Transcriptional and post-transcriptional control of eEF1A2 expression during myoblast diffrerentiation

GENOMICS, TRANSCRIPTOMICS AND PROTEOMICS

UDC 577.2 + 616-006

Òðàíñêðèïö³éíèé ³ ïîñòòðàíñêðèïö³éíèé êîíòðîëü

åêñïðåñ³¿ eEF1A2 ó ïðîöåñ³ äèôåðåíö³àö³¿ ì³îáëàñò³â

À. À. ³ñëîâóõ

_{, ². Ñ. Ãðîéñìàí}

_{, À. Â. ªëüñüêà}

_{, Á. Ñ. Íåãðóöüêèé}

_,

À. Í. Ïîëºñüêà

1_{Äåðæàâíà êëþ÷îâà ëàáîðàòîð³ÿ ìîëåêóëÿðíî¿ ³ êë³òèííî¿ á³îëî㳿} ²íñòèòóò ìîëåêóëÿðíî¿ á³îëî㳿 ³ ãåíåòèêè ÍÀÍ Óêðà¿íè

Âóë. Àêàäåì³êà Çàáîëîòíîãî, 150, Êè¿â, Óêðà¿íà, 03680

2_{Íàö³îíàëüíèé öåíòð íàóêîâèõ äîñë³äæåíü (CNRS) òà Êîì³ñàð³àò àòîìíî¿ åíåðãåòèêè (CEA)} Ñàêëå, Æèô-ñþð-²âåò, Ôðàíö³ÿ, F-91191

a.a.vislovukh@imbg.org.ua

Ó ïðîöåñ³ ïîñòíàòàëüíîãî ðîçâèòêó â íåéðîíàëüí³é ³ ì’ÿçîâ³é òêàíèíàõ â³äáóâàºòüñÿ çì³íà ³çîôîðìè À1 ôàêòîðà åëîíãàö³¿ òðàíñëÿö³¿ íà ³çîôîðìó À2. Ïåðåìèêàííÿ åêñïðåñ³¿ ³çîôîðì º æèòòºâî âàæëèâîþ ïî䳺þ, îñê³ëüêè ìóòàíòí³ ìèø³, ùî ì³ñòÿòü äåëåö³þ EEF1A2, ãèíóòü íà 28-é äåíü ï³ñëÿ íàðîäæåííÿ. Ìåõàí³çìè ³íã³áóâàííÿ À1 ³ ñòèìóëÿö³¿ À2 ïðîòÿãîì ïåðøèõ äí³â ïîñòíàòàëüíîãî ðîçâèòêó íåâ³äîì³. Íàÿâí³ñòü ñàéò³â çâ’ÿçóâàííÿ ì³êðîÐÍÊ ó 3'-íåòðàíñëüîâàíèõ ïîñë³äîâíîñòÿõ ìÐÍÊ ³çîôîðì ôàêòîðà åëîíãàö³¿ òðàíñëÿö³¿ ïåðåäáà÷ຠ³ñíóâàííÿ ïîñòòðàíñêðèïö³éíîãî êîíòðîëþ äàíîãî ïðîöåñó.Ìåòà. Ïå-ðåâ³ðèòè ìîæëèâ³ñòü ïîñòòðàíñêðèïö³éíî¿ ðåãóëÿö³¿ åêñïðåñ³¿ ³çîôîðì À1 ³ À2 ï³ä ÷àñ äèôåðåíö³àö³¿ ³ì-ìîðòàë³çîâàíèõ ì³îáëàñò³â ëþäèíè LHCN.Ìåòîäè. гâåíü åêñïðåñ³¿ ãåí³â âèçíà÷àëè ìåòîäîì ê³ëüê³ñíî¿ ÏËÐ, íàÿâí³ñòü ïîñòòðàíñêðèïö³éíîãî êîíòðîëþ äåòåêòóâàëè ìåòîäîì ðåïîðòåðíèõ ãåí³â. Ðåçóëüòà-òè. Âèêîðèñòîâóþ÷è ÿê ìîäåëü êë³òèííó ë³í³þ ³ììîðòàë³çîâàíèõ ì³îáëàñò³â ëþäèíè LHCN, ïîêàçàíî ³í-äóêö³þ ³çîôîðìè À2 ôàêòîðà åëîíãàö³¿ òðàíñëÿö³¿ eEF1 ïðè äèôåðåíö³àö³¿ ì³îáëàñò³â, íàÿâí³ñòü òðàíñ-êðèïö³éíîãî ³ ïîñòòðàíñòðàíñ-êðèïö³éíîãî êîíòðîëþ â äàíîìó ïðîöåñ³, à òàêîæ ïîòåíö³éíó ó÷àñòü ì³êðî-ÐÍÊ ó ïðîöåñ³ çì³íè åêñïðåñ³¿ ³çîôîðì.Âèñíîâêè. ²íäóêö³ÿ ³çîôîðìè À2 ïðîòÿãîì äèôåðåíö³àö³¿ ì³îáëà-ñò³â ìîæå â³äáóâàòèñÿ ÿê íà òðàíñêðèïö³éíîìó, òàê ³ íà ïîñòòðàíñêðèïö³éíîìó ð³âí³.

Êëþ÷îâ³ ñëîâà: eEF1A1, eEF1A2, ³ììîðòàë³çîâàí³ ì³îáëàñòè ëþäèíè LHCN, äèôåðåíö³àö³ÿ, ì³êðîÐÍÊ.

Âñòóï. Åâêàð³îòíèé ôàêòîð åëîíãàö³¿ òðàíñëÿö³¿

(eEF1A) º îäíèì ³ç êëþ÷îâèõ åëåìåíò³â á³ëêîâîãî ñèíòåçó [1]. Îñíîâíîþ ôóíêö³ºþ eEF1A º äîñòàâêà àì³íîàöèëüîâàíî¿ òÐÍÊ â À-ñàéò ðèáîñîìè [2]. ϳä ÷àñ åìáð³îãåíåçó ³çîôîðìà À1 (eEF1A1) º îñíîâíîþ ³ åêñïðåñóºòüñÿ â óñ³õ òêàíèíàõ. Àëå â ïðîöåñ³ ïîñò-íàòàëüíîãî ïåð³îäó ðîçâèòêó â äåÿêèõ òêàíèíàõ â³ä-áóâàºòüñÿ çíèæåííÿ ³ çóïèíêà åêñïðåñ³¿ ³çîôîðìè À1 ç ïàðàëåëüíîþ ³íäóêö³ºþ åêñïðåñ³¿ ³çîôîðìè À2 (eEF1A2) [3, 4]. Òàê³ çì³íè õàðàêòåðí³ ëèøå äëÿ òåðì³íàëüíî äèôåðåíö³éîâàíèõ êë³òèí, çîêðåìà, äëÿ íåéðîí³â, êàðä³îì³îöèò³â ³ ì³îöèò³â [5, 6]. Ïèòàííÿ ïðî ìåõàí³çì ³ ô³ç³îëîã³÷íå çíà÷åííÿ ïåðåìèêàííÿ

åêñïðåñ³¿ ³çîôîðì íà ñüîãîäí³øí³é äåíü çàëè-øàºòüñÿ áåç â³äïîâ³ä³. ³äñóòí³ñòü ³çîôîðìè À2, ñïðè÷èíåíà ÷àñòêîâîþ äåëåö³ºþ ãåíà EEF1A2,

çíàéäåíà ó ìóòàíòíèõ ìèøåéwst/wst, º ëåòàëüíîþ.

Ó òàêèõ ìèøåé ïîñòóïîâî ïîðóøóºòüñÿ åêñïðåñ³ÿ À1, àëå íà çàì³íó ¿é åêñïðåñ³ÿ À2 íå ç’ÿâëÿºòüñÿ. Ôåíîòèï äàíèõ ìèøåé õàðàêòåðèçóºòüñÿ çìåíøåí-íÿì ìàñè ò³ëà, òðåìîðîì, ïàòîëîã³ÿìè ì’ÿçîâî¿ ³ íå-ðâîâî¿ ñèñòåì. Íà 28-é äåíü ï³ñëÿ íàðîäæåííÿ ãîìî-çèãîòè çà öèì àëåëåì ïîìèðàþòü [7].

Çàçíà÷åí³ ³çîôîðìè íà 98 % º ãîìîëîã³÷íèìè. Àëå íåçâàæàþ÷è íà çíà÷íó ãîìîëîã³þ ³ ñõîæó åôåê-òèâí³ñòü òðàíñëÿö³¿, ö³ á³ëêè çíà÷íî â³äð³çíÿþòüñÿ çà íåêàíîí³÷íèìè ôóíêö³ÿìè [8, 9]. Ïîêàçàíî, ùî eEF1A1 ìຠïðîàïîïòè÷í³, òîä³ ÿê eEF1A2 –

àíòè-ISSN 0233–7657. Biopolymers and Cell. 2012. Vol. 28. N 6. P. 456–460 doi 10.7124/bc.000136

àïîïòè÷í³ âëàñòèâîñò³ [10]. ²ñíóþòü äàí³, ÿê³ ï³ä-òâåðäæóþòü ïðîòîîíêîãåííó ïðèðîäó eEF1A2 [11]. Íàÿâí³ñòü ³çîôîðìè À2 ïîêàçàíî â òêàíèíàõ ðàêó ìîëî÷íî¿ çàëîçè, ëåãåí³â òà ÿº÷íèê³â [12].

Íåçâàæàþ÷è íà âèñîêèé ð³âåíü ãîìîëî㳿 á³ëê³â, ìÐÍÊ, ÿê³ êîäóþòü ³çîôîðìè À1 ³ À2, çíà÷íî â³äð³ç-íÿþòüñÿ çà 3'- ³ 5'-íåòðàíñëüîâàíèìè ïîñë³äîâíîñòÿ-ìè (ÍÒÏ). Öå ñâ³ä÷èòü ïðî ìîæëèâ³ñòü ð³çíîãî êîíò-ðîëþ åêñïðåñ³¿ ³çîôîðì íà ïîñòòðàíñêðèïö³éíîìó ð³âí³. ̳êðîÐÍÊ º îäíèìè ç ãîëîâíèõ ãðàâö³â ó ñèñ-òåì³ ïîñòòðàíñêðèïö³éíîãî êîíòðîëþ [13]. Íàÿâ-í³ñòü ñàéò³â äëÿ ð³çíèõ ì³êðîÐÍÊ ó ÍÒÏ-ä³ëÿíêàõ ³çî-ôîðì íàâîäèòü íà äóìêó ñòîñîâíî ó÷àñò³ ïîñòòðàíñ-êðèïö³éíîãî êîíòðîëþ ïðè ïåðåìèêàíí³ åêñïðåñ³¿ ³çîôîðì ç À1 íà À2.  äàí³é ðîáîò³ åêñïåðèìåíòàëü-íî ï³äòâåðäæååêñïåðèìåíòàëü-íî ã³ïîòåçó íàÿâåêñïåðèìåíòàëü-íîñò³ ïîñòòðàíñêðèï-ö³éíîãî êîíòðîëþ ó ðàç³ ïåðåìèêàííÿ ³çîôîðì ôàê-òîðà åëîíãàö³¿ eEF1A ïðè äèôåðåíö³àö³¿ ì³îáëà-ñò³â, à òàêîæ ó÷àñòü ì³êðîÐÍÊ ó äàíîìó ïðîöåñ³.

Ìàòåð³àëè ³ ìåòîäè.Êóëüòóðà êë³òèí.

Êóëüòè-âóâàííÿ ³ äèôåðåíö³àö³þ ³ììîðòàë³çîâàíèõ ì³îáëà-ñò³â ëþäèíè LHCN ïðîâîäèëè çã³äíî ç îðèã³íàëü-íîþ ìåòîäèêîþ [14].

Ñèíòåç êÄÍÊ, ê³ëüê³ñíà ÏËÐ. Òîòàëüíó ÐÍÊ

âè-ä³ëÿëè ç (1–2)×106êë³òèí, âèêîðèñòîâóþ÷è

TRI-ðå-àãåíò («Sigma Chem. Co.», ÑØÀ). êÄÍÊ ñèíòåçó-âàëè çà äîïîìîãîþ çâîðîòíî¿ òðàíñêðèïòàçè Revert-Aid («Fermentas», Ëèòâà) òà îë³ãîÄÒ-ïðàéìåðà çã³ä-íî ç ðåêîìåíäàö³ÿìè âèðîáíèêà. ʳëüê³ñíó ÏËÐ

ïðîâîäèëè, ÿê îïèñàíî ðàí³øå [15]. ³äíîñíó ê³ëü-ê³ñòü ìÐÍÊ EEF1A1/A2 íîðìàë³çóâàëè äî ìÐÍÊ áå-òà-àêòèíó. ʳëüê³ñíèé ÏËÐ-àíàë³ç ì³êðîÐÍÊ çä³é-ñíþâàëè ³ç çàñòîñóâàííÿì êîìåðö³éíîãî íàáîðó Taq-Man®_{MicroRNA Assays («Applied Biosystems», ÑØÀ)}

çà ðåêîìåíäàö³ÿìè âèðîáíèêà.

Ìåòîä ðåïîðòåðíîãî ãåíà. Ïðîë³ôåðóþ÷³ àáî

äè-ôåðåíö³éîâàí³ êë³òèíè LHCN òðàíñô³êóâàëè 20 íã ïëàçì³äè, ÿêà ì³ñòèòü â³äêðèòó ðàìêó ç÷èòóâàííÿ ëþöèôåðàçè òà 3'-ÍÒÏ EEF1A1/A2, ç âèêîðèñòàí-íÿì ë³ïîôåêòàì³íó çã³äíî ç ³íñòðóêö³ºþ âèðîáíèêà. ×åðåç 24 ãîä ð³âåíü ëþöèôåðàçè âèì³ðþâàëè çà äî-ïîìîãîþ Dual-Luciferase®_{Reporter Assay System çà}

ïðîòîêîëîì âèðîáíèêà.

Ðåçóëüòàòè ³ îáãîâîðåííÿ. ²ììîðòàë³çîâàí³

ì³îáëàñòè ëþäèíè îòðèìóâàëè, òðàíñôîðìóþ÷è ¿õ òåëîìåðàçîþ ³ öèêë³í-çàëåæíþ ê³íàçîþ 4 [14]. Îò-ðèìàíà êë³òèííà ë³í³ÿ çäàòíà ÿê äî ïðîë³ôåðàö³¿ ó âèãëÿä³ íåäèôåðåíö³éîâàíèõ ì³îáëàñò³â, òàê ³ äî äèôåðåíö³àö³¿ ³ç ôîðìóâàííÿì ì³îöèò³â. Îñíîâíèì êðèòåð³ºì ìîæëèâîñò³ âèêîðèñòàííÿ êë³òèí LHCN äëÿ âèâ÷åííÿ ðåãóëÿö³¿ åêñïðåñ³¿ À1 ³ À2 º âçàºìî-çàì³íà åêñïðåñ³¿ ³çîôîðì ï³ñëÿ ïðîöåñó äèôåðåí-ö³àö³¿. Òîìó ìè ïîð³âíÿëè åêñïðåñ³þ ³çîôîðì â íå-äèôåðåíö³éîâàíèõ ì³îáëàñòàõ ³ íà 6-é äåíü äèôå-ðåíö³àö³¿. ϳñëÿ çàâåðøåííÿ äèôåðåíö³àö³¿ ñïî-ñòåð³ãàëè 20-ðàçîâå çá³ëüøåííÿ åêñïðåñ³¿ ìÐÍÊ ³çî-ôîðìè À2, ùî â³äîáðàæຠàíàëîã³÷íèé ïðîöåñ â îð-ãàí³çì³ [7] (ðèñ. 1). Âîäíî÷àñ ïîêàçàíî ìåíø çíà÷ó-ùå, í³æ ó EEF1A2, àëå ñòàòèñòè÷íî äîñòîâ³ðíå ï’ÿ-òèðàçîâå çðîñòàííÿ ìÐÍÊ ³çîôîðìè EEF1A1.

̳êðîÐÍÊ â³ä³ãðàþòü âàæëèâó ðîëü ó ïðîöåñ³ äèôåðåíö³àö³¿ ì³îáëàñò³â ³ ðîçâèòêó ïàòîëîã³é ì’ÿ-çîâî¿ ³ ñåðöåâî¿ òêàíèí [16]. Ùîá ïåðåâ³ðèòè ìîæ-ëèâó ó÷àñòü ì³êðîÐÍÊ ó ðåãóëÿö³¿ åêñïðåñ³¿ ³çî-ôîðì ïðè äèôåðåíö³àö³¿ ì³îáëàñò³â ìè ïðîàíàë³çó-âàëèin silicoñàéòè çâ’ÿçóâàííÿ ì³êðîÐÍÊ â 3'-ÍÒÏ

ìÐÍÊ EEF1A1/A2. Âñòàíîâëåíî, ùî â ìÐÍÊ ³çî-ôîðìè EEF1A1 ìîæóòü ³ñíóâàòè ñàéòè çâ’ÿçóâàííÿ mir-133, mir-543, mir-33, à â ìÐÍÊ EEF1A2 – ñàéòè çâ’ÿçóâàííÿ mir-744, mir-661, mir-675. Ñë³ä çàóâà-æèòè, ùî 3'-ÍÒÏ ìÐÍÊ EEF1A1 íà â³äì³íó â³ä EEF1A2 ì³ñòèòü ñàéò çâ’ÿçóâàííÿ äëÿ mir-133, êëþ-÷îâî¿ ì³êðîÐÍÊ ïðè äèôåðåíö³àö³¿ ì³îáëàñò³â [16].

Êîðåëÿö³ÿ çì³íè åêñïðåñ³¿ ì³êðîÐÍÊ ç ïîðóøåí-íÿì åêñïðåñ³¿ ö³ëüîâèõ á³ëê³â º îäíèì ³ç ôàêò³â, ùî

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Ðèñ. 1. Çì³íà åêñïðåñ³¿ ³çîôîðì ôàêòîðà åëîíãàö³¿ òðàíñëÿö³¿ eEF1 ï³ä ÷àñ äèôåðåí- ö³àö³¿ ì³îáëàñò³â. ʳëüê³ñíèé ÏËÐ-àíàë³ç ìÐÍÊ EEF1A1/A2 â ïðîë³ôåðóþ÷èõ ì³îáëàñòàõ (1) òà äèôåðåíö³éîâàíèõ

ñâ³ä÷èòü íà êîðèñòü íàÿâíîñò³ ïîñòòðàíñêðèïö³é-íîãî êîíòðîëþ äàíèõ á³ëê³â. ʳëüê³ñíèé ÏËÐ-àíà-ë³ç ð³âíÿ åêñïðåñ³¿ âñ³õ ì³êðîÐÍÊ, ïåðåäáà÷åíèõ á³î³íôîðìàòè÷íèì ìåòîäîì, âèÿâèâ 500-ðàçîâå çðî-ñòàííÿ âì³ñòó mir-133 (ðèñ. 2). Òàêå çíà÷íå ï³äâè-ùåííÿ ìîæå ñòàòè ïðè÷èíîþ â òîìó ÷èñë³ ³ ïîñò-òðàíñêðèïö³éíî¿ áëîêàäè ìÐÍÊ EEF1A1. Äëÿ ³çî-ôîðìè À2 âèÿâëåíî äâ³ ì³êðîÐÍÊ (744 ³ mir-661), åêñïðåñ³ÿ ÿêèõ çíèæóºòüñÿ â ïðîöåñ³ äèôåðåí-ö³àö³¿ íà 13 ³ 36 % â³äïîâ³äíî. Çìåíøåííÿ âì³ñòó öèõ ì³êðîÐÍÊ êîðåëþº ³ç çá³ëüøåííÿì ê³ëüêîñò³ ìÐÍÊ EEF1A2 ïðè äèôåðåíö³àö³¿ (ðèñ.1).

Äëÿ ï³äòâåðäæåííÿ ³ñíóâàííÿ ïîñòòðàíñêðèï-ö³éíîãî êîíòðîëþ ïåðåìèêàííÿ ³çîôîðì ï³ä ÷àñ

äè-ôåðåíö³àö³¿ ì³îáëàñò³â ìè çàñòîñóâàëè ìåòîä ðåïîð-òåðíèõ ãåí³â. Ïðîë³ôåðóþ÷³ ì³îáëàñòè òà äèôåðåí-ö³éîâàí³ ì³îöèòè ï³ñëÿ øåñòè äí³â äèôåðåíö³àö³¿ òðàíñô³êóâàëè ðåïîðòåðíîþ ïëàçì³äîþ, ùî ì³ñòèòü 3'-ÍÒÏ ìÐÍÊ À1 àáî À2 â³äïîâ³äíî. Ðåçóëüòàòè åêc-ïåðèìåíòó âèÿâèëè ìàéæå äâîðàçîâå çðîñòàííÿ ð³â-íÿ ëþöèôåðàçè ó ðàç³ 3'-ÍÒÏ ìÐÍÊ A2 (ðèñ. 3), ùî êîðåëþº ç îòðèìàíèìè ðàí³øå äàíèìè ñòîñîâíî çá³ëüøåííÿ ê³ëüêîñò³ ìÐÍÊ À2 òà çìåíøåííÿ ð³âíÿ åêñïðåñ³¿ ì³êðîÐÍÊ, ÿê³ ïîòåíö³éíî ðåãóëþþòü À2 (ðèñ. 1, 2). Âîäíî÷àñ çàô³êñîâàíî äåÿêó ïîçèòèâíó ïîñòòðàíñêðèïö³éíó ðåãóëÿö³þ åêñïðåñ³¿ ðåïîðòåð-íîãî ãåíà äëÿ A1 (ðèñ. 3).

20-Ðàçîâà çì³íà åêñïðåñ³¿ À2 íà ð³âí³ ìÐÍÊ ³ äâîðàçîâà – ó ðàç³ çàñòîñóâàííÿ ðåïîðòåðíîãî âåê-òîðà ç âèêîðèñòàííÿì 3'-ÍÒÏ A2 ìîæóòü ñâ³ä÷èòè ïðî ïåâíèé âíåñîê ïîñòòðàíñêðèïö³éíî¿ ðåãóëÿö³¿ ó ïîçèòèâíèé êîíòðîëü åêñïðåñ³¿ ìÐÍÊ ³çîôîðìè À2 â ïðîöåñ³ äèôåðåíö³àö³¿ ì³îáëàñò³â.

Îòæå, ñòèìóëÿö³ÿ åêñïðåñ³¿ ìÐÍÊ EEF1A2 ïðè äèôåðåíö³àö³¿ â³äáóâàºòüñÿ ÿê íà òðàíñêðèïö³éíî-ìó, òàê ³ íà ïîñòðàíñêðèïö³éíîìó ð³âí³. Ðàçîì ç òèì, ïðèãí³÷åííÿ ³çîôîðìè À1, åêñïðåñ³ÿ ÿêî¿ çä³éñíþ-ºòüñÿ â óñ³õ òêàíèíàõ íà äóæå âèñîêîìó ð³âí³ [14], âî÷åâèäü ïîòðåáóº á³ëüø ïîòóæíèõ, àí³æ ì³êðî-ÐÍÊ, çàñîá³â ðåãóëÿö³¿. ijéñíî, íàâ³òü âèñîêîïðåñîâàíà mir-133 ó íàøèõ äîñë³äàõ íå ³íã³áóº åêñ-ïðåñ³¿ EEF1A1, ùî óçãîäæóºòüñÿ ç ë³òåðàòóðíèìè äàíèìè [17].

Âèñíîâêè. Îòðèìàí³ ðåçóëüòàòè ñâ³ä÷àòü ïðî

òå, ùî ³íäóêö³ÿ åêñïðåñ³¿ EEF1A2 ïðè äèôåðåíö³à-ö³¿ êîíòðîëþºòüñÿ íà òðàíñêðèïö³éíîìó ³ ïîñò-òðàíñêðèïö³éíîìó ð³âíÿõ. Êð³ì òîãî, ï³äâèùåííÿ

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Ðèñ. 2. Åêñïðåñ³ÿ ì³êðîÐÍÊ, äëÿ ÿêèõ ïåðåäáà÷åíî ñàéòè çâ’ÿçóâàííÿ â 3'-ÍÒÏ ìÐÍÊ EEF1A1/A2, ï³ä ÷àñ äèôå-ðåíö³àö³¿ ì³îáëàñò³â. ʳëüê³ñíèé ÏËÐ-àíàë³ç ì³êðîÐÍÊ ó ïðîë³ôåðóþ÷èõ ì³î-áëàñòàõ (1) òà äèôåðåíö³éîâàíèõ

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Ðèñ. 3. Ïîñòòðàíñêðèïö³éíèé êîíòðîëü åêñïðåñ³¿ ³çîôîðì ôàêòîðà åëîíãàö³¿ òðàíñëÿö³¿ eEF1 ó ïðîöåñ³ äèôåðåíö³àö³¿ ì³îáëàñò³â. Ïðîë³ôåðóþ÷³ ì³îáëàñòè (1) àáî äèôåðåíö³éîâàí³ ì³îöèòè (2) LHCN òðàíñô³êóâàëè ðåïîðòåðíèìè ïëàçì³äàìè, ÿê³ ì³ñòÿòü â³ä-êðèòó ðàìêó ç÷èòóâàííÿ ëþöèôåðàçè ³ 3'-ÍÒÏ EEF1A1/A2: à– pSICHECK+ eEF1A1 3'- ÍÒÏ;á–pSICHECK+ eEF1A2 3'-ÍÒÏ;â– pSICHECK-2;t-òåñò Ñò’þäåíòà ϳñëÿ 24 ãîä ³íêóáàö³¿ âèì³ðþâàëè

åêñïðåñ³¿ ìÐÍÊ ³çîôîðìè À2 òà ðåïîðòåðíîãî ãåíà, ùî ì³ñòèòü 3'-ÍÒÏ ìÐÍÊ EEF1À2, êîðåëþº ç³ çíè-æåííÿì åêñïðåñ³¿ mir-744 ³ mir-661 – ì³êðîÐÍÊ, äëÿ ÿêèõ ïåðåäáà÷åí³ ñàéòè çâ’ÿçóâàííÿ â 3'-ÍÒÏ ìÐÍÊ EEF1À2. Öå íàâîäèòü íà äóìêó, ùî äàí³ ì³êðîÐÍÊ áåðóòü ó÷àñòü ó ïîñòòðàíñêðèïö³éí³é ðåãóëÿö³¿ åêñ-ïðåñ³¿ äàíî¿ ³çîôîðìè. Íåñïîä³âàíèì âèÿâèëîñÿ ï³ä-âèùåííÿ åêñïðåñ³¿ ³çîôîðìè À1 ï³ñëÿ äèôåðåíö³à-ö³¿ ì³îáëàñò³â, ùî íå º õàðàêòåðíèì äëÿ äàíîãî ïðî-öåñó in vivo. Ìîæëèâèì ïîÿñíåííÿì öüîãî º

øòó÷-í³ñòü óìîâ äëÿ äèôåðåíö³àö³¿, à ñàìå – çàñòîñóâàííÿ ³íñóë³íó ÿê îäíîãî ç îñíîâíèõ ñêëàäîâèõ ñåðåäîâè-ùå äëÿ äèôåðåíö³àö³¿. Ïîêàçàíî, ùî öåé ãîðìîí ñòè-ìóëþº åêñïðåñ³þ ìÐÍÊ, ÿê³ ì³ñòÿòü îë³ãîï³ðèì³äè-íîâèé òðàêò â 5'-ÍÒÏ [18]. Ñàìå äî òàêèõ ìÐÍÊ íà-ëåæèòü ìÐÍÊ EEF1A1 [19].

Îòðèìàí³ äàí³ º ñóòòºâèì âíåñêîì ó ðîçóì³ííÿ ôóíäàìåíòàëüíîãî ìåõàí³çìó äèôåðåíö³àö³¿ ì³îáëà-ñò³â ³ ìîæóòü äîïîìîãòè ó ðîçêðèòò³ ìåõàí³çì³â ðîç-âèòêó òàêèõ ïàòîëîã³é, ÿê ðàê, îñê³ëüêè ïîÿâà ³çî-ôîðìè À2 ó íåõàðàêòåðíèõ äëÿ íå¿ òêàíèíàõ ïðè-çâîäèòü äî çëîÿê³ñíîãî ïåðåðîäæåííÿ êë³òèíè [10].

Ïèòàííÿ ñòîñîâíî íåãàòèâíî¿ ðåãóëÿö³¿ eEF1A1 ó ì’ÿçîâ³é ³ íåðâîâ³é òêàíèíàõ çàëèøàºòüñÿ â³äêðè-òèì ³ ïîòðåáóº ïîäàëüøîãî âèâ÷åííÿ.

A. A. Vislovukh1

, I. S. Groisman2

, A. V. El’skaya1

, B. S. Negrutskii1

, A. N. Polesskaya2

Transcriptional and post-transcriptional control of eEF1A2 expression during myoblast diffrerentiation

1_{The State Key Laboratory of Molecular and Cellular Biology} Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 2_{CNRS FRE 3377 and Univ. Paris-Sud, CEA}

Saclay, F-91191 Gif-sur-Yvette, France Summary

During postnatal development, the switch of the expression from iso-form A1 to the isoiso-form A2 of eukaryotic translation elongation factor (eEF1A) is observed in neuronal and muscle tissues. The switch of the expression is a vital fundamental process, as mutant mice, with the par-tial EEF1A2 deletion dies on the 28thday after birth. Mechanism of the inhibition of A1 and stimulation of A2 expression during the first days of postnatal development is unknown. The existence of potential miRNA binding sites in the 3'UTR of mRNAs encoding the isoforms assumes a post-transcriptional control of abovementioned phenomenon.Aim. To check the possibility of post-transcriptional regulation of the isoforms A1 and A2 expression during differentiation of the human immorta-lized myoblasts cell line LHCN.Methods. The level of gene expression was quantified by qPCR, the existence of post-transcriptional

regula-tion was demonstrated with Dual-Luciferase®Reporter Assay.Results. Using immortalized human myoblasts cell line LHCN, the induction of isoform A2 of eEF1 during differentiation of myoblasts was shown. The existence of transcriptional and post-transcriptional control of the abo-vementioned process was confirmed. Downregulation of mir-661 and mir-744 that have binding sites in the 3' UTR of EEF1A2 mRNA, du-ring differentiation suggests a potential role of microRNAs in the eEF1A2 induction during myoblast differentiation.Conclusions. Induction of A2 isoform of eEF1 during differentiation of myoblasts occurs on transcrip-tional and post-transcriptranscrip-tional level.

Keywords: eEF1A1, eEF1A2, immortalized human myoblasts LHCN, differentiation, microRNA.

À. À. Âèñëîâóõ, ². Ñ. Ãðîéñìàí, À. Â. Åëüñêàÿ, Á. Ñ. Íåãðóöêèé, À. Í. Ïîëåññêàÿ

Òðàíñêðèïöèîííûé è ïîñòòðàíñêðèïöèîííûé êîíòðîëü ýêñïðåññèè eEF1A2 â ïðîöåññå äèôôåðåíöèàöèè ìèîáëàñòîâ Ðåçþìå

 ïðîöåññå ïîñòíàòàëüíîãî ðàçâèòèÿ â íåéðîíàëüíîé è ìûøå÷-íîé òêàíÿõ ïðîèñõîäèò èçìåíåíèå ýêñïðåññèè èçîôîðìû À1 ôàê-òîðà ýëîíãàöèè òðàíñëÿöèè íà ýêñïðåññèþ èçîôîðìû À2. Ïåðå-êëþ÷åíèå ýêñïðåññèè èçîôîðì ÿâëÿåòñÿ æèçíåííî âàæíûì ôóí-äàìåíòàëüíûì ïðîöåññîì, ïîñêîëüêó ìóòàíòíûå ìûøè, ñîäåð-æàùèå äåëåöèþ ÷àñòè ãåíà EEF1A2, ïîãèáàþò íà 28-é äåíü ïîñëå ðîæäåíèÿ. Ìåõàíèçìû èíãèáèðîâàíèÿ ýêñïðåññèè À1 è ñòèìóëÿ-öèè À2 â òå÷åíèå ïåðâûõ äíåé ïîñòíàòàëüíîãî ðàçâèòèÿ íåèçâå-ñòíû. Íàëè÷èå ïîòåíöèàëüíûõ ñàéòîâ ñâÿçûâàíèÿ ìèêðîÐÍÊ â 3'-íåòðàíñëèðóåìûõ ïîñëåäîâàòåëüíîñòÿõ ìÐÍÊ èçîôîðì ôàêòî-ðà ýëîíãàöèè òôàêòî-ðàíñëÿöèè ïðåäóñìàòðèâàåò âîçìîæíîñòü ïîñò-òðàíñêðèïöèîííîãî êîíòðîëÿ äàííîãî ïðîöåññà.Öåëü. Ïðîâåðèòü âîçìîæíîñòü ïîñòòðàíñêðèïöèîííîé ðåãóëÿöèè ýêñïðåññèè èçî-ôîðì À1 è À2 âî âðåìÿ äèôôåðåíöèàöèè èììîðòàëèçîâàííûõ ìèî-áëàñòîâ ÷åëîâåêà LHCN.Ìåòîäû. Óðîâåíü ýêñïðåññèè ãåíîâ îïðå-äåëÿëè ìåòîäîì êîëè÷åñòâåííîãî ÏËÐ, íàëè÷èå ïîñòòðàíñêðèï-öèîííîãî êîíòðîëÿ äåòåêòèðîâàëè ìåòîäîì ðåïîðòåðíèõ ãåíîâ.

Ðåçóëüòàòû. Èñïîëüçóÿ êëåòî÷íóþ ëèíèþ èììîðòàëèçîâàííûõ ìèîáëàñòîâ ÷åëîâåêà LHCN êàê ìîäåëü, ïîêàçàíà èíäóêöèÿ èçî-ôîðìû À2 ôàêòîðà ýëîíãàöèè òðàíñëÿöèè eEF1 ïðè äèôôåðåí-öèàöèè ìèîáëàñòîâ, íàëè÷èå òðàíñêðèïöèîííîãî è ïîñòòðàíñ-êðèïöèîííîãî êîíòðîëÿ â äàííîì ïðîöåññå, à òàêæå ïîòåíöèàëü-íîå ó÷àñòèå ìèêðîÐÍÊ â ïðîöåññå èçìåíåíèÿ ýêñïðåññèè èçîôîðì.

Âûâîäû. Èíäóêöèÿ èçîôîðìû À2 ïðè äèôôåðåíöèàöèè ìèîáëà-ñòîâ ìîæåò ïðîèñõîäèòü êàê íà òðàíñêðèïöèîííîì, òàê è íà ïîñòòðàíñêðèïöèîííîì óðîâíå.

Êëþ÷åâûå ñëîâà: eEF1A1, eEF1A2, èììîðòàëèçîâàííûå ìèî-áëàñòû ÷åëîâåêà LHCN, äèôôåðåíöèàöèÿ, ìèêðîÐÍÊ.

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