ka nucleus ribosome, e hana ana i kahi kīnā molekala parasite-specific i wehewehe ʻole ʻia i loko o nā cell ola manuahi. ʻike ʻia nā ʻano ola.
Ma hope o ke hoʻohālike ʻana i nā protein ribosomal canonical a me ka rRNA, ua ʻike mākou ʻaʻole hiki i nā ʻāpana ribosomal maʻamau ke wehewehe i nā ʻāpana ʻekolu o ke kiʻi cryo-EM. ʻElua o kēia mau ʻāpana he mau mole liʻiliʻi ka nui (Kiʻi 5, Kiʻi Hoʻohui 8). Ua hoʻopaʻa ʻia ka ʻāpana mua ma waena o nā protein ribosomal uL15 a me eL18 ma kahi kūlana i noho pinepine ʻia e ka C-terminus o eL18, i hoʻopōkole ʻia ma E. cuniculi. ʻOiai ʻaʻole hiki iā mākou ke hoʻoholo i ka ʻike o kēia mole, ua wehewehe maikaʻi ʻia ka nui a me ke ʻano o kēia mokupuni density e ke alo o nā mole spermidine. Hoʻopaʻa ʻia kona hoʻopaʻa ʻana i ka ribosome e nā mutations microsporidia-specific i loko o nā protein uL15 (Asp51 a me Arg56), kahi e hoʻonui ai i ka pilina o ka ribosome no kēia mole liʻiliʻi, no ka mea, ʻae lākou iā uL15 e kāʻei i ka mole liʻiliʻi i loko o kahi ʻano ribosomal. Kiʻi Hoʻohui 2). 8, ʻikepili hou 1, 2).
ʻO ke kiʻi Cryo-EM e hōʻike ana i ke alo o nā nucleotides ma waho o ka ribose i hoʻopaʻa ʻia i ka ribosome E. cuniculi. I loko o ka ribosome E. cuniculi, noho kēia nucleotide i kahi like me ka 25S rRNA A3186 nucleotide (Saccharomyces cerevisiae numbering) ma ka hapa nui o nā ribosome eukaryotic ʻē aʻe. b I loko o ke ʻano ribosomal o E. cuniculi, aia kēia nucleotide ma waena o nā protein ribosomal uL9 a me eL20, no laila e hoʻopaʻa ana i ka pilina ma waena o nā protein ʻelua. cd eL20 ka nānā ʻana i ka mālama ʻana i ke kaʻina ma waena o nā ʻano microsporidia. ʻO ka lāʻau phylogenetic o nā ʻano Microsporidia (c) a me ka hoʻonohonoho ʻana i nā kaʻina he nui o ka protein eL20 (d) e hōʻike ana ua mālama ʻia nā koena hoʻopaʻa nucleotide F170 a me K172 i ka hapa nui o Microsporidia maʻamau, koe wale nō ʻo S. lophii, koe wale nō ʻo Microsporidia lālā mua, ka mea i mālama i ka hoʻonui ES39L rRNA. e Hōʻike kēia kiʻi aia nā koena hoʻopaʻa nucleotide F170 a me K172 i loko o ka eL20 o ka genome microsporidia i hoʻemi nui ʻia, akā ʻaʻole i loko o nā eukaryotes ʻē aʻe. Ma keʻano holoʻokoʻa, hōʻike kēia ʻikepili ua hoʻomohala nā ribosomes Microsporidian i kahi kahua hoʻopaʻa nucleotide e ʻike ʻia e hoʻopaʻa i nā mole AMP a hoʻohana iā lākou e hoʻopaʻa i nā pilina protein-protein i loko o ke ʻano ribosomal. ʻO ka mālama kiʻekiʻe ʻana o kēia kahua hoʻopaʻa ma Microsporidia a me kona nele i nā eukaryotes ʻē aʻe e hōʻike ana e hiki i kēia kahua ke hāʻawi i kahi pono ola koho no Microsporidia. No laila, ʻaʻole ʻike ʻia ka ʻeke hoʻopaʻa nucleotide i loko o ka ribosome microsporidia he hiʻohiʻona degenerate a i ʻole ke ʻano hopena o ka hoʻohaʻahaʻa rRNA e like me ka mea i wehewehe mua ʻia, akā he mea hou evolutionary pono e hiki ai i ka microsporidia ribosome ke hoʻopaʻa pololei i nā mole liʻiliʻi, e hoʻohana ana iā lākou ma ke ʻano he mau poloka hale molekala. nā poloka hale no nā ribosomes. ʻO kēia ʻike ʻana e hana i ka microsporidia ribosome ʻo ia wale nō ka ribosome i ʻike ʻia e hoʻohana i hoʻokahi nucleotide ma ke ʻano he poloka hale kūkulu. f Ke ala evolutionary hypothetical i loaʻa mai ka hoʻopaʻa ʻana o ka nucleotide.
Aia ka lua o ka nui o ke kaumaha molekala haʻahaʻa ma ka pilina ma waena o nā protein ribosomal uL9 a me eL30 (Kiʻi 5a). Ua wehewehe mua ʻia kēia pilina ma ke ʻano o ka ribosome Saccharomyces cerevisiae ma ke ʻano he wahi hoʻopaʻa no ka nucleotide 25S o rRNA A3186 (ʻāpana o ka hoʻonui ʻana o ka rRNA ES39L)38. Ua hōʻike ʻia ma nā ribosome P. locustae ES39L degenerate, hoʻopaʻa kēia pilina i kahi nucleotide hoʻokahi i ʻike ʻole ʻia 31, a ua manaʻo ʻia he ʻano hope loa kēia nucleotide o ka rRNA, kahi o ka lōʻihi o ka rRNA he ~130-230 mau kumu. Hoʻemi ʻia ʻo ES39L i hoʻokahi nucleotide 32.43. Kākoʻo kā mākou mau kiʻi cryo-EM i ka manaʻo e hiki ke wehewehe ʻia ka nui e nā nucleotides. Eia nō naʻe, ua hōʻike ka hoʻonā kiʻekiʻe o kā mākou ʻano he mole extraribosomal kēia nucleotide, ʻo AMP paha (Kiʻi 5a, b).
A laila ua nīnau mākou inā ua ʻike ʻia kahi hoʻopaʻa nucleotide ma ka ribosome E. cuniculi a i ʻole ua ola mua ia. ʻOiai ʻo ka hoʻopaʻa ʻana o ka nucleotide e hoʻopili nui ʻia e nā koena Phe170 a me Lys172 i loko o ka protein ribosomal eL30, ua loiloi mākou i ka mālama ʻana o kēia mau koena i loko o 4396 eukaryotes ʻelele. E like me ke ʻano o uL15 ma luna, ua ʻike mākou ua mālama nui ʻia nā koena Phe170 a me Lys172 ma Microsporidia maʻamau wale nō, akā ʻaʻohe i loko o nā eukaryotes ʻē aʻe, me ka atypical Microsporidia Mitosporidium a me Amphiamblys, kahi i hoʻemi ʻole ʻia ai ka ʻāpana ES39L rRNA 44, 45, 46 (Kiʻi 5c). -e).
I ka hui pū ʻia ʻana, kākoʻo kēia mau ʻikepili i ka manaʻo ua hoʻomohala ʻo E. cuniculi a me nā microsporidia canonical ʻē aʻe i ka hiki ke hopu pono i nā helu he nui o nā metabolites liʻiliʻi i loko o ke ʻano ribosome e uku no ka emi ʻana o ka rRNA a me nā pae protein. I ka hana ʻana pēlā, ua hoʻomohala lākou i kahi hiki kū hoʻokahi e hoʻopaʻa i nā nucleotides ma waho o ka ribosome, e hōʻike ana e uku nā ʻano molekala parasitic ma ka hopu ʻana i nā metabolites liʻiliʻi he nui a hoʻohana iā lākou ma ke ʻano he mau mimics structural o nā ʻāpana RNA a me nā protein i hoʻohaʻahaʻa ʻia.
ʻO ke kolu o ka ʻāpana i hoʻohālikelike ʻole ʻia o kā mākou palapala ʻāina cryo-EM, i loaʻa i loko o ka subunit ribosomal nui. ʻO ka hoʻonā kiʻekiʻe (2.6 Å) o kā mākou palapala ʻāina e hōʻike ana he pili kēia density i nā protein me nā hui kūikawā o nā koena kaulahao ʻaoʻao nui, kahi i hiki ai iā mākou ke ʻike i kēia density ma ke ʻano he protein ribosomal i ʻike ʻole ʻia ma mua a mākou i ʻike ai Ua kapa ʻia ʻo msL2 (Microsporidia-specific protein L2) (nā ʻano hana, kiʻi 6). Ua hōʻike kā mākou hulina homology ua mālama ʻia ʻo msL2 i loko o ka clade Microsporidia o ka genus Encephaliter a me Orosporidium, akā ʻaʻohe i nā ʻano ʻē aʻe, me nā Microsporidia ʻē aʻe. I loko o ke ʻano ribosomal, noho ʻo msL2 i kahi hakahaka i hana ʻia e ka nalowale ʻana o ka ES31L rRNA i hoʻonui ʻia. I loko o kēia hakahaka, kōkua ʻo msL2 i ka hoʻopaʻa ʻana i ka pelu ʻana o ka rRNA a hiki ke uku no ka nalowale ʻana o ES31L (Kiʻi 6).
a ʻO ka nui o ka electron a me ke kumu hoʻohālike o ka protein ribosomal Microsporidia-specific msL2 i loaʻa i nā ribosomes E. cuniculi. b ʻO ka hapa nui o nā ribosomes eukaryotic, me ka ribosome 80S o Saccharomyces cerevisiae, ua nalowale ka hoʻonui ʻana o ka ES19L rRNA i ka hapa nui o nā ʻano Microsporidian. Hōʻike ka ʻano i hoʻokumu mua ʻia o ka ribosome V. necatrix microsporidia e uku ʻia ka nalowale o ES19L i loko o kēia mau parasites e ka ulu ʻana o ka protein ribosomal msL1 hou. Ma kēia haʻawina, ua ʻike mākou ua hoʻomohala pū ka ribosome E. cuniculi i kahi protein mimic RNA ribosomal hou ma ke ʻano he uku kūpono no ka nalowale ʻana o ES19L. Eia nō naʻe, ʻokoʻa nā kumu hoʻonohonoho a me ka ulu ʻana o msL2 (i kēia manawa i hōʻike ʻia ʻo ia ka protein ECU06_1135 hypothetical) a me msL1. c Hōʻike kēia ʻike ʻana o ka hanauna o nā protein msL1 a me msL2 ribosomal i pili ʻole i ka evolution inā hōʻiliʻili nā ribosome i nā mutations maikaʻi ʻole i loko o kā lākou rRNA, hiki iā lākou ke hoʻokō i nā pae like ʻole o ka ʻokoʻa o ka haku mele ʻana i loko o kahi ʻāpana liʻiliʻi o nā ʻano pili loa. Hiki i kēia ʻike ke kōkua i ka wehewehe ʻana i ke kumu a me ka ulu ʻana o ka ribosome mitochondrial, i ʻike ʻia no kāna rRNA i hoʻemi nui ʻia a me ka ʻokoʻa ʻē aʻe i ka haku mele protein ma waena o nā ʻano.
A laila ua hoʻohālikelike mākou i ka protein msL2 me ka protein msL1 i wehewehe mua ʻia, ʻo ia wale nō ka protein ribosomal microsporidia-specific i ʻike ʻia i loaʻa i loko o ka V. necatrix ribosome. Makemake mākou e hoʻāʻo inā pili ka msL1 a me msL2 i ka evolution. Ua hōʻike kā mākou loiloi e noho ana ʻo msL1 a me msL2 i ka lua like i loko o ke ʻano ribosomal, akā he ʻokoʻa nā ʻano mua a me nā ʻano tertiary, e hōʻike ana i ko lākou kumu evolutionary kūʻokoʻa (Kiʻi 6). No laila, ʻo kā mākou ʻike ʻana o msL2 e hāʻawi i ka hōʻike e hiki i nā hui o nā ʻano eukaryotic compact ke hoʻomohala kūʻokoʻa i nā protein ribosomal ʻokoʻa e uku no ka nalowale ʻana o nā ʻāpana rRNA. He mea koʻikoʻi kēia ʻike ʻana i ka hapa nui o nā ribosome eukaryotic cytoplasmic i loaʻa kahi protein invariant, me ka ʻohana like o 81 mau protein ribosomal. ʻO ka ʻike ʻia ʻana o msL1 a me msL2 i nā clades like ʻole o microsporidia i ka pane ʻana i ka nalowale ʻana o nā ʻāpana rRNA i hoʻonui ʻia e hōʻike ana i ka hōʻino ʻana o ke ʻano molekala o ka parasite e hoʻoulu ai i nā parasites e ʻimi i nā mutations compensatory, kahi e alakaʻi ai i ko lākou loaʻa ʻana i nā heluna parasite like ʻole. nā ʻano.
ʻO ka hope loa, i ka pau ʻana o kā mākou kumu hoʻohālike, ua hoʻohālikelike mākou i ka haku mele ʻana o ka ribosome E. cuniculi me ka mea i wānana ʻia mai ke kaʻina genome. Ua manaʻo mua ʻia kekahi mau protein ribosomal, me eL14, eL38, eL41, a me eS30, ua nalowale mai ka genome E. cuniculi ma muli o ka ʻike ʻole ʻia o kā lākou homologues mai ka genome E. cuniculi. Ua wānana ʻia hoʻi ka nalowale ʻana o nā protein ribosomal he nui i ka hapa nui o nā parasites intracellular i hoʻemi nui ʻia a me nā endosymbionts. No ka laʻana, ʻoiai ʻo ka hapa nui o nā bacteria ola manuahi e loaʻa ka ʻohana like o 54 mau protein ribosomal, he 11 wale nō o kēia mau ʻohana protein i loaʻa nā homologues i ʻike ʻia i kēlā me kēia genome i kālailai ʻia o nā bacteria i kaupalena ʻia e ka host. I ke kākoʻo ʻana i kēia manaʻo, ua ʻike ʻia ka nalowale ʻana o nā protein ribosomal ma ka hoʻokolohua ma V. necatrix a me P. locustae microsporidia, ka mea nele i nā protein eL38 a me eL4131,32.
Eia nō naʻe, hōʻike kā mākou mau ʻano hana ua nalowale wale ʻo eL38, eL41, a me eS30 i loko o ka ribosome E. cuniculi. Ua mālama ʻia ka protein eL14 a ua hōʻike kā mākou ʻano hana i ke kumu i hiki ʻole ai ke loaʻa kēia protein i ka ʻimi homology (Kiʻi 7). I loko o nā ribosome E. cuniculi, ua nalowale ka hapa nui o ka wahi hoʻopaʻa eL14 ma muli o ka hōʻino ʻia ʻana o ka ES39L i hoʻonui ʻia e rRNA. I ka loaʻa ʻole o ES39L, ua nalowale ka hapa nui o ka eL14 i kona ʻano lua, a he 18% wale nō o ka moʻo eL14 i like ma E. cuniculi lāua ʻo S. cerevisiae. He mea kupaianaha kēia mālama ʻana i ka moʻo maikaʻi ʻole no ka mea ʻo Saccharomyces cerevisiae a me Homo sapiens—nā meaola i ulu aʻe he 1.5 biliona mau makahiki—e kaʻana like ma mua o 51% o nā koena like ma eL14. ʻO kēia nalowale ʻano ʻē o ka mālama ʻana e wehewehe ana i ke kumu i hōʻike ʻia ai ʻo E. cuniculi eL14 i kēia manawa ʻo ia ka protein M970_061160 i manaʻo ʻia a ʻaʻole ʻo ia ka protein ribosomal eL1427.
a ua nalowale ka ribosome Microsporidia i ka hoʻonui ʻana o ka ES39L rRNA, kahi i hoʻopau hapa i ka pūnaewele hoʻopaʻa protein ribosomal eL14. I ka loaʻa ʻole o ES39L, ua nalowale ka protein microspore eL14 i ka hoʻonohonoho lua, kahi e lilo ai ka α-helix hoʻopaʻa rRNA mua i loko o kahi loop lōʻihi liʻiliʻi. b Hōʻike ka hoʻonohonoho ʻana o nā kaʻina he nui ua mālama nui ʻia ka protein eL14 i nā ʻano eukaryotic (57% ka ʻike kaʻina ma waena o ka yeast a me nā homologues kanaka), akā mālama maikaʻi ʻole ʻia a ʻokoʻa hoʻi i ka microsporidia (kahi ʻaʻole i ʻoi aku ma mua o 24% o nā koena i like me ka homologue eL14). mai S. cerevisiae a i ʻole H. sapiens). ʻO kēia mālama kaʻina maikaʻi ʻole a me ka loli o ka hoʻonohonoho lua e wehewehe ana i ke kumu i loaʻa ʻole ai ka homologue eL14 ma E. cuniculi a no ke aha i manaʻo ʻia ai ua nalowale kēia protein ma E. cuniculi. I ka hoʻohālikelike ʻana, ua hoʻopaʻa mua ʻia ʻo E. cuniculi eL14 ma ke ʻano he protein M970_061160 i manaʻo ʻia. Hōʻike kēia ʻike ʻana ua nui loa ka manaʻo o ka ʻokoʻa o ka genome microsporidia i kēia manawa: ʻo kekahi mau genes i manaʻo ʻia ua nalowale i loko o ka microsporidia ua mālama ʻia, ʻoiai ma nā ʻano i hoʻokaʻawale loa ʻia; akā, ua manaʻo ʻia kekahi e code no nā genes microsporidia no nā protein kikoʻī ilo (e laʻa, ka protein hypothetical M970_061160) e code maoli no nā protein like ʻole i loaʻa i nā eukaryotes ʻē aʻe.
Hōʻike kēia ʻike e hiki i ka denaturation rRNA ke alakaʻi i ka nalowale nui o ka mālama ʻana i ke kaʻina i nā protein ribosomal e pili ana, e hoʻolilo ana i kēia mau protein i mea hiki ʻole ke ʻike ʻia no nā ʻimi homology. No laila, hiki iā mākou ke hoʻonui i ke kiʻekiʻe maoli o ka hōʻino ʻana o ka molekala i nā meaola genome liʻiliʻi, ʻoiai kekahi mau protein i manaʻo ʻia ua nalowale e mau nei, ʻoiai i nā ʻano i hoʻololi nui ʻia.
Pehea e hiki ai i nā parasites ke mālama i ka hana o kā lākou mau mīkini molekala ma lalo o nā kūlana o ka hōʻemi nui ʻana o ka genome? Pane kā mākou noiʻi i kēia nīnau ma ka wehewehe ʻana i ke ʻano molekala paʻakikī (ribosome) o E. cuniculi, kahi meaola me kekahi o nā genomes eukaryotic liʻiliʻi loa.
Ua ʻike ʻia no kahi kokoke i iwakālua mau makahiki he ʻokoʻa pinepine nā molekala protein a me RNA i loko o nā parasite microbial mai kā lākou mau molekala homologous i loko o nā ʻano ola manuahi no ka mea nele lākou i nā kikowaena kaohi maikaʻi, ua hoʻemi ʻia i 50% o ko lākou nui i loko o nā microbes ola manuahi, a pēlā aku. . he nui nā mutations hoʻonāwaliwali e hoʻopilikia ai i ka pelu ʻana a me ka hana. No ka laʻana, ʻo nā ribosomes o nā meaola genome liʻiliʻi, me nā parasite intracellular he nui a me nā endosymbionts, e manaʻo ʻia e nele i kekahi mau protein ribosomal a hiki i ka hapakolu o nā nucleotides rRNA i hoʻohālikelike ʻia me nā ʻano ola manuahi 27, 29, 30, 49. Eia nō naʻe, ʻo ke ʻano o ka hana ʻana o kēia mau molekala i loko o ka parasite e mau nei i kahi mea pohihihi, i aʻo nui ʻia ma o ka genomics hoʻohālikelike.
Hōʻike kā mākou haʻawina e hiki i ke ʻano o nā macromolecules ke hōʻike i nā ʻano he nui o ka evolution i paʻakikī ke unuhi mai nā haʻawina genomic hoʻohālikelike kuʻuna o nā parasites intracellular a me nā meaola ʻē aʻe i kaupalena ʻia e ka host (Supplementary Fig. 7). No ka laʻana, hōʻike ka laʻana o ka protein eL14 hiki iā mākou ke hoʻonui i ke kiʻekiʻe maoli o ka hōʻino ʻana o ka mea hana molekala i nā ʻano parasitic. Ua manaʻoʻiʻo ʻia nā parasites encephalitic i kēia manawa he mau haneli o nā genes microsporidia-specific. Eia naʻe, hōʻike kā mākou mau hopena he mau ʻano like ʻole o kēia mau genes kikoʻī i maʻamau i nā eukaryotes ʻē aʻe. Eia kekahi, hōʻike ka laʻana o ka protein msL2 pehea mākou e nānā ʻole ai i nā protein ribosomal hou a hoʻohaʻahaʻa i ka ʻike o nā mīkini molekala parasitic. Hōʻike ka laʻana o nā molekala liʻiliʻi pehea mākou e nānā ʻole ai i nā hana hou akamai loa i nā ʻano molekala parasitic hiki ke hāʻawi iā lākou i ka hana olaola hou.
I ka hui pū ʻia ʻana, hoʻomaikaʻi kēia mau hopena i ko mākou ʻike ʻana i nā ʻokoʻa ma waena o nā ʻano molekala o nā meaola i kaupalena ʻia e ka mea hoʻokipa a me ko lākou mau hoa i nā meaola ola manuahi. Ke hōʻike nei mākou i nā mīkini molekala, i manaʻo ʻia no ka manawa lōʻihi e hoʻemi ʻia, e palaho ana, a i loaʻa i nā ʻano hoʻololi hoʻonāwaliwali like ʻole, akā, he mau hiʻohiʻona ʻano maʻamau i nānā ʻole ʻia.
Ma ka ʻaoʻao ʻē aʻe, ʻo nā ʻāpana rRNA non-bulky a me nā ʻāpana i hoʻohui ʻia a mākou i loaʻa ai i loko o nā ribosomes o E. cuniculi e hōʻike ana e hiki i ka hōʻemi ʻana o ka genome ke hoʻololi i kēlā mau ʻāpana o ka mīkini molekala kumu i mālama ʻia i nā ʻano ʻekolu o ke ola - ma hope o kahi o 3.5 biliona mau makahiki. ka ulu kūʻokoʻa o nā ʻano.
ʻO nā ʻāpana rRNA ʻole i hoʻopili ʻia a me nā ʻāpana i hoʻopili ʻia i loko o nā ribosomes E. cuniculi he mea hoihoi loa ia i ka mālamalama o nā haʻawina mua o nā molekala RNA i loko o nā bacteria endosymbiotic. No ka laʻana, i loko o ka aphid endosymbiont Buchnera aphidicola, ua hōʻike ʻia nā molekala rRNA a me tRNA he mau ʻano palekana i ka mahana ma muli o ka bias composition A+T a me kahi hapa nui o nā hui kumu non-canonical20,50. ʻO kēia mau loli i ka RNA, a me nā loli i nā molekala protein, ua manaʻo ʻia i kēia manawa ke kuleana no ka hilinaʻi nui ʻana o nā endosymbionts i nā hoa a me ka hiki ʻole o nā endosymbionts ke hoʻoili i ka wela 21, 23. ʻOiai he mau loli ʻokoʻa ke ʻano o ka microsporidia rRNA parasitic, ʻo ke ʻano o kēia mau loli e hōʻike ana he mau hiʻohiʻona maʻamau o nā molekala RNA i loko o nā meaola me nā genomes i hoʻemi ʻia.
Ma ka ʻaoʻao ʻē aʻe, hōʻike kā mākou mau ʻano hana ua hoʻomohala ka parasite microsporidia i kahi hiki kū hoʻokahi e kūʻē i nā ʻāpana rRNA a me nā protein i mālama nui ʻia, e hoʻomohala ana i ka hiki ke hoʻohana i nā metabolites liʻiliʻi he nui a loaʻa koke e like me nā hoʻohālike kūkulu o ka degenerate rRNA a me nā ʻāpana protein. Hoʻohaʻahaʻa ʻana o ke ʻano molekala. . Kākoʻo ʻia kēia manaʻo e ka ʻoiaʻiʻo e hoʻopaʻa nā mole liʻiliʻi e uku no ka nalowale ʻana o nā ʻāpana protein i loko o ka rRNA a me nā ribosomes o E. cuniculi i nā koena microsporidia-specific i loko o nā protein uL15 a me eL30. Hōʻike kēia i ka hoʻopaʻa ʻana o nā mole liʻiliʻi i nā ribosomes he huahana paha ia o ke koho maikaʻi, kahi i koho ʻia ai nā mutations Microsporidia-specific i loko o nā protein ribosomal no ko lākou hiki ke hoʻonui i ka pilina o nā ribosomes no nā mole liʻiliʻi, kahi e alakaʻi ai i nā meaola ribosomal ʻoi aku ka maikaʻi. Hōʻike ka ʻike ʻana i kahi hana hou akamai i ke ʻano molekala o nā parasites microbial a hāʻawi iā mākou i kahi ʻike maikaʻi aʻe i ke ʻano o ka mālama ʻana o nā ʻano molekala parasite i kā lākou hana me ka ʻole o ka evolution reductive.
I kēia manawa, ʻaʻole maopopo ka ʻike ʻana o kēia mau molekala liʻiliʻi. ʻAʻole maopopo ke kumu o ka ʻokoʻa o ke ʻano o kēia mau molekala liʻiliʻi i loko o ke ʻano ribosomal ma waena o nā ʻano microsporidia. ʻOi aku ka maopopo, ʻaʻole maopopo ke kumu e ʻike ʻia ai ka nakinaki nucleotide i loko o nā ribosomes o E. cuniculi lāua ʻo P. locustae, ʻaʻole i loko o nā ribosomes o V. necatrix, ʻoiai ke alo o ke koena F170 i loko o nā protein eL20 a me K172 o V. necatrix. Hiki ke hana ʻia kēia holoi ʻana e ke koena 43 uL6 (aia ma kahi kokoke i ka ʻeke nakinaki nucleotide), ʻo ia ka tyrosine ma V. necatrix a ʻaʻole ka threonine ma E. cuniculi lāua ʻo P. locustae. Hiki i ke kaulahao ʻaoʻao aromatic nui o Tyr43 ke hoʻopilikia i ka nakinaki nucleotide ma muli o ka steric overlap. Ma kekahi ʻaoʻao, ʻo ka holoi ʻana o ka nucleotide i ʻike ʻia ma muli paha o ka hoʻonā haʻahaʻa o ke kiʻi cryo-EM, ka mea e pale ai i ke kumu hoʻohālike o nā ʻāpana ribosomal V. necatrix.
Ma ka ʻaoʻao ʻē aʻe, hōʻike kā mākou hana he mana hana hou paha ke kaʻina hana o ka palaho genome. Ma ke ʻano kūikawā, hōʻike ka ʻano o ka ribosome E. cuniculi e hoʻokumu ka nalowale ʻana o ka rRNA a me nā ʻāpana protein i loko o ka microsporidia ribosome i ke kaomi evolutionary e paipai ana i nā loli i ke ʻano o ka ribosome. Loaʻa kēia mau ʻano like ʻole ma kahi mamao loa mai ka wahi hana o ka ribosome a ʻike ʻia e kōkua i ka mālama ʻana (a i ʻole ka hoʻihoʻi ʻana) i ka hui ribosome kūpono e hoʻopilikia ʻia e ka rRNA i hoʻemi ʻia. Hōʻike kēia ua ulu kahi hana hou nui o ka microsporidia ribosome i mea e pono ai ke pale i ka drift gene.
Malia paha ua hoʻākāka maikaʻi ʻia kēia e ka nakinaki ʻana o ka nucleotide, kahi i ʻike ʻole ʻia i nā meaola ʻē aʻe a hiki i kēia manawa. ʻO ka ʻoiaʻiʻo aia nā koena nakinaki nucleotide i loko o ka microsporidia maʻamau, akā ʻaʻole i loko o nā eukaryotes ʻē aʻe, e hōʻike ana ʻaʻole nā ​​​​​​wahi nakinaki nucleotide he mau relics e kali ana e nalo, a i ʻole ke kahua hope loa no ka rRNA e hoʻihoʻi ʻia i ke ʻano o nā nucleotides pākahi. Akā, me he mea lā he hiʻohiʻona pono kēia pūnaewele i hiki ke ulu aʻe ma luna o kekahi mau pōʻai o ke koho maikaʻi. ʻO nā wahi nakinaki nucleotide he huahana ʻaoʻao paha ia o ke koho kūlohelohe: i ka wā e hoʻohaʻahaʻa ʻia ai ʻo ES39L, ua koi ʻia nā microsporidia e ʻimi i ka uku e hoʻihoʻi i ka biogenesis ribosome maikaʻi loa i ka loaʻa ʻole o ES39L. No ka mea hiki i kēia nucleotide ke hoʻohālike i nā pilina molekala o ka nucleotide A3186 ma ES39L, lilo ka mole nucleotide i poloka hale o ka ribosome, ʻo ka nakinaki ʻana o ia mea i hoʻomaikaʻi hou ʻia e ka mutation o ka moʻo eL30.
E pili ana i ka ulu ʻana o nā molekala o nā parasites intracellular, hōʻike kā mākou noiʻi ʻaʻole hana like nā mana o ke koho kūlohelohe Darwinian a me ka genetic drift o ka genome decay, akā oscillate. ʻO ka mea mua, hoʻopau ka genetic drift i nā hiʻohiʻona koʻikoʻi o nā biomolecules, e hana ana i ka uku e pono nui ai. Ke hoʻokō nā parasites i kēia pono ma o ke koho kūlohelohe Darwinian e loaʻa i kā lākou macromolecules kahi manawa e hoʻomohala i kā lākou mau ʻano kupaianaha a hou loa. ʻO ka mea nui, ʻo ka ulu ʻana o nā wahi hoʻopaʻa nucleotide i loko o ka E. cuniculi ribosome e hōʻike ana ʻaʻole kēia ʻano pohō-e-loaʻa o ka ulu ʻana o ka molekala e hoʻopau wale i nā mutations deleterious, akā i kekahi manawa hāʻawi i nā hana hou loa i nā macromolecules parasitic.
He kūlike kēia manaʻo me ke kumumanaʻo kaulike neʻe ʻo Sewell Wright, kahi e ʻōlelo nei he ʻōnaehana koʻikoʻi o ke koho kūlohelohe e kaupalena ana i ka hiki o nā meaola ke hana hou51,52,53. Eia nō naʻe, inā hoʻopilikia ka genetic drift i ke koho kūlohelohe, hiki i kēia mau drift ke hana i nā loli ʻaʻole i kūpono iā lākou iho (a i ʻole hōʻino paha) akā alakaʻi i nā loli hou aʻe e hāʻawi ana i ke kūlana kiʻekiʻe a i ʻole ka hana olaola hou. Kākoʻo kā mākou papa hana i kēia manaʻo ma ka hōʻike ʻana i ke ʻano like o ka mutation e hōʻemi ana i ka pelu a me ka hana o kahi biomolecule e like me ke kumu nui no kona hoʻomaikaʻi ʻana. Ma ke kūlike me ke kumu hoʻohālike evolutionary lanakila-lanakila, hōʻike kā mākou noiʻi ʻo ka palaho genome, i manaʻo ʻia ma ke ʻano he hana degenerative, he mea hoʻokele nui hoʻi ia o ka hana hou, i kekahi manawa a malia paha e ʻae pinepine ana i nā macromolecules e loaʻa nā hana parasitic hou. hiki ke hoʻohana iā lākou.