Identification and functional analysis of an alternative promoter of human intersectin 1 gene

Intersectin 1 (ITSN1) gene encodes an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling, apoptosis and cytoskeleton rearrangements. Its expression is characterized by multiple alternative splicing. Alternative promoter usage is an additional wa...

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Datum:	2010
Hauptverfasser:	Kropyvko, S.V., Tsyba, L.O., Skrypkina, I.Ya., Rynditch, A.V.
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Sprache:	English
Veröffentlicht:	Інститут молекулярної біології і генетики НАН України 2010
Schriftenreihe:	Вiopolymers and Cell
Schlagworte:	Structure and Function of Biopolymers
Online Zugang:	http://dspace.nbuv.gov.ua/handle/123456789/153875
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spelling	irk-123456789-1538752019-07-06T20:26:08Z Identification and functional analysis of an alternative promoter of human intersectin 1 gene Kropyvko, S.V. Tsyba, L.O. Skrypkina, I.Ya. Rynditch, A.V. Structure and Function of Biopolymers Intersectin 1 (ITSN1) gene encodes an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling, apoptosis and cytoskeleton rearrangements. Its expression is characterized by multiple alternative splicing. Alternative promoter usage is an additional way to create diversity and flexibility in the regulation of gene expression. The aim of this study was to identify possible alternative promoters of ITSN1 gene. Methods. In silico prediction, 5' RACE, RT-PCR and reporter gene expression assay were used for identification and functional characterization of alternative promoter region. Results. We detected an alternative promoter of human ITSN1 gene which is located in intron 5 and generates 5' truncated transcripts containing in-frame ATG codon with strong Kozak sequence and could encode an N-terminally truncated isoforms lacking first EH domain. The region located 246–190 bp upstream of exon 6 is required for alternative promoter activity. ITSN1 transcripts generated from an alternative promoter were detected in human kidney, liver, lung and brain tissues. However, the level of their expression was significantly lower than that of major ITSN1 isoforms. Conclusion. The results obtained suggest that alternative promoter region located in intron 5 of ITSN1 gene functions as a weak promoter. Further experiments are required to clarify the role of 5' truncated ITSN1 transcripts. Ген інтерсектину 1 (ITSN1) кодує еволюційно консервативний адаптерний білок, причетний до клатрин-опосередкованого ендоцитозу, внутрішньоклітинної передачі сигналу, апоптозу та реорганізації цитоскелету. Його експресія пов‘язана з багатьмя подіями альтернативного сплайсингу. Додатковим способом досягнення різноманіття і тонкої регуляції експресії генів є пошук альтернативних промоторів. Мета роботи полягала у виявленні можливих альтернативних промоторів гена ITSN1. Методи. Застосоваано комп’ютерне передбачення, 5' RACE, RT-РCR і тест, оснований на аналізі експресії репортерного гена. Результати. Виявлено альтернативний промотор гена ITSN1 людини, локалізований у 5-му інтроні, внаслідок використання якого утворюються транскрипти з відкритою рамкою зчитування та консенсусною послідовністю Козак, здатні кодувати ITSN1-ізоформи без першого EH-домену. Визначено, що ділянка, розташована за 246–190 п. н. до початку 6-го екзона, необхідна для функціонування альтернативного промотору. Транскрипти ITSN1, що утворюються в результаті використання альтернативного промотору, знайдено в тканинах нирок, печінки, легень і мозку людини, проте рівень їхньої експресії значно нижчий порівняно з основними ізоформами ITSN1. Висновки. Отримані дані свідчать про те, що альтернативний промотор, локалізований у 5-му інтроні гена ITSN1 людини, функціонує як слабкий промотор. Подальші дослідження необхідні для з’ясування функції транскриптів ITSN1 з альтернативним 5'-кінцем. Ген интерсектина 1 (ITSN1) кодирует эволюционно консервативный адаптерный белок, участвующий в клатрин-опосредованном эндоцитозе, внутриклеточной передаче сигнала, апоптозе и реорганизации цитоскелета. Его экспрессия характеризуется многочисленными событиями альтернативного сплайсинга. Дополнительным способом достижения разнообразия и тонкой регуляции экспрессии генов является использование альтернативных промоторов. Цель данной работы состояла в обнаружении возможных альтернативных промоторов гена ITSN1. Методы. Применены компьютерное предсказание, 5' RACE, RT-PCR и тест, основанный на анализе экспрессии репортерного гена. Результаты. Нами выявлен альтернативный промотор гена ITSN1 человека, локализованный в 5-м интроне, в результате использования которого образуются транскрипты с открытой рамкой считывания и консенсусной последовательностью Козак, способные кодировать изоформы ITSN1 без первого ЕН-домена. Показано, что участок, находящийся за 246–190 п. н. до начала 6-го экзона, необходим для функционирования альтернативного промотора. Транскрипты ITSN1, полученные с альтернативного промотора, обнаружены в тканях почек, печени, легких и мозга человека, но уровень их экспрессии значительно ниже по сравнению с основными изоформами ITSN1. Выводы. Полученные результаты свидетельствуют о том, что альтернативный промотор, локализованный в 5-м интроне гена ITSN1, функционирует как слабый промотор. Дальнейшие исследования необходимы для выяснения функции транскриптов ITSN1 с альтернативным 5'-концом. 2010 Article Identification and functional analysis of an alternative promoter of human intersectin 1 gene / S.V. Kropyvko, L.O. Tsyba, I.Ya. Skrypkina, A.V. Rynditch // Вiopolymers and Cell. — 2010. — Т. 26, № 2. — С. 115-120. — Бібліогр.: 10 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00014D http://dspace.nbuv.gov.ua/handle/123456789/153875 577.214.5 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Structure and Function of Biopolymers Structure and Function of Biopolymers
spellingShingle	Structure and Function of Biopolymers Structure and Function of Biopolymers Kropyvko, S.V. Tsyba, L.O. Skrypkina, I.Ya. Rynditch, A.V. Identification and functional analysis of an alternative promoter of human intersectin 1 gene Вiopolymers and Cell
description	Intersectin 1 (ITSN1) gene encodes an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling, apoptosis and cytoskeleton rearrangements. Its expression is characterized by multiple alternative splicing. Alternative promoter usage is an additional way to create diversity and flexibility in the regulation of gene expression. The aim of this study was to identify possible alternative promoters of ITSN1 gene. Methods. In silico prediction, 5' RACE, RT-PCR and reporter gene expression assay were used for identification and functional characterization of alternative promoter region. Results. We detected an alternative promoter of human ITSN1 gene which is located in intron 5 and generates 5' truncated transcripts containing in-frame ATG codon with strong Kozak sequence and could encode an N-terminally truncated isoforms lacking first EH domain. The region located 246–190 bp upstream of exon 6 is required for alternative promoter activity. ITSN1 transcripts generated from an alternative promoter were detected in human kidney, liver, lung and brain tissues. However, the level of their expression was significantly lower than that of major ITSN1 isoforms. Conclusion. The results obtained suggest that alternative promoter region located in intron 5 of ITSN1 gene functions as a weak promoter. Further experiments are required to clarify the role of 5' truncated ITSN1 transcripts.
format	Article
author	Kropyvko, S.V. Tsyba, L.O. Skrypkina, I.Ya. Rynditch, A.V.
author_facet	Kropyvko, S.V. Tsyba, L.O. Skrypkina, I.Ya. Rynditch, A.V.
author_sort	Kropyvko, S.V.
title	Identification and functional analysis of an alternative promoter of human intersectin 1 gene
title_short	Identification and functional analysis of an alternative promoter of human intersectin 1 gene
title_full	Identification and functional analysis of an alternative promoter of human intersectin 1 gene
title_fullStr	Identification and functional analysis of an alternative promoter of human intersectin 1 gene
title_full_unstemmed	Identification and functional analysis of an alternative promoter of human intersectin 1 gene
title_sort	identification and functional analysis of an alternative promoter of human intersectin 1 gene
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2010
topic_facet	Structure and Function of Biopolymers
url	http://dspace.nbuv.gov.ua/handle/123456789/153875
citation_txt	Identification and functional analysis of an alternative promoter of human intersectin 1 gene / S.V. Kropyvko, L.O. Tsyba, I.Ya. Skrypkina, A.V. Rynditch // Вiopolymers and Cell. — 2010. — Т. 26, № 2. — С. 115-120. — Бібліогр.: 10 назв. — англ.
series	Вiopolymers and Cell
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first_indexed	2025-07-14T05:18:43Z
last_indexed	2025-07-14T05:18:43Z
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fulltext	STRUCTURE AND FUNCTION OF BIOPOLYMERS Identification and functional analysis of an alternative promoter of human intersectin 1 gene S. V. Kropyvko, L. O. Tsyba, I. Ya. Skrypkina, A. V. Rynditch Institute of Molecular Biology and Genetics NAS of Ukraine 150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680 rynditch@imbg.org.ua Aim. Intersectin 1 (ITSN1) gene encodes an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling, apoptosis and cytoskeleton rearrangements. Its expression is characterized by multiple alternative splicing. Alternative promoter usage is an additional way to create diversity and flexibility in the regulation of gene expression. The aim of this study was to identify possible alternative promoters of ITSN1 gene. Methods. In silico prediction, 5¢ RACE, RT-PCR and reporter gene expression assay were used for identification and functional characterization of alternative promoter region. Results. We detected an alternative promoter of human ITSN1 gene which is located in intron 5 and generates 5¢ truncated transcripts containing in-frame ATG codon with strong Kozak sequence and could encode an N-terminally truncated isoforms lacking first EH domain. The region located 246–190 bp upstream of exon 6 is required for alternative promoter activity. ITSN1 transcripts generated from an alternative promoter were detected in human kidney, liver, lung and brain tissues. However, the level of their expression was significantly lower than that of major ITSN1 isoforms. Conclusion. The results obtained suggest that alternative promoter region located in intron 5 of ITSN1 gene functions as a weak promoter. Further experiments are required to clarify the role of 5¢ truncated ITSN1 transcripts. Keywords: intersectin 1, alternative promoter, alternative splicing, 5¢ UTR, adaptor proteins. Introduction. Intersectin 1 (ITSN1) is a conserved adaptor protein implicated in clathrin-mediated endo- cytosis, apoptosis, signal transduction and cytoskele- ton organization (for a review see [1]). Its expression is characterized by multiple alternative splicing [2–4]. The major ITSN1 protein isoforms described in mam- mals are ubiquitously expressed short form, ITSN1-S, and the long form, ITSN1-L, that is mainly expressed in neurons. The short form consists of two N-terminal Eps15 homology domains (EH1 and EH2), a coiled- coil region and five Src homology domains (SH3 A– E). The long form contains C-terminal extension with a Dbl homology (DH), a pleckstrin homology (PH), and a C2 domains [1]. Recently, we described 15 additional ITSN1 transcriptional isoforms generated by different combinations of alternatively spliced exons [5]. Several lines of evidence suggested that a large fraction of human genes possesses multiple promoters which could be regulated in a different manner and complements alternative splicing in generating diffe- rent protein isoforms [6]. Here we report the identi- fication of an alternative promoter of ITSN1 gene which is located in intron 5 and generates 5¢ truncated ITSN1 transcripts. Materials and methods. RNA isolation and RT- PCR. Total RNA from human tissues was isolated as described previously [5]. cDNA was synthesized from 1 to 5 µg of total RNA using oligo(dT) primer and Expand Reverse Transcriptase («Roche», France). Five percent of the cDNA obtained was used as a template 115 ISSN 0233-7657. Biopolymers and Cell. 2010. Vol. 26. N 2 Ó Institute of Molecular Biology and Genetics NAS of Ukraine, 2010 for PCR as described previously [5]. The following oli- gonucleotides were used for expression analysis of transcripts generated from an alternative promoter: exon 5, forward – 543-ATCAGCTACCCTCTGCACTTCC-564; exon 9, reverse – 1041-TGAGCCTGTGGTAAACTTGACTGC-1018; AP210, forward – 35122238-GTTCTGTCTTCAGGTTGAGTC-35122258; b-actin, forward – 5¢-GAAATCGTGCGTGACATTAAG-3¢; b-actin, reverse – 5¢-AAGCATTTGCGGTGGACGATGGAG-3¢; GAPDH, forward – 5¢-TGAAGGTCGGAGTCAACGGATTTGGT-3¢; GAPDH, reverse – 5¢-CATGTGGGCCATGAGGTCCACCAC-3¢. For the detection of alternative splicing events in the 5¢ UTR the following oligonucleotides were used: exon 1, forward – 2-AGCAAGCTTGGGAGCGAAGGAGGT-25; exon 1à, forward – 35061642-TTCCCAAATGCGGCATCTGTGT-35061663; exon 1b, reverse – 35088748-GGGGTGTGAAGTGATCAACTCA-35088769; exon 6, reverse – 730-AGCTGCTGTGGGAACAGAAGAT-709. Nucleotide positions for oligonucleotides are based on the ITSN1-L and ITSN1-S cDNAs and genomic se- quence of chromosome 21 (GenBank accession num- bers NM_001001132.1, NM_003024.2 and NC_ 000021.8). 5¢ RACE was performed using human fetal brain and lung total RNAs and the 5¢/3¢ RACE kit («Roche») according to the manufacturer’s instructions. The pri- mers used for 5¢ RACE were 5¢-GGCCACATCAAATG ACTGT-3¢ and 5¢-ATTTCTTGCCTTTGGGTGGTC-3¢. Cell culture and transfection. The cell lines HeLa and HEK293 were grown at 37 oC in 5 % CO2 in Dul- becco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 50 U/ml penicillin and 100 mg/ml streptomycin. The cells were transfected using JetPEI (Polyplus Transfection). Cells grown in 12-well plates at 60–70 % confluence were transfected in duplicate with 3 mg reporter gene constructs and 300 ng of Renilla luciferase expression vector, pRL-TK. Cells were collected 42 h after transfection. Cell extracts were assayed for Firefly and Renilla luciferase activities using the Dual Luciferase Reporter Assay System («Promega», USA) and VICTOR3 Mul- tilabel Counter 1420 («PerkinElmer», USA). Relative luciferase activities were determined as the ratio of Firefly luciferase activity of each sample to Renilla lu- ciferase activity. Plasmids. For Dual Luciferase Reporter Assay, PCR products corresponding to the regions of intron 5 of ITSN1 gene were subcloned upstream the luciferase gene in pGL3-basic expression vector («Promega»). All PCR-generated DNA fragments were sequenced to confirm fidelity. Results and Discussion. Identification of alterna- tive transcription start sites in the human ITSN1 gene. To search the putative alternative promoters of ITSN1 gene we analyzed database of transcription start sites, DBTSS, based on experimentally determined 5¢ end clones [7] and found two clones with putative trans- cription start sites located in introns 5 and 12. Then we performed 5¢ RACEs on human fetal lung and brain total RNAs with antisense primers within exons 6 and 13. Discrete bands were obtained only with primers specific to exon 6. 5¢ ends of RACE products were located 343, 323 and 285 bp directly upstream of the exon 6 (Fig. 1, A). To verify the obtained results and more precisely map possible transcription start sites, RT-PCRs with different sense primers located at positions 36, 210, 338, 415 and 526 bp upstream of exon 6 and antisense primer within exon 9 were performed. The results suggest that alternative transcription start sites are located at positions between 285–415 bp upstream of exon 6 (Fig. 1, B). The 5¢ end of the longest 5¢ RACE product was designated as +1. Sequence analysis revealed the alternative in-frame ATG codon located in exon 6 (at position +347 bp) that contains strong Kozak sequence (GGTATGG) and could be considered as good candidate for the initiation of the translation. The resulting ORF could encode an N-terminally truncated version of ITSN1 that lacks first EH domain. Functional conformation of alternative promoter region using reporter gene analysis. To determine whether the putative alternative promoter region has functional promoter activity, three fragments of the 116 KROPYVKO S. V. ET AL. intron 5 spanning the region from –1157 to +153 bp were cloned into the promoterless pGL3-basic vector upstream of the firefly luciferase coding sequence (Fig. 2, A). Alternative promoter activity was analyzed in HeLa and HEK293 cells in comparison with control promoter of the herpes simplex virus thymidine kinase (pTK-Luc) and major ITSN1 promoter located up- stream of exon 1 (–997 +86 bp). The results of the luciferase assays showed that all three alternative promoter constructs were able to drive reporter gene expression in both cell lines. In HEK293 cells the activity of alternative promoter constructs was higher than in HeLa, however it was approximately 3,5-fold lower than the activity of TK promoter and 1,8-fold lower than that of the major ITSN1 promoter. In HeLa cells alternative promoter activity was 10-fold lower than the activity of the major promoter. Mapping the regions of promoter activity. We ge- nerated a series of deletion constructs in order to loca- lize regulatory sequence elements and the minimal pro- moter region. Deletion of the region located between +97 and –23 bp did not result in significant decrease of luciferase activity (Fig. 2, B). When HEK293 cells were transfected with pAP-283D1, pAP-283D2, pAP- 667D8, pAP-667D6 and pAP-1157D7 constructs, the promoter activity was slightly increased, thereby sug- gesting the presence of a negative regulatory element located between –675 and –23 bp. However, deletion of the segment between +97 and +153 bp caused mar- ked decreases of luciferase activity. These results imp- ly that the +97 to +153 bp region is required for alter- native ITSN1 promoter activity. Analysis of expression of ITSN1 transcripts gene- rated from an alternative promoter. The mammalian ITSN1 gene produces two major isoforms, ITSN1-S and ITSN1-L [1]. To analyze the expression of ITSN1 transcripts produced from an alternative promoter, nested RT-PCRs using a sense primer of intron 5 and ANALYSIS OF AN ALTERNATIVE PROMOTER OF HUMAN INTERSECTIN 1 GENE 117 Fig. 1. A – scheme of ITSN1 gene with domain organization of short isoform (exons of ITSN1 gene are numbered; partial sequence of intron 5 and exon 6 of human ITSN1 gene is shown; sequence of exon 6 is underlined; positions of 5' ends of RACE products as well as transcription start site predicted by DBTSS are indicated as arrows above the sequence; primers for 5' end mapping are indicated by arrows below the sequence; consensus Kozak sequence is boxed; region required for promoter activity (D4) is indicated by figure arrows above the sequence); B – mapping of the 3' end of ITSN1 transcripts generated from an alternative promoter using RT-PCR with AP415 and AP526 primers (control reactions without reverse transcriptase (–RT) are shown); C – analysis of expression of ITSN1 transcripts produced from the major (I) and alternative (II) promoters and control GAPDH gene (III) in fetal and adult human tissues; wk – week of fetus antisense primers specific for the short and long ITSN1 isoforms were performed. PCR products were detected only with primers specific to ITSN1-S isoform. Sub- sequent cloning and sequencing of these products showed that they represent two different splice variants of ITSN1-S (Fig. 3). A first transcript was amplified from fetal kidney. It lacks exons 25 and 26 that encode the SH3C domain involved in interaction with proline- rich motifs of dynamin 1, synaptojanin 1, SOS1, WNK-kinase and other proteins [8–10]. The second transcript was detected in fetal lung tissue and was characterized by unusual for ITSN1 combination of alternatively spliced exons. It contains elongated exon 12 due to the use of an alternative 3¢ splicing site located 22 nt upstream of the 5¢ end of exon 12 and lacks exons 25, 26 and 27. This combination of alter- native splicing events shifts the open reading frame and introduces stop codons. To examine further the expression of the ITSN1 transcripts, RT-PCR was performed on total RNAs from different tissues using primers specific for the amplification of ITSN1 mRNAs originating from the major and alternative promoters. The expression of ITSN1 transcripts generated from an alternative promoter was significantly lower than that of major ITSN1 isoforms. RT-PCR products of transcripts pro- duced from an alternative promoter were observed in fetal kidney, liver, lung and brain, as well as in adult kidney and ovary (Fig. 1, C). However, a second step of PCR amplification with nested primers revealed the presence of these transcripts in all tissues tested indica- ting the low level of their expression (data not shown). RT-PCR analysis and results of luciferase assays sug- gest that identified alternative promoter region func- tions as a weak promoter. We could not use Western blot analysis to study the expression of ITSN1 proteins encoded by transcripts from an alternative promoter since the molecular weight of these proteins coincides with that of alternatively spliced ITSN1-22a isoform. Alternative splicing affecting the 5¢ UTR of ITSN1 mRNA. 5¢ untranslated region (5¢ UTR) of human ITSN1 mRNA produced from the major promoter con- 118 KROPYVKO S. V. ET AL. Fig. 2. A – activity of ITSN1 alternative promoter in HeLa and HEK293 cells (cells were transfected with sequentially deleted reported constructs of the –1157 +153 bp sequence; transient transfection and luciferase assays were performed in triplicate; the data were normalized to renilla luciferase activity and are shown as relative activities compared to that for pTK-Luc; the mean values and standard deviations were calculated from three experiments); B – mapping of ITSN1 alternative promoter region (deletion constructs of ITSN1 alternative promoter region used for luciferase assay in cell line HEK293 are indicated) sists of two exons and could start from seven different transcriptional start sites according to CAGE-tags da- tabase. While analyzing GenBank Database we found two ESTs that had high homology with human ITSN1 gene and carried two insertions of 62 and 187 bp lo- cated between exons 1 and 2. The insertions are gene- rated due to addition of two exons of 62 and 125 bp that were designated as exons 1a and 1b (Fig. 4, A). The combination of exons 1a and 1b resulted in an insertion of 187 bp. Start-codon of ITSN1 is located in exon 2 and addition of two supplementary exons expands the total length of 5¢ UTR of ITSN1 mRNA and potentially could affect translation efficiency, mRNA transport and stability. To explore tissue specificity of ITSN1 transcripts with two supplementary exons, we performed RT-PCR and found that these isoforms are widely expressed in the majority of tissues without any significant tissue specificity (Fig. 4). Our further experiments showed that exons 1a and 1b are predominantly spliced in a mutually exclusive fashion. Using primers within exons 1a and 6 we obtained PCR product of 700 bp that corresponds to the isoform containing exon 1a, but failed to amplify the 825-bp PCR product containing both exons 1a and 1b (Fig. 4, B). To confirm these findings, we performed RT-PCR using sense primer within exon 1 and antisense primer specific to exon 1b. The results indicated the presence of the products of 269 bp representing the transcript with exon 1b and absence of the 331-bp product corresponding to the isoforms containing exons 1a and 1b (Fig. 4, C). Thus, we identified alternative promoter of ITSN1 gene that generates novel transcript variants and the- refore diversifies functions of endocytic adapter pro- tein ITSN1. The data concerning alternative splicing events within 5¢ UTR could be also important for fur- ther understanding of ITSN1 regulation at posttrans- criptional level. 119 ANALYSIS OF AN ALTERNATIVE PROMOTER OF HUMAN INTERSECTIN 1 GENE Fig. 3. Schematic representation of the ITSN1 transcripts produced from an alternative promoter (I, II) and domain structure of the two main isoforms, short (ITSN1-S) and long (ITSN1-L). ITSN1 exons are shown as black and white boxes and numbered above. Stop codons are marked by asterisks Fig. 4. A – schematic representation of splicing events affected 5' UTR of human ITSN1 mRNA; B – analysis of expression of ITSN1 isoforms containing exon 1a using primers specific for exons 1a and 6 (BI); C – analysis of expression of ITSN1 isoforms containing exon 1b using primers specific for exons 1 and 1b (CI); B, C (II) – analysis of expression of control genes GAPDH and b-actin C. Â. Êðî ïèâ êî, Ë. Î. Öèáà, ². ß. Ñêðèïê³íà, À. Â. Ðèí äè÷ ²äåí òèô³êàö³ÿ òà ôóíêö³îíàëü íèé àíàë³ç àëü òåð íà òèâ íî ãî ïðî ìî òîðó ãåíà ³íòåð ñåê òè íó 1 ëþ äè íè Ðå çþ ìå Ìåòà. Ãåí ³íòåð ñåê òè íó 1 (ITSN1) êîäóº åâî ëþö³éíî êîí ñåð âà - òèâíèé àäàï òåð íèé á³ëîê, ïðè ÷åò íèé äî êëàò ðèí-îïî ñå ðåä êî - âàíîãî åí äî öè òîçó, âíóòð³øíüîêë³òèííî¿ ïå ðå äà÷³ ñèã íà ëó, àïîï òîçó òà ðå îð ãàí³çàö³¿ öè òîñ êå ëå òó. Éîãî åêñïðåñ³ÿ ïî â‘ÿ - çàíà ç áà ãàòü ìÿ ïîä³ÿìè àëü òåð íà òèâ íî ãî ñïëàé ñèí ãó. Äî äàò - êîâèì ñïî ñî áîì äî ñÿã íåí íÿ ð³çíî ìàí³òòÿ ³ òîí êî¿ ðå ãó ëÿö³¿ åêñïðåñ³¿ ãåí³â º ïîøóê àëü òåð íà òèâ íèõ ïðî ìî òîð³â. Ìåòà ðî- áîòè ïî ëÿ ãà ëà ó âè ÿâ ëåíí³ ìîæ ëè âèõ àëü òåð íà òèâ íèõ ïðî ìî - òîð³â ãåíà ITSN1. Ìå òî äè. Çàñ òî ñî âà à íî êîì ï’þ òåð íå ïå ðåä- áà ÷åí íÿ, 5' RACE, RT-ÐCR ³ òåñò, îñíî âà íèé íà àíàë³ç³ åêñïðå- ñ³¿ ðå ïîð òåð íî ãî ãåíà. Ðå çóëü òà òè. Âè ÿâ ëå íî àëü òåð íà òèâ íèé ïðî ìî òîð ãåíà ITSN1 ëþ äè íè, ëî êàë³çî âà íèé ó 5-ìó ³íòðîí³, âíàñë³äîê âèêîðèñòàííÿ ÿêîãî óòâî ðþ þòü ñÿ òðàíñ êðèï òè ç â³äêðè òîþ ðàì êîþ ç÷è òó âàííÿ òà êîí ñåí ñóñ íîþ ïîñë³äîâ- í³ñòþ Êî çàê, çäàòí³ êî äó âà òè ITSN1-³çî ôîð ìè áåç ïåð øî ãî EH-äî ìå íó. Âèç íà ÷å íî, ùî ä³ëÿí êà, ðîç òà øîâàíà çà 246–190 ï. í. äî ïî ÷àò êó 6-ãî åê çî íà, íå îáõ³äíà äëÿ ôóíêö³îíó âàí íÿ àëü - òåð íà òèâ íî ãî ïðî ìî òî ðó. Òðà íñêðèï òè ITSN1, ùî óòâî ðþ - þòü ñÿ â ðå çóëü òàò³ âè êî ðèñ òàí íÿ àëüòåð íà òèâ íî ãî ïðî ìî- òîðó, çíàé äå íî â òêà íè íàõ íè ðîê, ïå÷³íêè, ëå ãåíü ³ ìîç êó ëþ äè - íè, ïðî òå ð³âåíü ¿õíüî¿ åêñïðåñ³¿ çíà÷ íî íè æ÷èé ïîð³âíÿ íî ç îñíîâ íè ìè ³çî ôîð ìà ìè ITSN1. Âèñ íîâêè. Îòðè ìàí³ äàí³ ñâ³ä- ÷àòü ïðî òå, ùî àëü òåð íà òèâ íèé ïðî ìî òîð, ëî êàë³çî âà íèé ó 5-ìó ³íòðîí³ ãåíà ITSN1 ëþ äè íè, ôóíêö³îíóº ÿê ñëàá êèé ïðî ìî - òîð. Ïî äàëüø³ äîñë³äæåí íÿ íå îáõ³äí³ äëÿ ç’ÿñó âàí íÿ ôóíêö³¿ òðàíñ êðèïò³â ITSN1 ç àëü òåð íà òèâ íèì 5'-ê³íöåì. Êëþ ÷îâ³ ñëî âà: ³íòåð ñåê òèí 1, àëü òåð íà òèâ íèé ïðî ìî òîð, àëü òåð íà òèâ íèé ñïëàé ñèíã, àäàï òåðí³ á³ëêè, 5’ UTR. Ñ. Â. Êðî ïèâ êî, Ë. A. Öèáà, È. ß. Ñêðèï êè íà, À. Â. Ðûí äè÷ Èäåí òè ôè êà öèÿ è ôóíê öè î íàëü íûé àíà ëèç àëü òåð íà òèâ íî ãî ïðî ìî òî ðà ãåíà èí òåð ñåê òè íà 1 ÷å ëî âå êà Ðå çþ ìå Öåëü. Ãåí èí òåð ñåê òè íà 1 (ITSN1) êî äè ðó åò ýâî ëþ öè îí íî êîí - ñåð âà òèâ íûé àäàï òåð íûé áå ëîê, ó÷àñ òâó þ ùèé â êëàò ðèí-îïî- ñðå äî âàí íîì ýí äî öè òî çå, âíóò ðèê ëå òî÷ íîé ïå ðå äà ÷å ñèã íà ëà, àïîï òî çå è ðå îð ãà íè çà öèè öè òîñ êå ëå òà. Åãî ýêñ ïðåñ ñèÿ õà ðàê - òå ðè çó åò ñÿ ìíî ãî ÷èñ ëåí íû ìè ñî áû òè ÿ ìè àëü òåð íà òèâ íî ãî ñïëàé ñèí ãà. Äî ïîë íè òåëü íûì ñïî ñî áîì äîñ òè æå íèÿ ðàç íî îá - ðàçèÿ è òîí êîé ðå ãó ëÿ öèè ýêñ ïðåñ ñèè ãå íîâ ÿâ ëÿ åò ñÿ èñ ïîëü çî - âà íèå àëü òåð íà òèâ íûõ ïðî ìî òî ðîâ. Öåëü äàí íîé ðà áî òû ñî- ñòî ÿ ëà â îá íà ðó æå íèè âîç ìîæ íûõ àëü òåð íà òèâ íûõ ïðî ìî òî - ðîâ ãåíà ITSN1. Ìå òî äû. Ïðè ìå íåíû êîì ïüþ òåð íîå ïðåä ñêà - çà íèå, 5' RACE, RT-PCR è òåñò, îñíî âàí íûé íà àíà ëè çå ýêñ- ïðå ññèè ðå ïîð òåð íî ãî ãåíà. Ðå çóëü òà òû. Íàìè âûÿâëåí àëü - òåð íà òèâ íûé ïðî ìî òîð ãåíà ITSN1 ÷å ëî âå êà, ëî êà ëè çî âàí íûé â 5-ì èí òðî íå, â ðå çóëü òà òå èñ ïîëü çî âà íèÿ êî òî ðî ãî îá ðà çó - þò ñÿ òðàíñ êðèï òû ñ îò êðû òîé ðàì êîé ñ÷è òû âà íèÿ è êîí ñåí - ñóñ íîé ïî ñëå äî âà òåëü íîñ òüþ Êî çàê, ñïîñîáíûå êî äè ðî âàòü èçî ôîð ìû ITSN1 áåç ïåð âî ãî ÅÍ-äî ìåíà. Ïî êà çà íî, ÷òî ó÷àñ - òîê, íà õî äÿ ùèé ñÿ çà 246–190 ï. í. äî íà ÷à ëà 6-ãî ýê çî íà, íå îá - õî äèì äëÿ ôóíê öè î íè ðî âà íèÿ àëü òåð íà òèâ íî ãî ïðî ìî òî ðà. Òðà íñêðèï òû ITSN1, ïî ëó ÷åí íûå ñ àëü òåð íà òèâ íî ãî ïðî ìî òî - ðà, îá íà ðó æå íû â òêà íÿõ ïî ÷åê, ïå ÷å íè, ëåãêèõ è ìîç ãà ÷å ëî âå - êà, íî óðî âåíü èõ ýêñ ïðåñ ñèè çíà ÷è òåëü íî íèæå ïî ñðàâ íå íèþ ñ îñíîâ íû ìè èçîôîð ìà ìè ITSN1. Âû âî äû. Ïî ëó ÷åí íûå ðå çóëü òà - òû ñâè äå ò åëüñòâó þò î òîì, ÷òî àëü òåð íà òèâ íûé ïðî ìî òîð, ëî êà ëè çî âàí íûé â 5-ì èí òðî íå ãåíà ITSN1, ôóíê öè î íè ðó åò êàê ñëà áûé ïðî ìî òîð. 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Identification and functional analysis of an alternative promoter of human intersectin 1 gene

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