Biomedical Chemistry: Research and Methods 2019, 2(8), e00108

Inhibition of Caspase-2 Activity in Human Jurkat T-Cell Lymphoma Cells by Splice Switching Oligonucleotide to its pre-mRNA

D.D. Zhdanov1,2*, A.A. Plyasova1, Yu.A. Gladilina1, M.V. Pokrovskaya1, S.S. Alexandrova1, N.N. Sokolov1

1Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia; *e-mail: zhdanovdd@mail.ru
2Peoples Friendship University of Russia, 6 Miklukho-Maklaya str., Moscow, 117198 Russia

Keywords: caspase-2; alternative splicing; splice switching oligonucleotide; enzymatic activity

DOI:10.18097/BMCRM00108

The whole version of this paper is available in Russian.

Caspase-2 is a key enzyme thinvolved in induction of apoptosis. The caspase-2 level is regulated by alternative splicing (AS) of its mRNA. The aim of this work was to determine the ability of an oligonucleotide complementary to Casp-2 pre-mRNA to induce AS. This oligonucleotide blocked the binding of splicing-regulating proteins to their sites at the end of exon 9 of Casp-2 pre-mRNA, leading to induction of AS of Casp-2 mRNA. The decrease in expression of full-size active splice-variant (Casp-2L) and the increase the expression of a shortened variant (Casp-2S) was demonstrated in human T-cell lymphoma Jurkat cell line. The expression level of total Casp-2 remained unchanged. Disproportion of splice variants of Casp-2 led to inhibition of enzymatic activity of caspase-2.
Figure 1. Hypothetical scheme of Casp-2 AS as a result of SSO action. Hypothetical scheme of Casp-2 mRNA AS (A). Inclusion of exon 9 in mRNA transcript leads to the formation of preliminary stop-codon and to the synthesis of truncated Casp-2S splice-variant. Schematic presentation of splicing-regulating protein SF2/ASF (shown as white ellipse) and SRp30c (shown as grey ellipse), which bound to splicing cites (shown in the black frames) on Casp-2 pre-mRNA (В). Base-pairing of SSO to Casp-2 pre-mRNA blocks the binding of both SF2/ASF and SRp30c to their functional sites (C). Such interaction results in induction of AS and inclusion of exon 9.
Figure 2. Efficiency of transfection of Jurkat cells. Representative flow cytometry diagrams of cells within 96 h after transfection with SSO (A) or control 30-mer oligonucleotide (B). Efficiency of transfection (C). MFI of Cy-5.5-positive cells. A.U. – Arbitrary units.
Figure 3. Induction of AS of Casp-2 mRNA and the inhibition of activity of caspase 2 as a result of cell transfection with SSO. mRNA levels of total Casp-2 (A) and its splice-variants: Casp-2L (B) and Casp-2S (C), which were determined by real-time RT-PCR. Enzymatic activity of caspase 2 in transfected cells. * p ≤ 0.05 vs. cells transfected with control 30-mer oligonucleotide.

CLOSE
Table 1. Oligonucleotides used for the transfection of Jurkat cells.

CLOSE
Table 2. Primers used for real-time RT-PCR.

FUNDING

The work was performed in the framework of the Program for Basic Research of the State Academies of Sciences for 2013–2020.

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