DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS

The DR3 «death receptor» plays an important role in the initiation of apoptosis, proliferation, or inflammation. This receptor is shown to be involved in various diseases, including infectious conditions. Different variants of mRNA DR3 are formed as a result of alternative splicing. These variant tr...

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Autores principales:	V. D. Cvetkova, N. A. Sakharnov, D. I. Knyazev, O. V. Utkin, N. V. Neumoina, N. F. Brusnigina
Formato:	article
Lenguaje:	RU
Publicado:	SPb RAACI 2016
Materias:	dr3/lard alternative splicing mrna variants infectious mononucleosis real-time pcr Immunologic diseases. Allergy RC581-607
Acceso en línea:	https://doaj.org/article/7157b68f4a474e82aa99183da806dfa0
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spelling	oai:doaj.org-article:7157b68f4a474e82aa99183da806dfa02021-11-18T08:03:45ZDETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS1563-06252313-741X10.15789/1563-0625-2016-2-139-150https://doaj.org/article/7157b68f4a474e82aa99183da806dfa02016-04-01T00:00:00Zhttps://www.mimmun.ru/mimmun/article/view/997https://doaj.org/toc/1563-0625https://doaj.org/toc/2313-741XThe DR3 «death receptor» plays an important role in the initiation of apoptosis, proliferation, or inflammation. This receptor is shown to be involved in various diseases, including infectious conditions. Different variants of mRNA DR3 are formed as a result of alternative splicing. These variant transcripts encode membrane and soluble forms of the receptor which have different functions. Features of their expression and contribution of individual DR3 variants to the immune pathogenesis of infectious mononucleosis (IM) are poorely understood.The purpose of this work was to develop, validate and test the techniques of DR3 gene expression assays, as well as to evaluate the DR3 mRNA splice variants by means of real-time RT-PCR and RT-PCR in the IM patients.The original version of real-time RT-PCR allowed to determine relative amounts of DR3 mRNA, DR3 membrane variants (LARD1a + LARD8), and ratios of mRNAs encoding membrane and soluble forms of the receptor. The technique proved to be specific and sensitive (a semi-quantitative detection limit = 34-35 cycles) when tested in healthy volunteers and patients with acute infectious mononucleosis (AIM). Lower expression levels were shown for two alternative membrane variants of DR3 mRNA (LARD1b and DR3beta) thus regarding these isoforms as minor fractions. The relative levels of total DR3 mRNA expression were decreased in patients with AIM, as compared to healthy volunteers, whereas mRNA expression of membrane receptor variants did not differ between IM and controls.To determine a qualitative contribution of either LARD1a and LARD8 variants into the expression of membrane forms of DR3, a two-step «nested» version of RT-PCR has been developed. It was shown that, in majority of control and IM samples, both main LARD1a, and alternative LARD8 membrane forms are contributing to mRNA expression of membrane DR3 variants.The presented methods for evaluation of expression and occurrence of DR3 mRNA variants allow to characterize changes in the expression and function of this receptor associated with infectious mononucleosis. Further investigations are required to test the technique in larger number of samples.V. D. CvetkovaN. A. SakharnovD. I. KnyazevO. V. UtkinN. V. NeumoinaN. F. BrusniginaSPb RAACIarticledr3/lardalternative splicingmrna variantsinfectious mononucleosisreal-time pcrImmunologic diseases. AllergyRC581-607RUMedicinskaâ Immunologiâ, Vol 18, Iss 2, Pp 139-150 (2016)
institution	DOAJ
collection	DOAJ
language	RU
topic	dr3/lard alternative splicing mrna variants infectious mononucleosis real-time pcr Immunologic diseases. Allergy RC581-607
spellingShingle	dr3/lard alternative splicing mrna variants infectious mononucleosis real-time pcr Immunologic diseases. Allergy RC581-607 V. D. Cvetkova N. A. Sakharnov D. I. Knyazev O. V. Utkin N. V. Neumoina N. F. Brusnigina DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS
description	The DR3 «death receptor» plays an important role in the initiation of apoptosis, proliferation, or inflammation. This receptor is shown to be involved in various diseases, including infectious conditions. Different variants of mRNA DR3 are formed as a result of alternative splicing. These variant transcripts encode membrane and soluble forms of the receptor which have different functions. Features of their expression and contribution of individual DR3 variants to the immune pathogenesis of infectious mononucleosis (IM) are poorely understood.The purpose of this work was to develop, validate and test the techniques of DR3 gene expression assays, as well as to evaluate the DR3 mRNA splice variants by means of real-time RT-PCR and RT-PCR in the IM patients.The original version of real-time RT-PCR allowed to determine relative amounts of DR3 mRNA, DR3 membrane variants (LARD1a + LARD8), and ratios of mRNAs encoding membrane and soluble forms of the receptor. The technique proved to be specific and sensitive (a semi-quantitative detection limit = 34-35 cycles) when tested in healthy volunteers and patients with acute infectious mononucleosis (AIM). Lower expression levels were shown for two alternative membrane variants of DR3 mRNA (LARD1b and DR3beta) thus regarding these isoforms as minor fractions. The relative levels of total DR3 mRNA expression were decreased in patients with AIM, as compared to healthy volunteers, whereas mRNA expression of membrane receptor variants did not differ between IM and controls.To determine a qualitative contribution of either LARD1a and LARD8 variants into the expression of membrane forms of DR3, a two-step «nested» version of RT-PCR has been developed. It was shown that, in majority of control and IM samples, both main LARD1a, and alternative LARD8 membrane forms are contributing to mRNA expression of membrane DR3 variants.The presented methods for evaluation of expression and occurrence of DR3 mRNA variants allow to characterize changes in the expression and function of this receptor associated with infectious mononucleosis. Further investigations are required to test the technique in larger number of samples.
format	article
author	V. D. Cvetkova N. A. Sakharnov D. I. Knyazev O. V. Utkin N. V. Neumoina N. F. Brusnigina
author_facet	V. D. Cvetkova N. A. Sakharnov D. I. Knyazev O. V. Utkin N. V. Neumoina N. F. Brusnigina
author_sort	V. D. Cvetkova
title	DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS
title_short	DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS
title_full	DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS
title_fullStr	DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS
title_full_unstemmed	DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS
title_sort	determination of level expression of mrna splicing variants for dr3 in blood cells in infectious mononucleosis
publisher	SPb RAACI
publishDate	2016
url	https://doaj.org/article/7157b68f4a474e82aa99183da806dfa0
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DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS

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